RESUMO
Published studies have shown that cognitive deficit is a characteristic manifestation of neurodegenerative disease in diabetes. However, there is no effective prevention and treatment for this diabetes-associated behavior disorder. In the present study, we attempted to elucidate the effect of zeaxanthin on cognitive deficit and the change in the hippocampus correlated with cognitive decline in diabetic rats. Diabetic rats in this study were induced by high-fat diet and low-dose streptozocin (STZ), cognitive ability of rats were evaluated use morris water maze (MWM) and morphology change in hippocampus was assessed by cresyl violet stain. Moreover, we detected the expression of phosphorylated serine/threonine kinase (p-AKT) and Cleaved caspase-3, and the activity of nuclear factor-κB (NF-κB) use western-blot (WB). Results displayed that supplementation with zeaxanthin reduce blood glucose, improve cognitive deficit, survive neural cell, increase p-AKT level, inhibit Cleaved caspase-3 level and NF-κB nuclear transcription in hippocampus. This study demonstrated that zeaxanthin ameliorate diabetes-related cognitive deficit may by means of protecting neural cell from hyperglycemia involved in AKT/NF-κB signaling pathway. This study may provide a potential therapeutic approach for the prevention of diabetes- associated cognitive deficit.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/enzimologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Nootrópicos/farmacologia , Zeaxantinas/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/psicologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Nootrópicos/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Zeaxantinas/químicaRESUMO
We have found that Fas/FasL-mediated "extrinsic" pathway promoted cell apoptosis induced by renal ischemic injury. This study is to elucidate the upstream mechanism regulating FasL-induced extrinsic pathway during renal ischemia/reperfusion. Results demonstrated that when SIRT2 was activated by renal ischemia/reperfusion, activated SIRT2 could bind to and deacetylate FOXO3a, promoting FOXO3a nuclear translocation which resulted in an increase of nuclear FOXO3a along with FasL expression and activation of caspase8 and caspase3, triggering cell apoptosis during renal ischemia/reperfusion. The administration of SIRT2 inhibitor AGK2 prior to renal ischemia decreased significantly the number of apoptotic renal tubular cells and alleviated ultrastructure injury. These results indicate that inhibition of FOXO3a deacetylation might be a promising therapeutic approach for renal ischemia /reperfusion injury.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Apoptose/fisiologia , Proteína Ligante Fas/fisiologia , Proteína Forkhead Box O3/metabolismo , Isquemia/patologia , Rim/irrigação sanguínea , Traumatismo por Reperfusão/patologia , Sirtuína 2/fisiologia , Acetilação , Animais , Núcleo Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Furanos/farmacologia , Isquemia/metabolismo , Rim/patologia , Masculino , Processamento de Proteína Pós-Traducional , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismoRESUMO
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/Siah1 signaling pathway has been recognized as a sensor of nitric oxide (NO). It is associated with a variety of injurious conditions, suggesting its therapeutic potential for spinal cord injury (SCI). Sivelestat sodium (SIV), a neutrophil elastase (NE) inhibitor initially used to treat acute lung injury, has been known to protect against compression-induced and ischemic SCI. However, little is known about the relationship between the GAPDH/Siah1 cascade and SIV. Thus, we aimed to assess the role of GAPDH/Siah1 cascade in traumatic SCI and its possible link with SIV. Rats were assigned to four groups: sham group, SCI group, 5-mg/kg SIV group, and 10-mg/kg SIV. The traumatic SCI was induced by dropping a 10-g impactor from a height of 25mm on the dorsal surface of T9 and T10. SIV was injected intraperitoneally immediately after surgery. Our results showed that the nuclear translocation of GAPDH was induced together with the nuclear translocation of Siah1 and the formation of the GAPDH/Siah1 complex in the spinal cord after traumatic SCI. However, the activation of the GAPDH/Siah1 cascade was attenuated by treatment with SIV. We also found that SIV suppressed apoptosis, NE and inducible nitric oxide synthase (iNOS) protein expressions, the number of NE and iNOS immunostained cells, the production of interleukin (IL)-1ß and tumor necrosis factor-alpha (TNF-α), and the activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) signaling in the spinal cord. The behavioral tests showed that SIV promoted functional recovery after traumatic SCI as reflected in the sustained increase in the Basso-Beattie-Bresnahan (BBB) scores throughout the observation period. In conclusion, our results reveal GAPDH/Siah1 as a novel signaling pathway during the progression of SCI, which can be blocked by SIV.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicina/análogos & derivados , Fármacos Neuroprotetores/farmacologia , Proteínas Nucleares/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Sulfonamidas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Glicina/farmacologia , Interleucina-1beta/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-ß-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action.
Assuntos
Antivirais/administração & dosagem , Núcleo Celular/metabolismo , Euphorbia/química , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Extratos Vegetais/administração & dosagem , Internalização do Vírus/efeitos dos fármacosRESUMO
Disabled-1 (Dab1) is an essential intracellular protein in the Reelin pathway. It has a nuclear localization signal (NLS; hereafter referred to as "NLS1") and 2 nuclear export signals, and shuttles between the nucleus and the cytoplasm. In this study, we found that Dab1 has an additional unidentified NLS, and that the Dab1 NLS1 mutant could translocate to the nucleus in an unconventional ATP/temperature-dependent and cytoplasmic factor/RanGTP gradient-independent manner. Additional mutations in the NLS1 mutant revealed that K(67) and K(69) are important for the nuclear transport. Furthermore, an excess of the intracellular domain of the Reelin receptors inhibited the nuclear translocation of Dab1. An in utero electroporation study showed that a large amount of Dab1 in the cytoplasm in migrating neurons inhibited the migration, and that forced transport of Dab1 into the nucleus attenuated this inhibitory effect. In addition, rescue experiments using yotari, an autosomal recessive mutant of dab1, revealed that cells expressing Dab1 NLS1 mutant tend to distribute at more superficial positions than those expressing wild-type Dab1. Taken together, these findings suggest that Dab1 has at least 2 NLSs, and that the regulation of the subcellular localization of Dab1 is important for the proper migration of excitatory neurons.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Citoplasma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Citoplasma/patologia , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , Receptores Notch/metabolismo , Proteína Reelina , Temperatura , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Urate has emerged as a promising target for neuroprotection based on epidemiological observations, preclinical models, and early clinical trial results in multiple neurologic diseases, including Parkinson's disease (PD). This study investigates the astrocytic mechanism of urate's neuroprotective effect. Targeted biochemical screens of conditioned medium from urate- versus vehicle-treated astrocytes identified markedly elevated glutathione (GSH) concentrations as a candidate mediator of urate's astrocyte-dependent neuroprotective effects. Urate treatment also induced the nuclear translocation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and transcriptional activation of its key target genes in primary astrocytic cultures. Urate's neuroprotective effect was attenuated when GSH was depleted in the conditioned media either by targeting its synthesis or release by astrocytes. Overall, these results implicate GSH as the extracellular astrocytic factor mediating the protective effect of urate in a cellular model of PD. These results also show that urate can employ a novel indirect neuroprotective mechanism via induction of the Nrf2 signaling pathway, a master regulator of the response to oxidative stress, in astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Glutationa/metabolismo , Fármacos Neuroprotetores/farmacologia , Ácido Úrico/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Meios de Cultivo Condicionados , Avaliação Pré-Clínica de Medicamentos , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Insufficient production of neurotrophic factors is implicated in the pathogenesis of various neurodegenerative disorders. The aim of the present study was to evaluate the potential of N-propargyl caffeate amide (PACA) to enhance nerve growth factor (NGF)-induced neurite outgrowth and the underlying mechanisms. We discovered that PACA not only potentiated NGF-induced neurite outgrowth but also attenuated 6-hydroxydopamine (6-OHDA) neurotoxicity in dopaminergic PC12 cells and primary rat midbrain neurons. To identify the PACA-binding proteins, we introduced a biotin tag to the covalent PACA-protein adducts via "click chemistry" alkyne-azido cycloaddition. As a result, kelch-like ECH-associated protein 1 (Keap1) was isolated as the predominant protein from PACA treated PC12 cells. We demonstrated that the formation of PACA-Keap1 conjugates induced the nuclear translocation of transcription factor Nrf2 and the expression of antioxidant heme oxygenase-1 (HO-1). Importantly, specific HO-1 inhibitor SnPP diminished the neuroprotective and neuritogenic activities of PACA. Moreover, PACA attenuated 6-OHDA-induced production of neurotoxic reactive oxygen species and reactive nitrogen species. PACA also preserved mitochondrial membrane integrity and enhanced the cellular resistance against 6-OHDA neurotoxicity. These results suggest that PACA may exhibit neuroprotective and neuritogenic activities via activating the Nrf2/HO-1 antioxidant pathway.
Assuntos
Ácidos Cafeicos/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Ácidos Cafeicos/síntese química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Dopamina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/patologia , Mesencéfalo/fisiopatologia , Neuritos/patologia , Neuritos/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Fármacos Neuroprotetores/síntese química , Óxido Nítrico/metabolismo , Oxidopamina/toxicidade , Células PC12 , Ratos , Superóxidos/metabolismoRESUMO
Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/ß-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3ß and, subsequently, induced the nuclear accumulation of the ß-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/ß-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/ß-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Osteoblastos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antígenos de Diferenciação/biossíntese , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoblastos/citologia , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/prevenção & controle , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , RNA Mensageiro/biossíntese , Ratos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Via de Sinalização Wnt/fisiologiaRESUMO
Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcεRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcεRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcεRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcεRI-induced cytokine secretion.
Assuntos
Células da Medula Óssea/metabolismo , Mastócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Tirosina Quinase da Agamaglobulinemia , Animais , Células da Medula Óssea/citologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocinas/genética , Citocinas/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Tirosina Quinases/genética , Receptores de IgE/genéticaRESUMO
Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Azadirachta/química , Núcleo Celular/metabolismo , Inseticidas/farmacologia , Limoninas/farmacologia , Receptores do Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Núcleo Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inseticidas/química , Limoninas/química , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Receptores do Ácido Retinoico/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células U937RESUMO
The activation of cAMP response element binding protein (CREB) and transcription of CRE-targeted genes play critical roles in various physiological and pathological conditions. Transducers of regulated CREB activity (TORCs) represent a new family of conserved CREB coactivators that promote the activation of CRE-targeted genes. Tanshinone IIA (TSA) has been proven to protect the brain against focal ischemia injury. However, little is known regarding the underlying mechanisms. Herein, we examined the activity-dependent nuclear translocation of TORC1 (transducer of regulated CREB activity 1) and the expression of TORC1, phosphorylated CREB (pCREB) and BDNF (brain-derived neurotrophic factor) at the early time of ischemic stroke as well as after the treatment with TSA. We observed a bimodal increase in pCREB, TORC1 and BDNF protein levels and transient nuclear accumulation of TORC1 in the acute stage of ischemia. Compared with vehicle group, TSA (20mg/kg) dramatically lessened neurological deficits scores, brain water contents and infarct sizes, significantly enhanced nuclear accumulation of TORC1 and upregulated the expression of TORC1, pCREB and BDNF (P<0.05). Collectively, the present results suggest that TSA protects rat brain from pristine ischemic damage in cerebral cortex, which might be correlated with induced nuclear translocation of TORC1 and upregulated expression of TORC1, pCREB and BDNF.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fármacos Neuroprotetores/farmacologia , Fenantrenos/farmacologia , Acidente Vascular Cerebral/metabolismo , Fatores de Transcrição/metabolismo , Abietanos , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Masculino , Testes Neuropsicológicos , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia , Fatores de Transcrição/genética , Regulação para CimaRESUMO
The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.
Assuntos
Capsídeo/metabolismo , Membrana Celular , Herpesvirus Humano 1/metabolismo , Proteína Quinase C/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/química , Diglicerídeos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Herpes Simples/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Rede trans-Golgi/ultraestruturaRESUMO
Astrocytes in the hypothalamus of poorly controlled diabetic rats are reduced in number, due to increased apoptosis and decreased proliferation, and undergo morphological changes, including a decrease in projections. These changes are associated with modifications in synaptic proteins and most likely affect neuroendocrine signalling and function. The present study aimed to determine the intracellular mechanisms underlying this increase in hypothalamic cell death. Adult male Wistar rats were injected with streptozotocin (70 mg/kg, i.p) and controls received vehicle. Rats were killed at 1, 4, 6 and 8 weeks after diabetes onset (glycaemia > 300 mg/dl). Cell death, as detected by enzyme-linked immunosorbent assay, increased at 4 weeks of diabetes. Immunohistochemistry and terminal dUTP nick-end labelling (TUNEL) assays indicated that these cells corresponded to glial fibrillary acidic protein (GFAP) positive cells. No significant change in fragmentation of caspases 2, 3, 6, 7, 8, 9, or 12 was observed with western blot analysis. However, enzymatic assays indicated that caspase 3 activity increased significantly after 1 week of diabetes and decreased below control levels thereafter. In the hypothalamus, cell bodies lining the third ventricle, fibres radiating from the third ventricle and GFAP positive cells expressed fragmented caspase 3, with this labelling increasing at 1 week of diabetes. However, because no nuclear labelling was observed and this increase in activity did not correlate temporally with the increased cell death, this caspase may not be involved in astrocyte death. By contrast, nuclear translocation of apoptosis inducing factor (AIF) increased significantly in astrocytes in parallel with the increase in death and AIF was found in TUNEL positive cells. Thus, nuclear translocation of AIF could underlie the increased death, whereas fragmentation of caspase 3 could be associated with the morphological changes found in hypothalamic astrocytes of diabetic rats.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Fator de Indução de Apoptose/metabolismo , Apoptose/fisiologia , Astrócitos/metabolismo , Astrócitos/patologia , Diabetes Mellitus Experimental/metabolismo , Hipotálamo/citologia , Animais , Astrócitos/citologia , Glicemia/metabolismo , Caspases/metabolismo , Ativação Enzimática , Proteína Glial Fibrilar Ácida/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Marcação In Situ das Extremidades Cortadas , Insulina/metabolismo , Masculino , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.
Assuntos
Acetilglucosamina/química , Regulação da Expressão Gênica , Proteína HMGN1 , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Simulação por Computador , Citoplasma/metabolismo , Proteína HMGN1/química , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Humanos , Dados de Sequência Molecular , Nucleossomos/metabolismo , Fosforilação , Ligação Proteica , Alinhamento de SequênciaRESUMO
Although butein (3,4,2',4'-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-kappaB pathway, we investigated in detail the effect of this chalcone on NF-kappaB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner; suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-beta. We also found that butein blocked the phosphorylation and degradation of IkappaBalpha by inhibiting IkappaBalpha kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-kappaB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-kappaB activation pathway.
Assuntos
Chalconas/metabolismo , Cisteína/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/antagonistas & inibidores , Fator de Transcrição RelA/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/fisiologia , Carcinógenos/metabolismo , Chalconas/química , Ativação Enzimática , Genes Reporter , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/fisiologia , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Leptin binding to its functional receptor stimulates JAK-STAT-signaling pathway, which finally results in activation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (STAT) family, namely of STAT3. Here we report for the first time that systemic treatment with leptin (5 mg/kg; intraperitoneal injection) also increased the number of nuclear STAT5 signals in the hypothalamus. In particular, the entire arcuate nucleus (ARC), the ventral premammilary nucleus (PMV), and the supraoptic nucleus (SO) showed an enhanced nuclear STAT5 translocation in response to leptin when compared to saline, 120 min after the respective injection. Co-localization studies revealed that a high percentage of those STAT5-responsive cells proved to be neurons. In addition, some astrocytes within the ARC showed nuclear STAT5 signals. The functional relevance of leptin-induced nuclear STAT5 activation in hypothalamic cells still has to be determined.
Assuntos
Núcleo Celular/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Neurônios/metabolismo , Fator de Transcrição STAT5/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Regulação do Apetite/efeitos dos fármacos , Regulação do Apetite/fisiologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Núcleo Celular/efeitos dos fármacos , Hipotálamo/anatomia & histologia , Hipotálamo/efeitos dos fármacos , Injeções Intraperitoneais , Leptina/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Transcrição STAT5/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Núcleo Supraóptico/efeitos dos fármacos , Núcleo Supraóptico/metabolismoRESUMO
Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-kappaB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-kappaB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-kappaB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1beta and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-kappaB activation, IkappaBalpha degradation, and IkappaB Kinase complex activity. CFTR-KO and wild-type mice were pretreated with parthenolide or vehicle alone then challenged intratracheally with LPS. Bronchoalveolar lavage was performed 3, 6, and 8 h later. Parthenolide pretreatment inhibited PMN influx as well as cytokine and chemokine production. This was also associated with inhibition of IkappaBalpha degradation and NF-kappaB activation. We thus conclude that parthenolide inhibits IkappaB kinase, resulting in stabilization of cytoplasmic IkappaBalpha, which in turn leads to inhibition of NF-kappaB translocation and attenuation of subsequent inflammatory responses. IkappaB kinase may be a good target, and parthenolide and/or feverfew might be promising treatments for the excessive inflammation in CF.
Assuntos
Fibrose Cística/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Inflamação/metabolismo , NF-kappa B/metabolismo , Sesquiterpenos/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Proteína Oncogênica p65(gag-jun)/química , Serina/química , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Avidina/química , Avidina/metabolismo , Núcleo Celular/química , Células Cultivadas , Cães , Glicosilação , Camundongos , Estrutura Molecular , Células NIH 3T3 , Sinais de Localização Nuclear/síntese química , Fosforilação , Serina/metabolismo , Relação Estrutura-AtividadeRESUMO
Inappropriate upregulation of cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) has been implicated in the pathogenesis of various types of cancer. In the present study, we investigated the effects of hirsutenone, a diarylheptanoid isolated from the medicinal plant Alnus hirsuta var. sibirica, on the expression of COX-2 and MMP-9 induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in MCF10A human breast epithelial cells. Treatment of MCF10A cells with TPA led to the expression of COX-2 and MMP-9. Hirsutenone at 12 microM inhibited the TPA-induced COX-2 expression at both the transcriptional and posttranscriptional levels. Hirsutenone also suppressed the synthesis of prostaglandin E(2), one of the major products of COX-2, and its catalytic activity. The upregulation of MMP-9 by TPA was also significantly reduced by hirsutenone. Likewise, hirsutenone attenuated the invasiveness and motility of MCF10A cells stimulated with TPA. Hirsutenone blocked the TPA-induced DNA binding of nuclear factor kappa B (NF-kappaB) and translocation of p65, the functionally active NF-kappaB subunit, to the nucleus. The luciferase reporter gene assay revealed that hirsutenone abrogated the transcriptional activity of NF-kappaB. Treatment of MCF10A cells with N-alpha-Tosyl-l-phenylalanine chloromethyl ketone, a specific inhibitor of NF-kappaB, reduced the TPA-induced expression of COX-2 and MMP-9. In summary, hirsutenone inhibits the TPA-induced upregulation of COX-2 and MMP-9 in human breast epithelial cells, possibly by targeting NF-kappaB, which may contribute to its chemopreventive effects.
Assuntos
Catecóis/farmacologia , Ciclo-Oxigenase 2/metabolismo , Diarileptanoides/farmacologia , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/citologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Dinoprostona/biossíntese , Células Epiteliais/citologia , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Humanos , Estrutura Molecular , Transcrição Gênica , Regulação para CimaRESUMO
This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF kappaB antagonist assay (inhibition of IL-1- induced NF kappaB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQtrade mark 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC method is described along with two-point (minimum-maximum) and multiple point titration statistical methods. As a ratio of compartment intensities that tend to change proportionally, FLIN was resistant to photobleaching errors. Two-point minimum-maximum statistical analyses yielded the following: a Z' of 0.174 with the data as n = 320 independent well samples; Z' by row data in a range of 0.393-0.933, with a mean of 0.766; by-plate Z' data of 0.310, 0.443, 0.545, and 0.794; and by-plate means of columns Z' data of 0.879, 0.927, 0.945, and 0.963. The mean 50% inhibitory concentration (IC50) for IL-1 receptor antagonist over all experiments was 213 ng/ml. The combined IC50 coefficients of variation (CVs) were 0.74%, 0.85%, 2.09%, and 2.52% for the four plates. Repeatability IC50 CVs were as follows: day to day 3.0%, row to row 8.0%, plate to plate 2.8%, and day to day 0.6%. The number of cells required for statistically resolvable differences in dose concentrations, plotted in a family of FLIN sigma/deltamicro (SD/range) curves and tabulated, demonstrated cell-by-cell assay precision with our combined sigma/deltamicro = 0.32 that required approximately 10-fold fewer cells than in a previously reported NF kappaB assay with sigma/deltamicro = 1.52. To better understand the relationship between cell-by-cell measurements and IC50 precision, 500 Monte Carlo simulations with varying cell-measurement SDs were used to explore three-, five-, seven-, and 11-point model titrations. The reductions in deltaIC50 90% confidence intervals from 11- to three-point titrations were 10-fold with the previously reported sigma/deltamicro = 1.52 and twofold with our sigma/deltamicro = 0.32. With these normalized parameters, this report provides a common statistical foundation, independent of the assay details, for evaluating the performance of imaging data on any instrument.