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1.
Inflammopharmacology ; 32(1): 561-573, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37921960

RESUMO

Nitro-conjugated linoleic acid (NO2-CLA) has been observed to manifest salutary signaling responses, including anti-inflammatory and antioxidant properties. Here, the authors have explored the influence and underlying mechanisms of NO2-CLA on the proinflammatory reaction of murine macrophages that were challenged with lipopolysaccharide (LPS) derived from Prevotella intermedia, a putative periodontopathic bacterium. Treatment of LPS-activated RAW264.7 cells with NO2-CLA notably dampened the secretion of iNOS-derived NO, IL-1ß and IL-6 as well as their gene expressions and significantly enhanced the markers for M2 macrophage polarization. NO2-CLA promoted the HO-1 expression in cells challenged with LPS, and tin protoporphyrin IX, an HO-1 inhibitor, significantly reversed the NO2-CLA-mediated attenuation of NO secretion, but not IL-1ß or IL-6. We found that cells treated with NO2-CLA significantly increased mRNA expression of PPAR-γ compared to control cells, and NO2-CLA significantly reverted the decrease in PPAR-γ mRNA caused by LPS. Nonetheless, antagonists to PPAR-γ were unable to reverse the NO2-CLA-mediated suppression of inflammatory mediators. In addition, NO2-CLA did not alter the p38 and JNK activation elicited by LPS. Both NF-κB reporter activity and IκB-α degradation caused by LPS were notably diminished by NO2-CLA. NO2-CLA was observed to interrupt the nuclear translocation and DNA binding of p50 subunits caused by LPS with no obvious alterations in p65 subunits. Further, NO2-CLA attenuated the phosphorylation of STAT1/3 elicited in response to LPS. We propose that NO2-CLA could be considered as a possible strategy for the therapy of periodontal disease, although additional researches are certainly required to confirm this.


Assuntos
Ácidos Linoleicos Conjugados , Lipopolissacarídeos , Animais , Camundongos , Lipopolissacarídeos/farmacologia , Prevotella intermedia/química , Interleucina-6/metabolismo , Ácidos Linoleicos Conjugados/farmacologia , Ácidos Linoleicos Conjugados/metabolismo , Dióxido de Nitrogênio/metabolismo , Dióxido de Nitrogênio/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/farmacologia , Macrófagos , RNA Mensageiro/metabolismo
2.
Cells ; 12(3)2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36766836

RESUMO

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1ß), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.


Assuntos
Dióxido de Nitrogênio , Fator A de Crescimento do Endotélio Vascular , Humanos , Dióxido de Nitrogênio/metabolismo , Dióxido de Nitrogênio/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Ependimogliais/metabolismo , Gliose/metabolismo , Estresse Oxidativo , Hipóxia/metabolismo , Inflamação/metabolismo , RNA Mensageiro/metabolismo
3.
Microbiol Spectr ; 10(6): e0324722, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36326495

RESUMO

Phosphopantetheinyl transferases (PPTases) play important roles in activating apo-acyl carrier proteins (apo-ACPs) and apo-peptidyl carrier proteins (apo-PCPs) in both primary and secondary metabolism. PPTases catalyze the posttranslational modifications of those carrier proteins by covalent attachment of the 4'-phosphopantetheine group to a conserved serine residue. The protein-protein interactions between a PPTase and a cognate acyl or peptidyl carrier protein have important regulatory functions in microbial biosynthesis, but the molecular mechanism underlying their specific recognition remains elusive. In this study, we identified a new rishirilide biosynthetic gene cluster with a rare in-cluster PPTase from Streptomyces xanthophaeus no2. The function of this Sfp-type PPTase, SxrX, in rishirilide production was confirmed using genetic mutagenesis and biochemical characterization. We applied molecular modeling and site-directed mutagenesis to identify key residues mediating the protein-protein interaction between SxrX and its cognate ACP. In addition, six natural products were isolated from wild-type S. xanthophaeus no2 and the ΔsxrX mutant, including rishirilide A and lupinacidin A, that exhibited antimicrobial and anticancer activities, respectively. SxrX is the first Sfp-type PPTase identified from an aromatic polyketide biosynthetic gene cluster and shown to be responsible for high-level production of rishirilide derivatives. IMPORTANCE Genome mining has been a vital means for natural product drug discovery in the postgenomic era. The rishirilide-type polyketides have attracted attention due to their potent bioactivity, but the poor robustness of production hosts has limited further research and development. This study not only identifies a hyperproducer of rishirilides but also reveals a rare, in-cluster PPTase SxrX that plays an important role in boosting rishirilide biosynthesis. Experimental and computational investigations revealed new insights on the protein-protein interaction between SxrX and its cognate ACP with wide implications for understanding polyketide biosynthesis.


Assuntos
Proteínas de Bactérias , Dióxido de Nitrogênio , Dióxido de Nitrogênio/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo
4.
Nutr Res ; 106: 35-46, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36126528

RESUMO

The hypothesis of the present study was that nitro-fatty acids (NO2-FAs) would suppress inflammation associated with periodontal disease. To test this hypothesis, we investigated the influence of nitrooleic acid, a prototypical NO2-FA, on the inflammatory response of murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated the etiology of different types of periodontal diseases. LPS was prepared from P. intermedia cells by using phenol-water protocol. Culture supernatants were assayed for nitric oxide (NO), interleukin-1ß (IL-1ß), and IL-6. Real-time polymerase chain reaction and immunoblotting analyses were performed to quantify messenger RNA and protein expression, respectively. The secreted embryonic alkaline phosphatase reporter assay was performed to measure NF-κB activation. The transcription factor assay kit was used to measure DNA-binding of NF-κB subunits. Findings obtained from the present study revealed that nitrooleic acid suppresses the generation and messenger RNA expression of inducible NO synthase-derived NO, IL-1ß, and IL-6 in RAW264.7 cells activated with P. intermedia LPS and promotes macrophage polarization toward anti-inflammatory M2 phenotype. We also found that nitrooleic acid exerts its effect via heme oxygenase-1 induction and suppression of NF-κB signaling. The inhibition of NO and proinflammatory cytokine production by nitrooleic acid was independent from PPAR-γ, JNK, p38, and STAT1/3. Nitrooleic acid may represent a novel class of agent as a host modulator which has therapeutic benefit in periodontal disease, though more work is needed to confirm this.


Assuntos
Lipopolissacarídeos , Doenças Periodontais , Fosfatase Alcalina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , DNA , Ácidos Graxos/farmacologia , Heme Oxigenase-1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Dióxido de Nitrogênio/metabolismo , Dióxido de Nitrogênio/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/farmacologia , Fenóis/farmacologia , Prevotella intermedia/genética , Prevotella intermedia/metabolismo , RNA Mensageiro , Água/metabolismo , Água/farmacologia
5.
Appl Environ Microbiol ; 88(16): e0102322, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35916514

RESUMO

Linear nitramines are potentially carcinogenic environmental contaminants. The NnlA enzyme from Variovorax sp. strain JS1663 degrades the nitramine N-nitroglycine (NNG)-a natural product produced by some bacteria-to glyoxylate and nitrite (NO2-). Ammonium (NH4+) was predicted as the third product of this reaction. A source of nonheme FeII was shown to be required for initiation of NnlA activity. However, the role of this FeII for NnlA activity was unclear. This study reveals that NnlA contains a b-type heme cofactor. Reduction of this heme-either by a nonheme iron source or dithionite-is required to initiate NnlA activity. Therefore, FeII is not an essential substrate for holoenzyme activity. Our data show that reduced NnlA (FeII-NnlA) catalyzes at least 100 turnovers and does not require O2. Finally, NH4+ was verified as the third product, accounting for the complete nitrogen mass balance. Size exclusion chromatography showed that NnlA is a dimer in solution. Additionally, FeII-NnlA is oxidized by O2 and NO2- and stably binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with heme-binding PAS domains. Furthermore, a structural homology model of NnlA was generated using the PAS domain from Pseudomonas aeruginosa Aer2 as a template. The structural homology model suggested His73 is the axial ligand of the NnlA heme. Site-directed mutagenesis of His73 to alanine decreased the heme occupancy of NnlA and eliminated NNG activity, validating the homology model. We conclude that NnlA forms a homodimeric heme-binding PAS domain protein that requires reduction for initiation of the activity. IMPORTANCE Linear nitramines are potential carcinogens. These compounds result from environmental degradation of high-energy cyclic nitramines and as by-products of carbon capture technologies. Mechanistic understanding of the biodegradation of these compounds is critical to inform strategies for their remediation. Biodegradation of NNG by NnlA from Variovorax sp. strain JS 1663 requires nonheme iron, but its role is unclear. This study shows that nonheme iron is unnecessary. Instead, our study reveals that NnlA contains a heme cofactor, the reduction of which is critical for activating NNG degradation activity. These studies constrain the proposals for NnlA reaction mechanisms, thereby informing mechanistic studies of degradation of anthropogenic nitramine contaminants. In addition, these results will inform future work to design biocatalysts to degrade these nitramine contaminants.


Assuntos
Heme , Dióxido de Nitrogênio , Compostos Ferrosos/metabolismo , Heme/metabolismo , Proteínas Ligantes de Grupo Heme , Ferro/metabolismo , Óxido Nítrico/metabolismo , Dióxido de Nitrogênio/metabolismo
6.
Chem Commun (Camb) ; 57(34): 4162-4165, 2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33908442

RESUMO

Two mitochondria-localized Ru(ii) complexes with photo-labile ligands were reported to exert one- and two-photon activatable anticancer activity through a dual-function mechanism, i.e. mitochondrial DNA covalent binding after photo-induced ligand dissociation and photo-catalyzed NADH depletion, thus displaying good activity towards cisplatin-resistant cancer cells under both normoxic and hypoxic conditions.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , DNA Mitocondrial/efeitos dos fármacos , NAD/antagonistas & inibidores , Dióxido de Nitrogênio/metabolismo , Rutênio/farmacologia , Células A549 , Antineoplásicos/química , Antineoplásicos/metabolismo , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Dano ao DNA , DNA Mitocondrial/metabolismo , Humanos , Ligantes , Estrutura Molecular , NAD/análise , NAD/metabolismo , Processos Fotoquímicos , Fótons , Rutênio/química , Rutênio/metabolismo
7.
Mol Pharm ; 16(10): 4223-4229, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31536368

RESUMO

The effects of spherical nucleic acid (SNA) gold nanoparticle conjugates on the activation of macrophages in vitro and release of cytokines in vivo were explored. Herein, we show that G-quadruplexes, the formation of which is enhanced on gold nanoparticle surfaces, elicit an increase in cytokine release from mouse and human macrophages and induce the upregulation of activation receptors as well as NO2 production in vitro. Moreover, these G-rich SNAs can induce cytokine release when injected intravenously, though there were no severe, long-term effects observed. These results further reinforce the notion that nucleic acid sequence and structure play an important role in how SNAs interact in biological milieu and highlight a key design parameter.


Assuntos
Quadruplex G , Ouro/química , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Nanopartículas Metálicas/administração & dosagem , Ácidos Nucleicos/química , Animais , Células Cultivadas , Citocinas/metabolismo , Humanos , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos C57BL , Dióxido de Nitrogênio/metabolismo , Ácidos Nucleicos/metabolismo
8.
Sci Rep ; 8(1): 15418, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337623

RESUMO

Cold Atmospheric Plasma (CAP) is an ionized gas with a near room temperature. CAP is a controllable source for reactive species, neutral particles, electromagnetic field and UV radiation. CAP showed the promising application in cancer treatment through the demonstration in vitro and in vivo. In this study, we first demonstrate the existence of an activation state on the CAP-treated cancer cells, which drastically decreases the threshold of cell vulnerability to the cytotoxicity of the CAP-originated reactive species such as H2O2 and NO2-. The cytotoxicity of CAP treatment is still dependent on the CAP-originated reactive species. The activation state of cancer cells will not cause noticeable cytotoxicity. This activation is an instantaneous process, started even just 2 s after the CAP treatment begins. The noticeable activation on the cancer cells starts 10-20 s during the CAP treatment. In contrast, the de-sensitization of activation takes 5 hours after the CAP treatment. The CAP-based cell activation explains the mechanism by which direct CAP treatment causes a much stronger cytotoxicity over the cancer cells compared with an indirect CAP treatment do, which is a key to understand what the effect of CAP on cancer cells.


Assuntos
Adenocarcinoma/metabolismo , Sobrevivência Celular , Peróxido de Hidrogênio/metabolismo , Dióxido de Nitrogênio/metabolismo , Óxidos de Nitrogênio/metabolismo , Neoplasias Pancreáticas/metabolismo , Gases em Plasma/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Temperatura Baixa , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas
9.
J Neuroimmunol ; 302: 10-19, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27956075

RESUMO

Chronic and acute central nervous system (CNS) inflammation are contributors toward neurological injury associated with head trauma, stroke, infection, Parkinsons or Alzheimers disease. CNS inflammatory illnesses can also contribute toward risk of developing glioblastoma multiforme (GBM). With growing public interest in complementary and alternative medicines (CAMs), we conduct a high throughput (HTP) screening of >1400 natural herbs, plants and over the counter (OTC) products for anti-inflammatory effects on lipopolysaccharide (LPS)/interferon gamma (IFNγ) activated C6 glioma cells. Validation studies were performed showing a pro-inflammatory profile of [LPS 3 µg/ml/ IFNγ 3 ng/ml] consistent with greater release [>8.5 fold] of MCP-1, NO2-, cytokine-induced neutrophil chemo-attractants (CINC) 1, CINC 2a and CINC3. The data show no changes to the following, IL-13, TNF-a, fracktaline, leptin, LIX, GM-CSF, ICAM1, L-Selectin, activin A, agrin, IL-1α, MIP-3a, B72/CD86, NGF, IL-1b, MMP-8, IL-1 R6, PDGF-AA, IL-2, IL-4, prolactin R, RAGE, IL-6, Thymus Chemokine-1, CNTF,IL-10 or TIMP-1. A HTP screening was conducted, where we employ an in vitro efficacy index (iEI) defined as the ratio of toxicity (LC50)/anti-inflammatory potency (IC50). The iEI was precautionary to ensure biological effects were occurring in fully viable cells (ratio > 3.8) independent of toxicity. Using NO2- as a guideline molecule, the data show that 1.77% (25 of 1410 tested) had anti-inflammatory effects with iEI ratios >3.8 and IC50s <250µg/ml. These include reference drugs (hydrocortisone, dexamethasone N6-(1-iminoethyl)-l-lysine and NSAIDS: diclofenac, tolfenamic acid), a histone deacetylase inhibitor (apicidin) and the following natural products; Ashwaganda (Withania somnifera), Elecampagne Root (Inula helenium), Feverfew (Tanacetum parthenium), Green Tea (Camellia sinensis), Turmeric Root (Curcuma longa) Ganthoda (Valeriana wallichii), Tansy (Tanacetum vulgare), Maddar Root (Rubia tinctoria), Red Sandle wood (Pterocarpus santalinus), Bay Leaf (Laurus nobilis, Lauraceae), quercetin, cardamonin, fisetin, EGCG, biochanin A, galangin, apigenin and curcumin. The herb with the largest iEI was Ashwaganda where the IC50/LC50 was 11.1/>1750.0µg/ml, and the compound with the greatest iEI was quercetin where the IC50/LC50 was 10.0/>363.6µg/ml. These substances also downregulate the production of iNOS expression and attenuate CINC-3 release. In summary, this HTP screening provides guideline information about the efficacy of natural products that could prevent inflammatory processes associated with neurodegenerative disease and aggressive glioma tumor growth.


Assuntos
Produtos Biológicos/farmacologia , Quimiocina CXCL2/metabolismo , Citocinas/metabolismo , Glioma/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Dióxido de Nitrogênio/metabolismo , Animais , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quimiocina CXCL2/antagonistas & inibidores , Citocinas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Glioma/tratamento farmacológico , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Neutrófilos , Dióxido de Nitrogênio/antagonistas & inibidores , Ratos
10.
Int Arch Allergy Immunol ; 171(1): 27-35, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27820923

RESUMO

Asthma and allergies are both major global health problems with an increasing prevalence, and environmental data implicate an influence of air pollutants on their development. The present study focuses on the influence of nitrogen dioxide (NO2) and the major allergen of the house dust mite Der p 1 on human nasal epithelial cells of nonallergic patients in vitro. Nasal epithelial mucosa samples of 11 donors were harvested during nasal air passage surgery and cultured as an air-liquid interface. Exposure to 0.1, 1 and 10 ppm NO2 or synthetic air as a control was performed for 1 h. Subsequently, the cells were exposed to Der p 1 for 24 h. The release of interleukin (IL)-6 and IL-8 was measured by ELISA, and the production of IL-6 mRNA and IL-8 mRNA was measured by RT-PCR. NO2 exposure resulted in a concentration-dependent release of IL-6, but not IL-8 release. The coexposure of 0.1 ppm NO2 and Der p 1, or 1 ppm NO2 and Der p 1 significantly increased both IL-6 and IL-8 release. Exposure to NO2, Der p 1, or their combination, did not significantly influence the production of IL-6 or IL-8 mRNA. In conclusion, NO2 increases the release of inflammatory cytokines in human nasal epithelial cells, especially in coexposure with Der p 1, as a mechanism of allergotoxicology.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Dióxido de Nitrogênio/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Hipersensibilidade/genética , Imunização , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo
11.
Plant Signal Behav ; 11(7): e1197464, 2016 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-27301959

RESUMO

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr(36) of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.


Assuntos
Nicotiana/metabolismo , Nitratos/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Dióxido de Nitrogênio/metabolismo , Folhas de Planta/genética , Proteínas de Plantas/genética , Nicotiana/genética , Vacúolos/metabolismo
12.
Bioprocess Biosyst Eng ; 38(8): 1447-53, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25759162

RESUMO

Some of the noxious atmospheric pollutants such as nitrogen and sulfur dioxides come from the fossil fuel combustion. Biodesulfurization and biodenitrogenation are processes which remove those pollutants through the action of microorganisms. The ability of sulfur and nitrogen removal by the strain Rhodococcus erythropolis ATCC 4277 was tested in a biphasic system containing different heavy gas oil concentrations in a batch reactor. Heavy gas oil is an important fraction of petroleum, because after passing through, the vacuum distillation is incorporated into diesel oil. This strain was able to remove about 40% of the nitrogen and sulfur present in the gas heavy oil. Additionally, no growth inhibition occurred even when in the presence of pure heavy gas oil. Results present in this work are considered relevant for the development of biocatalytic processes for nitrogen and sulfur removal toward building feasible industrial applications.


Assuntos
Gases/metabolismo , Dióxido de Nitrogênio/metabolismo , Petróleo , Rhodococcus/crescimento & desenvolvimento , Dióxido de Enxofre/metabolismo
13.
Free Radic Biol Med ; 69: 172-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24447894

RESUMO

The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10(5) and (1.1±0.1)×10(6) M(-1) s(-1), respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10(6) M(-1) s(-1) at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10(7) M(-1) s(-1) at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly-most probably via reaction at the solvent-accessible heme edge-whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.


Assuntos
Citocromos c/metabolismo , Ferro/química , Dióxido de Nitrogênio/química , Oxirredução , Aminoácidos/química , Citocromos c/química , Heme/química , Heme/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Dióxido de Nitrogênio/metabolismo , Radiólise de Impulso , Triptofano/análogos & derivados , Triptofano/química , Tirosina/análogos & derivados , Tirosina/química
14.
Plant Physiol Biochem ; 67: 154-61, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23562799

RESUMO

In this study we aimed to determine and elucidate the effect of ambient air pollution on the foliar antioxidant system and stable carbon and nitrogen isotopes of white willow (Salix alba L.). We grew white willow in uniform potting soil in the near vicinity of sixteen air quality monitoring stations in Belgium where nitrogen dioxide (NO2), ozone, sulfur dioxide and particulate matter concentrations were continuously measured. The trees were exposed to ambient air during six months (April-September 2011), and, thereafter, the degree of lipid peroxidation and foliar content of antioxidant molecules (ascorbate, glutathione, polyphenols, flavonoids), antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, peroxidase) and foliar stable carbon (δ(13)C) and nitrogen (δ(15)N) isotopes were measured. We found that lipid peroxidation was caused by air pollution stress, arising from high ambient NO2 concentrations, as shown by an increased amount of malondialdehyde. The antioxidant system was activated by increasing the amount of polyphenols at monitoring stations with a high atmospheric NO2 and low O3 concentration, while no increase of key enzymes (e.g., ascorbate, glutathione) was observed. The δ(13)C also decreased with increasing NO2 concentrations and decreasing O3 concentrations, probably reflecting a decreased net photosynthesis and/or a concomitant decrease of (13)CO2 in the atmosphere. Shade also influenced foliar δ(13)C and the content of leaf ascorbate and glutathione.


Assuntos
Poluição do Ar/efeitos adversos , Antioxidantes/metabolismo , Isótopos de Carbono/metabolismo , Isótopos de Nitrogênio/metabolismo , Salix/efeitos dos fármacos , Salix/metabolismo , Ácido Ascórbico/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Dióxido de Nitrogênio/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo
15.
Free Radic Biol Med ; 57: 221-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23123410

RESUMO

Ras GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. Similar to the action of guanine-nucleotide exchange factors, the rate of GDP dissociation can be greatly enhanced by the reaction of Ras with small-molecule redox agents, such as nitrogen dioxide, which can promote Ras activation. Nitrogen dioxide is an autoxidation product of nitric oxide and can react with an accessible cysteine of Ras to cause oxidation of the bound guanine nucleotide to facilitate Ras guanine nucleotide dissociation. Glutathione has also been reported to modify Ras and alter its activity. To elucidate the mechanism by which glutathione alters Ras guanine nucleotide binding properties, we performed NMR, top-down and bottom-up mass spectrometry, and biochemical analyses of glutathiolated Ras. We determined that treatment of H-Ras, lacking the nonconserved hypervariable region, with oxidized glutathione results in glutathiolation specifically at cysteine 118. However, glutathiolation does not alter Ras structure or biochemical properties. Rather, changes in guanine nucleotide binding properties and Ras activity occur upon exposure of Ras to free radicals, presumably through the generation of a cysteine 118 thiyl radical. Interestingly, Ras glutathiolation protects Ras from further free radical-mediated activation events. Therefore, glutathiolation does not affect Ras activity unless Ras is modified by glutathione through a radical-mediated mechanism.


Assuntos
Glutationa/química , Glutationa/metabolismo , Proteínas ras/metabolismo , Ativação Enzimática , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Óxido Nítrico/química , Dióxido de Nitrogênio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Espécies Reativas de Oxigênio/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo
16.
Environ Monit Assess ; 184(5): 3095-107, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21713481

RESUMO

Conventional blanket application of nitrogen (N) fertilizer results in more loss of N from soil system and emission of nitrous oxide, a greenhouse gas (GHG). The leaf color chart (LCC) can be used for real-time N management and synchronizing N application with crop demand to reduce GHG emission. A 1-year study was carried out to evaluate the impact of conventional and LCC-based urea application on emission of nitrous oxide, methane, and carbon dioxide in a rice-wheat system of the Indo-Gangetic Plains of India. Treatments consisted of LCC scores of ≤4 and 5 for rice and wheat and were compared with conventional fixed-time N splitting schedule. The LCC-based urea application reduced nitrous oxide emission in rice and wheat. Application of 120 kg N per hectare at LCC ≤ 4 decreased nitrous oxide emission by 16% and methane by 11% over the conventional split application of urea in rice. However, application of N at LCC ≤ 5 increased nitrous oxide emission by 11% over the LCC ≤ 4 treatment in rice. Wheat reduction of nitrous oxide at LCC ≤ 4 was 18% as compared to the conventional method. Application of LCC-based N did not affect carbon dioxide emission from soil in rice and wheat. The global warming potential (GWP) were 12,395 and 13,692 kg CO(2) ha(-1) in LCC ≤ 4 and conventional urea application, respectively. Total carbon fixed in conventional urea application in rice-wheat system was 4.89 Mg C ha(-1) and it increased to 5.54 Mg C ha(-1) in LCC-based urea application (LCC ≤ 4). The study showed that LCC-based urea application can reduce GWP of a rice-wheat system by 10.5%.


Assuntos
Poluição do Ar/prevenção & controle , Monitoramento Ambiental/métodos , Oryza/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimento , Ureia/química , Agricultura , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Aquecimento Global/prevenção & controle , Efeito Estufa/prevenção & controle , Metano/análise , Metano/metabolismo , Nitrogênio/metabolismo , Ciclo do Nitrogênio , Dióxido de Nitrogênio/análise , Dióxido de Nitrogênio/metabolismo , Óxido Nitroso/análise , Óxido Nitroso/metabolismo , Oryza/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Triticum/metabolismo
17.
Toxicol In Vitro ; 26(1): 81-5, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22056765

RESUMO

Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins.


Assuntos
Heme/metabolismo , Nitritos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Hemina/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Dióxido de Nitrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Peroxidases/metabolismo
18.
Org Biomol Chem ; 9(10): 3733-45, 2011 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-21451865

RESUMO

Collision induced dissociation (CID) of sodiated peptide derivatives containing a nitrate ester functionality was used to regiospecifically generate three isomeric radicals of the model peptide Bz-Ala-Gly-OMe corresponding to radicals formed at: C(α) of the alanine residue [4+Na](+); C(α) of the glycine residue [5+Na](+); and the side chain of alanine [6+Na](+). The ion-molecule reactions of these peptide radicals were examined to model oxidative damage to peptides and to probe whether the radical sites maintain their integrity or whether they isomerise via intramolecular hydrogen atom transfer (HAT). Only [6+Na](+) is reactive towards O(2), forming the peroxyl radical [7+Na](+), which loses O(2), HO˙ and HO(2)˙ under CID. The radical ion [7 + Na](+) abstracts a hydrogen atom from 4-fluorothiophenol to form the hydroperoxide [8+Na](+), which upon CID fragments via the combined loss of HO˙ and CH(2)O. In contrast, all three of the isomeric sodiated radicals react with NO˙ and NO(2)˙ to form adducts. CID of the NO adducts only regenerates the radicals via NO˙ loss, thus providing no structural information. In contrast, CID of the NO(2) adducts gives rise to a range of product ions and the spectra are different for each of the three adducts, suggesting that the isomeric radicals [4+Na](+), [5+Na](+) and [6+Na](+) are produced as discrete species. Finally, CID of the NO(2) adducts was used to probe the rearrangement of the radicals [4+Na](+), [5+Na](+) and [6+Na](+) prior to their reaction with NO(2)˙: [6 + Na](+) rearranges to a mixture of [4+Na](+) and [5+Na](+) while [5+Na](+) rearranges to [4+Na](+).


Assuntos
Modelos Biológicos , Estresse Oxidativo , Oxigênio/química , Peptídeos/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Dióxido de Nitrogênio/química , Dióxido de Nitrogênio/metabolismo , Oxigênio/metabolismo , Peptídeos/metabolismo , Peróxidos/química , Peróxidos/metabolismo , Estereoisomerismo , Especificidade por Substrato
19.
Methods Mol Biol ; 720: 409-25, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21318889

RESUMO

Helicobacter pylori is a Gram-negative bacteria that infects the human stomach of half of the world's -population. Colonization is followed by infiltration of the gastric mucosa by lymphocytes and myeloid cells. These cells are activated by various bacterial factors, causing them to produce immune/inflammatory mediators, including reactive nitrogen species and polyamines that contribute to cellular damage and the pathogenesis of H. pylori-associated gastric cancer. In vitro experiments have revealed that H. pylori induces macrophage polyamine production by upregulation of the arginase 2/ornithine decarboxylase (ODC) metabolic pathway and enhances hydrogen peroxide synthesis through the activity of spermidine oxidase (SMO). In this chapter, we present a survey of the methods used to analyze the induction and the role of the enzymes related to polyamine metabolism, i.e., arginase, ODC, and SMO in H. pylori-infected macrophages.


Assuntos
Bioquímica/métodos , Infecções por Helicobacter/metabolismo , Helicobacter pylori/fisiologia , Poliaminas/metabolismo , Acetiltransferases/metabolismo , Animais , Apoptose , Arginase/metabolismo , Arsenicais/metabolismo , Células Cultivadas , Ensaios Enzimáticos , Infecções por Helicobacter/enzimologia , Humanos , Immunoblotting , Luciferases/metabolismo , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Dióxido de Nitrogênio/metabolismo , Ornitina Descarboxilase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Poliamina Oxidase
20.
Free Radic Biol Med ; 50(1): 196-205, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21034811

RESUMO

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Oxidantes/metabolismo , Ácido Peroxinitroso/metabolismo , Catálise , Avaliação de Medicamentos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/farmacologia , Ácido Hipocloroso/farmacologia , Técnicas In Vitro , Dióxido de Nitrogênio/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Taurina/análogos & derivados , Taurina/metabolismo , Taurina/farmacologia
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