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1.
Cell Chem Biol ; 24(11): 1321-1335.e5, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-28943355

RESUMO

NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.


Assuntos
Boro/química , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Células da Medula Óssea/citologia , Boro/farmacologia , Compostos de Boro/química , Compostos de Boro/metabolismo , Compostos de Boro/farmacologia , Cálcio/metabolismo , Células Cultivadas , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Conformação Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Relação Estrutura-Atividade
2.
MAGMA ; 30(2): 153-163, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27785640

RESUMO

OBJECTIVES: In the present study, we have tested whether MRI T1 relaxation time is a sensitive marker to detect early stages of amyloidosis and gliosis in the young 5xFAD transgenic mouse, a well-established animal model for Alzheimer's disease. MATERIALS AND METHODS: 5xFAD and wild-type mice were imaged in a 4.7 T Varian horizontal bore MRI system to generate T1 quantitative maps using the spin-echo multi-slice sequence. Following immunostaining for glial fibrillary acidic protein, Iba-1, and amyloid-ß, T1 and area fraction of staining were quantified in the posterior parietal and primary somatosensory cortex and corpus callosum. RESULTS: In comparison with age-matched wild-type mice, we observed first signs of amyloidosis in 2.5-month-old 5xFAD mice, and development of gliosis in 5-month-old 5xFAD mice. In contrast, MRI T1 relaxation times of young, i.e., 2.5- and 5-month-old, 5xFAD mice were not significantly different to those of age-matched wild-type controls. Furthermore, although disease progression was detectable by increased amyloid-ß load in the brain of 5-month-old 5xFAD mice compared with 2.5-month-old 5xFAD mice, MRI T1 relaxation time did not change. CONCLUSIONS: In summary, our data suggest that MRI T1 relaxation time is neither a sensitive measure of disease onset nor progression at early stages in the 5xFAD mouse transgenic mouse model.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Imageamento por Ressonância Magnética , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Corpo Caloso/diagnóstico por imagem , Modelos Animais de Doenças , Progressão da Doença , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Córtex Sensório-Motor/diagnóstico por imagem
3.
PLoS One ; 11(9): e0162497, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27598576

RESUMO

Microglial priming and enhanced reactivity to secondary insults cause substantial neuronal damage and are hallmarks of brain aging, traumatic brain injury and neurodegenerative diseases. It is, thus, of particular interest to identify mechanisms involved in microglial priming. Here, we demonstrate that priming of microglia with interferon-γ (IFN γ) substantially enhanced production of reactive oxygen species (ROS) following stimulation of microglia with ATP. Priming of microglial ROS production was substantially reduced by inhibition of p38 MAPK activity with SB203580, by increases in intracellular glutathione levels with N-Acetyl-L-cysteine, by blockade of NADPH oxidase subunit NOX2 activity with gp91ds-tat or by inhibition of nitric oxide production with L-NAME. Together, our data indicate that priming of microglial ROS production involves reduction of intracellular glutathione levels, upregulation of NADPH oxidase subunit NOX2 and increases in nitric oxide production, and suggest that these simultaneously occurring processes result in enhanced production of neurotoxic peroxynitrite. Furthermore, IFNγ-induced priming of microglial ROS production was reduced upon blockade of Kir2.1 inward rectifier K+ channels with ML133. Inhibitory effects of ML133 on microglial priming were mediated via regulation of intracellular glutathione levels and nitric oxide production. These data suggest that microglial Kir2.1 channels may represent novel therapeutic targets to inhibit excessive ROS production by primed microglia in brain pathology.


Assuntos
Interferon gama/farmacologia , Microglia/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glutationa/agonistas , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Glicoproteínas/farmacologia , Imidazóis/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Microglia/citologia , Microglia/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/agonistas , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Peroxinitroso/agonistas , Ácido Peroxinitroso/antagonistas & inibidores , Ácido Peroxinitroso/biossíntese , Fenantrolinas/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Piridinas/farmacologia , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Nat Commun ; 7: 12504, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27509875

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase-1 (COX-1) and COX-2 enzymes. The NLRP3 inflammasome is a multi-protein complex responsible for the processing of the proinflammatory cytokine interleukin-1ß and is implicated in many inflammatory diseases. Here we show that several clinically approved and widely used NSAIDs of the fenamate class are effective and selective inhibitors of the NLRP3 inflammasome via inhibition of the volume-regulated anion channel in macrophages, independently of COX enzymes. Flufenamic acid and mefenamic acid are efficacious in NLRP3-dependent rodent models of inflammation in air pouch and peritoneum. We also show therapeutic effects of fenamates using a model of amyloid beta induced memory loss and a transgenic mouse model of Alzheimer's disease. These data suggest that fenamate NSAIDs could be repurposed as NLRP3 inflammasome inhibitors and Alzheimer's disease therapeutics.


Assuntos
Doença de Alzheimer/prevenção & controle , Anti-Inflamatórios não Esteroides/farmacologia , Ácido Flufenâmico/farmacologia , Inflamassomos/metabolismo , Ácido Mefenâmico/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doença de Alzheimer/metabolismo , Animais , Células da Medula Óssea/metabolismo , Morte Celular , Canais de Cloreto/metabolismo , Cisteína/metabolismo , Feminino , Genótipo , Inflamação , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reconhecimento Visual de Modelos/efeitos dos fármacos , Ratos
5.
NMR Biomed ; 23(2): 163-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19757478

RESUMO

The aim of this work was to investigate the effect of experimental conditions on the visibility of polyamines. In solution the chemical shift of the three groups of peaks (at approximately 1.8, 2.1 and 3.1 ppm) were found to be pH dependent. Relaxation times in aqueous solution at pH 7.0, 298 K and 11.74 T were measured to be: putrescine (T(1) = 2.49 s, T(2) = 2.07 s), spermidine (T(1) = 1.27 s, T(2) = 1.05 s) and spermine (T(1) = 1.02 s, T(2) = 0.82 s). Simple spin-echo sequences could not be used to measure T(2) as the spins also experience phase evolution from homonuclear coupling which imposes a modulation on the T(2) decay curve. This modulation is eliminated by using CPMG sequences with an echo spacing of <500 micros. Relaxation times for spermine in solution in presence of metal ions and protein showed that metal ions had little effect on T(2); however, addition of 15 mg/ml bovine serum albumin reduced T(2) of spermine (0.41 s at 298 K and 0.19 s at 277 K) but was not as short as the T(2) of the polyamine peak in prostatic tissue (0.03 s at 277 K). The MR visibility of polyamines in prostate cell extracts, PC-3 xenograft (intact as well as extracted) and intact human prostatic tissues were investigated. Polyamines were not detected in methanol/chloroform extracts, but were visible in perchloric acid extracts of prostate tumour cells. No polyamines were detected in the HR MAS spectra of three samples of whole PC-3 xenograft tissue studied. In summary, the chemical shift of polyamine species is pH dependent, while protein binding causes peak broadening and reduction in T(2). Perchloric acid extraction improves visibility of intracellular polyamines, but whole tissue polyamines are not seen in xenografts without epithelial/ ductal structure.


Assuntos
Extratos Celulares/química , Espectroscopia de Ressonância Magnética , Espermina/análise , Extratos de Tecidos/química , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Masculino , Metaboloma , Camundongos , Putrescina/química , Soluções , Espermidina/química , Espermina/química , Transplante Heterólogo
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