RESUMO
By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours âafter the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H2O2, and O2-), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress, and microstructural damage in Chagas disease.
Assuntos
2,4-Dinitrofenol , Cardiomiopatia Chagásica , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Trypanosoma cruzi , Animais , 2,4-Dinitrofenol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Trypanosoma cruzi/efeitos dos fármacos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Desacopladores/farmacologia , Desacopladores/toxicidade , Camundongos , Miocárdio/patologia , Miocárdio/metabolismo , Nitroimidazóis/farmacologia , Doença Aguda , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Miocardite/parasitologia , Miocardite/metabolismo , Miocardite/tratamento farmacológico , Miocardite/patologia , Miocardite/induzido quimicamente , Doença de Chagas/tratamento farmacológico , Doença de Chagas/metabolismo , Doença de Chagas/patologia , Doença de Chagas/parasitologiaRESUMO
2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20 µM DNP (or vehicle) for 4 days. Significant increases in the proportion of nestin- and ß-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.
Assuntos
2,4-Dinitrofenol/farmacologia , Corpos Embrioides/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/citologia , 2,4-Dinitrofenol/química , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Proteína Glial Fibrilar Ácida , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina/metabolismo , Neurônios/metabolismo , Consumo de Oxigênio , Tretinoína/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg(2+)ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi>ADP·AlF(4)(-)>ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.
Assuntos
2,4-Dinitrofenol/farmacologia , Corantes/farmacologia , Lisina/metabolismo , Subfragmentos de Miosina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Fluorescência , Corantes Fluorescentes , Sequências Hélice-Alça-Hélice , Subfragmentos de Miosina/química , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , CoelhosRESUMO
Preventing the harm caused by nerve degeneration is a major challenge in neurodegenerative diseases and in various forms of trauma to the nervous system. The aim of the current work was to investigate the effects of systemic administration of 2,4-dinitrophenol (DNP), a compound with newly recognized neuroprotective properties, on sciatic-nerve degeneration following a crush injury. Sciatic-nerve injury was induced by unilateral application of an aneurysm clip. Four groups of mice were used: uninjured, injured treated with vehicle (PBS), injured treated with two intraperitoneal doses of DNP (0.06 mg DNP/kg every 24 h), and injured treated with four doses of DNP (every 12 h). Animals were sacrificed 48 h post injury and both injured and uninjured (contralateral) sciatic nerves were processed for light and electron microscopy. Morphometric, ultrastructural, and immunohistochemical analysis of injured nerves established that DNP prevented axonal degeneration, blocked cytoskeletal disintegration, and preserved the immunoreactivity of amyloid precursor protein (APP) and Neuregulin 1 (Nrg1), proteins implicated in neuronal survival and myelination. Functional tests revealed preservation of limb function following injury in DNP-treated animals. Results indicate that DNP prevents nerve degeneration and suggest that it may be a useful small-molecule adjuvant in the development of novel therapeutic approaches in nerve injury.
Assuntos
2,4-Dinitrofenol/farmacologia , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Degeneração Walleriana/tratamento farmacológico , 2,4-Dinitrofenol/uso terapêutico , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Esquema de Medicação , Feminino , Camundongos , Neuregulina-1/efeitos dos fármacos , Neuregulina-1/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/complicações , Neuropatia Ciática/fisiopatologia , Degeneração Walleriana/fisiopatologia , Degeneração Walleriana/prevenção & controleRESUMO
2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.
Assuntos
2,4-Dinitrofenol/farmacologia , AMP Cíclico/genética , Hipocampo/metabolismo , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Hipocampo/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Microcin J25 targets the RNA polymerase as well as bacterial membranes. Because there is scarce information on the relationship between the uptake and the activity, a fluorescent microcin J25-derivative was used to further characterize its mechanism of action. METHODS: MccJ25 I13K was labeled with FITC and its uptake by sensitive cells was assessed by fluorescence measurements from supernatants of MccJ25-Escherichia coli suspensions. The interaction of the peptide with bacterial membranes was investigated by fluorescence resonance energy transfer. Oxygen consumption was measured with Clark-type electrode. RNA synthesis was evaluated in vivo by incorporation of [3H]uridine. ROS production was assayed by measuring the fluorescence emission of the ROS-sensitive probe 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. RESULTS: The protonophore 2,4-dinitrophenol decreased 80% of the MccJ25 uptake and prevented inhibition of transcriptional activity, the antibiotic intracellular target. On the other hand, peptide binding to bacterial membranes was not affected and antibacterial activity remained nearly unchanged. Proton gradient dissipation by protonophore accelerated cell oxygen consumption rates and enhanced MccJ25-related reactive oxygen species overproduction. GENERAL SIGNIFICANCE: The deleterious reactive oxygen species would be produced as a consequence of the minor fraction of MccJ25 that interacts with the bacterial plasma membrane from the periplasmic side. These results show the first evidence of the mechanism underlying ROS production in sensitive bacteria.
Assuntos
Bacteriocinas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 2,4-Dinitrofenol/farmacologia , Substituição de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação , Consumo de Oxigênio/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Força Próton-Motriz , Desacopladores/farmacologiaRESUMO
We have previously characterized a number of small molecule organic compounds that prevent the aggregation of the beta-amyloid peptide and its neurotoxicity in hippocampal neuronal cultures. We have now evaluated the effects of such compounds on amyloid precursor protein (APP) accumulation in the CTb immortalized cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down's syndrome. Compared to a non-trisomic cortical cell line (CNh), CTb cells overexpress APP and exhibit slightly elevated resting intracellular Ca2+ levels ([Ca2+] inverted exclamation mark). Here, we show that the compounds 2,4-dinitrophenol, 3-nitrophenol and 4-anisidine decreased intracellular accumulation of APP in CTb cells. Those compounds were non-toxic to the cells, and slightly increased the basal [Ca2+] inverted exclamation mark. Results indicate that the compounds tested can be leads for the development of drugs to decrease intracellular vesicular accumulation of APP in trisomic cells.
Assuntos
2,4-Dinitrofenol/farmacologia , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Compostos de Anilina/farmacologia , Síndrome de Down/metabolismo , Nitrofenóis/farmacologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , CamundongosRESUMO
Caloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals.
Assuntos
2,4-Dinitrofenol/farmacologia , Metabolismo Energético/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Desacopladores/farmacologia , Animais , Glicemia/metabolismo , Respiração Celular/efeitos dos fármacos , Feminino , Insulina/sangue , Camundongos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Triglicerídeos/sangueRESUMO
We have evaluated the induction of the flocculent phenotype of Kloeckera apiculata by glucose mc1 and propose a pathway involved in carbohydrate flocculation induction. Pulses of glucose were given to cells growing in glucose-poor medium (2 g l(-1)) and the flocculation percentage was measured. To elucidate the mechanism involved in flocculation induction, cycloheximide was injected into the cultures 120 min before the glucose pulse. 2,4-Dinitrophenol or cAMP was added to the media instead, or simultaneously with glucose, while a protein kinase A (PKA) inhibitor was added 30 min before the glucose pulse. With 20 and 50 g l(-1) glucose pulse, the yeast flocculation percentage arises to 55 and 65%, respectively. The quantity of proteins and the reflocculating capacity of a lectinic protein extract from the yeast cell wall increase as the concentration of glucose pulse was higher. Cycloheximide prevented the glucose-induced flocculation, while cAMP or 2,4-dinitrophenol increased it 4- and 5-fold, respectively. PKA inhibitor completely prevented the glucose induction flocculation. The flocculent phenotype of K. apiculata mc1 was induced by glucose and the mechanism seems to imply de novo protein (lectin) synthesis via the PKA transduction pathway. This work contributes to the elucidation of the mechanism involved in flocculation induction by glucose of a non-Saccharomyces wine yeast, K. apiculata, which has not been reported. The induction of flocculation by glucose could be a biotechnological tool for the early removal of the indigenous microorganisms from the grape must before the inoculation of a selected starter strain to conduct the alcohol fermentation.
Assuntos
Adesão Celular , Glucose/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/fisiologia , 2,4-Dinitrofenol/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloeximida/farmacologia , Floculação , Proteínas Fúngicas/metabolismo , Lectinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Saccharomycetales/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
One of the earliest manifestations of Alzheimer's disease (AD) is the characteristic inability of affected individuals to form new memories. Memory impairment appears to significantly predate the death of nerve cells, implying that neuronal dysfunction is responsible for the pathophysiology of early stage AD. Mounting evidence now indicates that soluble oligomers of the amyloid-beta peptide (Abeta) are the main neurotoxins that lead to early neuronal dysfunction and memory deficits in AD. Cyclic AMP (cAMP) is a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory. Among other actions, cAMP triggers the phosphorylation and activation of the cAMP responsive element binding protein (CREB), a transcription factor that regulates the expression of genes that are important for long-term memory. Here, we discuss recent evidence suggesting that cAMP enhancing compounds may find applications as neurocognitive enhancers in AD and in other neurological disorders, as well as possible roles of cAMP in the regulation of neuronal regeneration. In particular, we review recent results showing that low concentrations of 2,4-dinitrophenol (DNP) upregulate neuronal cAMP and tau levels, promote neurite outgrowth and neuronal differentiation and block the oligomerization and neurotoxicity of Abeta. Possible implications of these findings in the development of novel therapeutic approaches in AD are discussed.