RESUMEN
We investigated the effects of shogaol, which has an α, ß-unsaturated carbonyl group, and gingerol, which does not, on primary-cultured microglia to understand how the α, ß-unsaturated carbonyl interacts with Kelch-like ECH-associated protein (Keap)1. Shogaol (1⯵M) but not the same concentration of gingerol significantly increased heme oxygenase (HO)-1 protein levels in cultured microglia without cytotoxicity. In addition, shogaol suppressed the release of the inflammation marker nitric oxide induced by 30 U/ml thrombin treatment. A docking simulation suggested that the α, ß-unsaturated carbonyl of shogaol but not gingerol interacts with Keap1. Nuclear import of nuclear factor E2-related factor 2 and increased binding of the HO-1 E2 enhancer support the docking-simulation prediction. The transcription inhibitor actinomycin D (0.1⯵g/ml) markedly blocked the increase of HO-1 mRNA levels by shogaol. To evaluate whether the α, ß-unsaturated carbonyl can be used for intracerebral hemorrhage (ICH) therapy, we investigated the effect of shogaol on an in vivo mouse ICH model. Intracerebroventricular injection of 0.2 nmol shogaol increased striatal HO-1 protein levels and rescued ICH-induced neuron loss. Thus, the α, ß-unsaturated carbonyl is necessary for the interaction of compounds, such as shogaol, with Keap1, and these findings may be useful for screening novel ICH therapeutic agents that increase HO-1 expression.
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Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/genética , Catecoles/farmacología , Hemorragia Cerebral/complicaciones , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Fármacos Neuroprotectores/farmacología , Animales , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Relación Dosis-Respuesta a Droga , Alcoholes Grasos/farmacología , Hemo-Oxigenasa 1/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacosRESUMEN
In a canine rapid atrial stimulation model of atrial fibrillation (AF), we have demonstrated an increased production of reactive oxygen species (ROS) along with electrical and structural remodeling. In the present study, we hypothesized that antioxidants can suppress atrial remodeling canines with AF. We therefore evaluated the effect of febuxostat, a xanthine oxidase (XO) inhibitor and a pure antioxidant, on atrial remodeling.AF was produced by performing a 3-week rapid atrial pacing (400 bpm) in 13 dogs divided into three groups: pacing + febuxostat group (n = 5; atrial pacing with 50 mg/day of febuxostat (administration); pacing control group (n = 5; atrial pacing without any drug administration); and non-pacing group (n = 3). Electrophysiological studies were conducted in the first 2 groups every week. Atrial tissue fibrosis was evaluated by Azan and immunofluorescent staining of fibronectin. Oxidative stress was evaluated by DHE and FCF-DA staining.Shortening of the refractory period and increase in AF inducibility appeared gradually in the pacing control group, but such changes were suppressed in the pacing + febuxostat group (P = 0.05). The pacing control group showed increase in fibrosis, which was suppressed in the febuxostat group. In DHE and DCF-DA staining, the pacing control group showed an increase in oxidative stress, which was suppressed in the pacing + febuxostat group. The pacing control group exhibited fibronectin expression, which was suppressed in the pacing + febuxostat group.The antioxidant effect of febuxostat may achieve an inhibition of new-onset AF in canines.
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Fibrilación Atrial/fisiopatología , Febuxostat/farmacología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Xantina Oxidasa/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/veterinaria , Remodelación Atrial/efectos de los fármacos , Modelos Animales de Enfermedad , Perros , Ecocardiografía , Febuxostat/administración & dosificación , Femenino , Fibronectinas/efectos de los fármacos , Fibronectinas/metabolismo , Fibrosis/patología , Supresores de la Gota/farmacología , Atrios Cardíacos/diagnóstico por imagen , Atrios Cardíacos/patología , Hemodinámica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8 M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Anguilas/metabolismo , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Secuencia de Aminoácidos , Animales , Factor Natriurético Atrial/química , Factor Natriurético Atrial/genética , Anguilas/genética , Atrios Cardíacos/química , Ventrículos Cardíacos/química , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/química , Péptido Natriurético Encefálico/genética , ARN Mensajero/genética , Vesículas Secretoras/química , Vesículas Secretoras/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Aliskiren, a direct renin inhibitor is expected to achieve sufficient suppression of renin-angiotensin system. We evaluated the effect of aliskiren on the electrical and structural remodeling in a canine atrial fibrillation (AF) model. Twenty-eight dogs were divided into three groups: (1) pacing control group (n = 12), with continuous atrial rapid pacing for 3 or 6 weeks, (2) pacing + aliskiren group (n = 12), with oral aliskiren (30 mg/kg/day), and (3) sham group (n = 4), no pacing nor drug administration. Electrophysiological properties and AF inducibility were evaluated every week. After the protocol, the left atrial tissue was sampled for the further histological and mRNA analysis. The electrical remodeling, AF inducibility, the left atrial enlargement and interstitial fibrosis were observed in pacing control group and were more prominent in the 6-week protocol (vs. 3 week, p < 0.05). The mRNA expressions of matricellular proteins exhibited upregulation in 3-week pacing control, but these upregulations became insignificant in 6 weeks. In contrast, collagen type 3 exhibited significant upregulation in 6 week but not in 3-week protocol. These changes were suppressed in the pacing + aliskiren group. Aliskiren suppressed the atrial remodeling in a canine AF model. This effect was accompanied by the suppression of tissue fibrosis.
Asunto(s)
Amidas/administración & dosificación , Fibrilación Atrial/terapia , Estimulación Cardíaca Artificial/métodos , Fumaratos/administración & dosificación , Atrios Cardíacos/fisiopatología , Sistema Renina-Angiotensina/efectos de los fármacos , Renina/administración & dosificación , Animales , Modelos Animales de Enfermedad , Perros , Ecocardiografía , Femenino , Fibrosis , Expresión Génica , HemodinámicaRESUMEN
EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Factores de Ribosilacion-ADP/biosíntesis , Empalme Alternativo/fisiología , Encéfalo/metabolismo , Variación Genética/fisiología , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/análisis , Factores de Ribosilacion-ADP/genética , Animales , Encéfalo/citología , Química Encefálica/fisiología , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/genética , Cobayas , Masculino , Ratones , Ratones Endogámicos C57BL , Conejos , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is one of the most potent angiogenesis stimulators. VEGF binds to VEGF receptor 1 (VEGFR1), inducing angiogenesis through the receptor's tyrosine kinase domain (TK), but the mechanism is not well understood. We investigated the role of VEGFR1 tyrosine kinase signaling in angiogenesis using the ischemic hind limb model. Relative to control mice, blood flow recovery was significantly impaired in mice treated with VEGFA-neutralizing antibody. VEGFR1 tyrosine kinase knockout mice (TK-/-) had delayed blood flow recovery from ischemia and impaired angiogenesis, and this phenotype was unaffected by treatment with a VEGFR2 inhibitor. Compared to wild type mice (WT), TK-/- mice had no change in the plasma level of VEGF, but the plasma levels of stromal-derived cell factor 1 (SDF-1) and stem cell factor, as well as the bone marrow (BM) level of pro-matrix metalloproteinase-9 (pro-MMP-9), were significantly reduced. The recruitment of cells expressing VEGFR1 and C-X-C chemokine receptor type 4 (CXCR4) into peripheral blood and ischemic muscles was also suppressed. Furthermore, WT transplanted with TK-/- BM significantly impaired blood flow recovery more than WT transplanted with WT BM. These results suggest that VEGFR1-TK signaling facilitates angiogenesis by recruiting CXCR4+VEGFR1+ cells from BM.
Asunto(s)
Miembro Posterior/metabolismo , Isquemia/sangre , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Anticuerpos Neutralizantes/farmacología , Velocidad del Flujo Sanguíneo , Médula Ósea/metabolismo , Trasplante de Médula Ósea , Quimiocina CXCL12/sangre , Quimiocina CXCL12/genética , Regulación de la Expresión Génica , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Isquemia/genética , Isquemia/patología , Masculino , Metaloproteinasa 9 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Recuperación de la Función , Transducción de Señal , Factor de Células Madre/sangre , Factor de Células Madre/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIMS: Thromboxane A2 (TXA2) induces platelet adhesion through thromboxane prostanoid (TP) receptor. Platelets contain many pro-angiogenic factors and are recruited to the site of vascular injury. However, the cellular and molecular mechanisms of platelet-dependent angiogenesis, especially the involvement of TP signalling, have not been fully elucidated. The present study hypothesized that TP-dependent platelet adhesion would contribute to angiogenesis in a mouse hindlimb ischaemic model. METHODS AND RESULTS: Blood flow recovery was suppressed by the TXA2 receptor antagonist (S-1452) and the TXA2 synthase inhibitor (OKY-046) compared with control mice. TP knockout mice (TP(-/-)) showed delayed blood flow recovery from ischaemia and impaired angiogenesis compared with wild-type (WT) mice and prostacyclin receptor knockout mice (IP(-/-)). Selective platelet adhesion to ischaemic endothelial cells (ECs) via P-selectin was identified in WT and IP(-/-), but not in TP(-/-), via in vivo microscopy. IF analysis showed that P-selectin glycoprotein ligand-1 (PSGL-1) co-localized with endothelial CD31 in ischaemic muscle in WT and IP(-/-) but not diminished in TP(-/-). Platelet-rich plasma levels of stromal cell-derived factor-1 and VEGF were increased after ischaemia in WT, and suppressed by antibody against P-selectin in WT but not in TP(-/-). Furthermore, the blood flow recovery was suppressed by neutralizing antibodies against VEGF or C-X-C chemokine receptor type 4 in WT and IP(-/-) but not in TP(-/-). CONCLUSION: These results indicated that TP signalling facilitates ischaemia-induced angiogenesis via P-selectin-mediated platelet adhesion to PSGL-1 on the ECs at ischaemic sites and the supply of pro-angiogenic factors by the adherent platelets.
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Plaquetas/efectos de los fármacos , Células Endoteliales/metabolismo , Isquemia Miocárdica/metabolismo , Adhesividad Plaquetaria/efectos de los fármacos , Tromboxano A2/farmacología , Animales , Plaquetas/citología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones Noqueados , Adhesividad Plaquetaria/genética , Receptores de Tromboxano A2 y Prostaglandina H2/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Compensatory lung growth models have been widely used to investigate alveolization because the remaining lung can be kept intact and volume loss can be controlled. Vascular endothelial growth factor (VEGF) plays an important role in blood formation during lung growth and repair, but the precise mechanisms involved are poorly understood; therefore, the aim of this study was to investigate the role of VEGF signaling in compensatory lung growth. After left pneumonectomy, the right lung weight was higher in VEGF transgenic mice than wild-type (WT) mice. Compensatory lung growth was suppressed significantly in mice injected with a VEGF neutralizing antibody and in VEGF receptor-1 tyrosine kinase-deficient mice (TK(-/-) mice). The mobilization of progenitor cells expressing VEGFR1(+) cells from bone marrow and the recruitment of these cells to lung tissue were also suppressed in the TK(-/-) mice. WT mice transplanted with bone marrow from TK(-/-)transgenic GFP(+) mice had significantly lower numbers of GFP(+)/aquaporin 5(+), GFP(+)/surfactant protein A(+), and GFP(+)/VEGFR1(+) cells than WT mice transplanted with bone marrow from WTGFP(+) mice. The GFP(+)/VEGFR1(+) cells also co-stained for aquaporin 5 and surfactant protein A. Overall, these results suggest that VEGF signaling contributes to compensatory lung growth by mobilizing VEGFR1(+) cells.
Asunto(s)
Pulmón/metabolismo , Pulmón/fisiología , Neumonectomía , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células de la Médula Ósea , Citocinas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Pulmón/química , Pulmón/cirugía , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos/fisiología , Proteínas Tirosina Quinasas/metabolismo , Alveolos Pulmonares/citología , Factor A de Crecimiento Endotelial Vascular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/análisis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Type I phosphatidylinositol 4-phosphate 5 kinase γ (PIP5KIγ) constitutes a major pathway for the generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) that regulates a variety of neuronal functions at both presynaptic and postsynaptic compartments. In this study, we examined the expression and localization of PIP5KIγ in the adult mouse retina. RT-PCR analysis revealed that PIP5KIγ_v2 was predominantly expressed in the retina while PIP5KIγ_v3 was also expressed faintly. Immunostaining of the adult mouse retina revealed intense PIP5KIγ-immunoreactivity in the inner and outer plexiform layers in a punctate manner. In the photoreceptor ribbon synapse, PIP5KIγ was highly concentrated at the periactive zone. These findings suggest that PIP5KIγ, especially PIP5KIγ_i2, is localized at the periactive zone, a functionally suitable compartment for the endocytosis of synaptic vesicles in photoreceptor ribbon synapses.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Células Fotorreceptoras/metabolismo , Sinapsis/metabolismo , Oxidorreductasas de Alcohol , Animales , Calbindinas/metabolismo , Clatrina/metabolismo , Proteínas Co-Represoras , Proteínas de Unión al ADN/metabolismo , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Células Fotorreceptoras/ultraestructura , Proteína Quinasa C/metabolismo , Retina/citología , Sinapsis/ultraestructura , Talina/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
BACKGROUND: This study was designed to investigate the role of a primary hyperoxidative stress in myocardial electrical remodeling using heterozygous heart/muscle-specific manganese superoxide dismutase-deficient (H/M-Sod2(+/-)) mice treated with L-buthionine-sulfoximine (BSO). METHODS AND RESULTS: Both H/M-Sod2(+/-)and wild-type (WT) mice were treated with intra-peritoneal BSO or saline for 7 days, and divided into 4 groups: H/M-Sod2(+/-)+BSO, WT+BSO, H/M-Sod2(+/-)control, and WT control. The ventricular effective refractory period (ERP) and the monophasic action potential duration (MAPD) were determined. Levels of oxidative stress, potassium channel-related molecules, and K(+)channel-interacting protein-2 (KChIP2) were also evaluated. The H/M-Sod2(+/-)+BSO group exhibited markedly prolonged MAPD20, MAPD90 and ERP in comparison with the other groups (MAPD20: 14 ± 1 vs. 11 ± 1 ms, MAPD90: 77 ± 7 vs. 58 ± 4 ms, ERP: 61 ± 6 vs. 41 ± 3 ms, H/M-Sod2(+/-)+BSO vs. WT control; P<0.05). Mitochondrial superoxide and hydrogen peroxide formation in the myocardium increased in the H/M-Sod2(+/-)+BSO group in comparison with the WT+BSO group (P<0.05). Real-time RT-PCR and Western blotting revealed that Kv4.2 expression was downregulated in both BSO-treated groups, whereas KChIP2 expression was downregulated only in the H/M-Sod2(+/-)+BSO group (P<0.05). CONCLUSIONS: BSO treatment caused hyperoxidative stress in the myocardium of H/M-Sod2(+/-)mice. Changes in the expression and function of potassium channels were considered to be involved in the mechanism of electrical remodeling in this model.
Asunto(s)
Regulación hacia Abajo , Proteínas de Interacción con los Canales Kv/biosíntesis , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Estrés Oxidativo , Canales de Potasio Shal/biosíntesis , Superóxidos/metabolismo , Animales , Antimetabolitos/farmacología , Butionina Sulfoximina/farmacología , Peróxido de Hidrógeno/metabolismo , Proteínas de Interacción con los Canales Kv/genética , Ratones , Ratones Noqueados , Miocitos Cardíacos , Canales de Potasio Shal/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Endosomal trafficking mediated by Rab11 and Arf6 small GTPases is essential for various neuronal functions. Family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1, also termed Eferin, is a dual effector for Rab11 and Arf6 and implicated in endosomal trafficking during cytokinesis. To understand the neuronal functions of FIP3, we first showed the widespread neuronal expression of FIP3 mRNA in adult mouse brain by in situ hybridization. Immunohistochemical analysis showed the association of FIP3 with a subpopulation of endosomes labeled with EEA1 and syntaxin 12 in hippocampal neurons. Immunoblot analysis showed the progressive increase of FIP3 with a peak around postnatal day 15 during hippocampal development. Furthermore, knockdown of endogenous FIP3 decreased the total dendritic length of cultured hippocampal neurons with a concomitant increase in the number of short (<40µm) primary dendrites. Together, FIP3 is suggested to regulate dendritic formation possibly through Rab11- and Arf6-mediated endosomal trafficking.
Asunto(s)
Proteínas Portadoras/metabolismo , Dendritas/fisiología , Endosomas/metabolismo , Hipocampo/crecimiento & desarrollo , Neuronas/fisiología , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Células COS , Proteínas Portadoras/genética , Tamaño de la Célula , Células Cultivadas , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Células HeLa , Hipocampo/citología , Hipocampo/fisiología , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Proteínas Qa-SNARE/metabolismo , ARN Mensajero/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
AIMS: We evaluated the effect of carvedilol, a beta-blocker with anti-oxidative action, against the atrial fibrillation (AF) inducibility, the development of atrial remodeling and the oxidative stress markers in a canine AF model. METHODS AND RESULTS: AF model was produced by performing 6-week rapid atrial stimulation in 15 dogs. The animals were divided into the following three groups: (I) pacing + carvedilol group (n=5); (II) pacing control group (n=5); and (III) non-pacing group (n=5). AF inducibility was gradually increased along the time course in the pacing control group. In the pacing + carvedilol group, the AF inducibility was suppressed especially in the latter phase of protocol in comparison with the pacing control group. Although carvedilol has beta-blocking effect, pacing control and pacing + carvedilol groups did not exhibit difference in the heart rate (177±13 vs. 155±13 bpm, P=0.08). On 8-hydroxy-2'-deoxyguanosine (8-OHdG), dihydroethidium and dichlorodihydrofluorescein diacetate staining, enhanced oxidative stress was observed in the atrial tissue in the pacing control, but not in the pacing + carvedilol group. CONCLUSIONS: Carvedilol suppressed AF inducibility and oxidative stress in the canine AF model.
RESUMEN
Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)γ is one of the phosphoinositide kinases that produce phosphatidylinositol 4,5-bisphosphate, which is a critical regulator of cell adhesion formation, actin dynamics and membrane trafficking. Here, we examined the functional roles of PIP5KIγ in radial neuronal migration during cortical formation. Reverse transcription-polymerase chain reaction analysis revealed that PIP5KIγ_v2/v6 and PIP5KIγ_v3 were expressed throughout cortical development with distinct expression patterns. In situ hybridisation analysis showed that PIP5KIγ mRNA was expressed throughout the cortical layers. Immunohistochemical analysis revealed that PIP5KIγ was localised in a punctate manner in the radial glia and migrating neuroblasts. Knockdown of PIP5KIγ using in utero electroporation disturbed the radial neuronal migration and recruitment of talin and focal adhesion kinase to puncta beneath the plasma membrane. The same inhibitory effect on neuronal migration was observed by overexpression of a catalytically inactive mutant of PIP5KIγ_v2 but not PIP5KIγ_v1 or PIP5KIγ_v3. These findings suggest an essential role of PIP5KIγ, particularly PIP5KIγ_i2, in neuronal migration, possibly through recruitment of adhesion components to the plasma membrane.
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Movimiento Celular , Corteza Cerebral/embriología , Células-Madre Neurales/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Células Ependimogliales/enzimología , Células Ependimogliales/metabolismo , Ratones , Ratones Endogámicos ICR , Células-Madre Neurales/metabolismoRESUMEN
The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined the immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and ß-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABA(A) Rs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, while BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/biosíntesis , Retina/metabolismo , Sinapsis/metabolismo , Animales , Western Blotting , Femenino , Factores de Intercambio de Guanina Nucleótido/análisis , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía InmunoelectrónicaRESUMEN
This is Part I of a two-part paper documenting the development of a novel asymmetric flow control method to improve the stability of a high-pressure-ratio turbocharger centrifugal compressor. Part I focuses on the nonaxisymmetrical flow in a centrifugal compressor induced by the nonaxisymmetrical geometry of the volute while Part II describes the development of an asymmetric flow control method to avoid the stall on the basis of the characteristic of nonaxisymmetrical flow. To understand the asymmetries, experimental measurements and corresponding numerical simulation were carried out. The static pressure was measured by probes at different circumferential and stream-wise positions to gain insights about the asymmetries. The experimental results show that there is an evident nonaxisymmetrical flow pattern throughout the compressor due to the asymmetric geometry of the overhung volute. The static pressure field in the diffuser is distorted at approximately 90 deg in the rotational direction of the volute tongue throughout the diffuser. The magnitude of this distortion slightly varies with the rotational speed. The magnitude of the static pressure distortion in the impeller is a function of the rotational speed. There is a significant phase shift between the static pressure distributions at the leading edge of the splitter blades and the impeller outlet. The numerical steady state simulation neglects the aforementioned unsteady effects found in the experiments and cannot predict the phase shift, however, a detailed asymmetric flow field structure is obviously obtained.
RESUMEN
This is part II of a two-part paper involving the development of an asymmetrical flow control method to widen the operating range of a turbocharger centrifugal compressor with high-pressure ratio. A nonaxisymmetrical self-recirculation casing treatment (SRCT) as an instance of asymmetrical flow control method is presented. Experimental and numerical methods were used to investigate the impact of nonaxisymmetrical SRCT on the surge point of the centrifugal compressor. First, the influence of the geometry of a symmetric SRCT on the compressor performance was studied by means of numerical simulation. The key parameter of the SRCT was found to be the distance from the main blade leading edge to the rear groove (Sr). Next, several arrangements of a nonaxisymmetrical SRCT were designed, based on flow analysis presented in part I. Then, a series of experiments were carried out to analyze the influence of nonaxisymmetrical SRCT on the compressor performance. Results show that the nonaxisymmetrical SRCT has a certain influence on the performance and has a larger potential for stability improvement than the traditional symmetric SRCT. For the investigated SRCT, the surge flow rate of the compressor with the nonaxisymmetrical SRCTs is about 10% lower than that of the compressor with symmetric SRCT. The largest surge margin (smallest surge flow rate) can be obtained when the phase of the largest Sr is coincident with the phase of the minimum static pressure in the vicinity of the leading edge of the splitter blades.
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Homeodomain-only protein X (HOPX)-ß promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to investigate the mechanistic roles of HOPX-ß promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-ß promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and protein levels were measured in relation to HOPX-ß promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-ß promoter methylation and clinical relevance of CRC patients was determined. HOPX-ß promoter methylation is cancer-specific and frequently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, invasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-ß promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio= 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-ß promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients.
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Neoplasias Colorrectales/genética , Silenciador del Gen , Proteínas de Homeodominio/genética , Proteínas Supresoras de Tumor/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Islas de CpG , Metilación de ADN , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estadificación de Neoplasias , Neovascularización Patológica/genética , Pronóstico , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.
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Proteínas de Uniones Estrechas/química , Proteínas de Uniones Estrechas/metabolismo , Red trans-Golgi/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Masculino , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Células 3T3 NIH , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Uniones Estrechas/antagonistas & inhibidores , Proteínas de Uniones Estrechas/genética , Distribución Tisular , Técnicas del Sistema de Dos Híbridos , Red trans-Golgi/ultraestructuraRESUMEN
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Polimorfismo Genético , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Animales , Retículo Endoplásmico/metabolismo , HidrólisisRESUMEN
OBJECTIVE: One of the hallmarks of inflammation is lymphangiogesis that drains the interstitial fluids. During chronic inflammation, angiogenesis is induced by a variety of inflammatory mediators, such as prostaglandins (PGs). However, it remains unknown whether they enhance lymphangiogenesis. We examined the roles of cyclooxygenase-2 (COX-2) and PGE2 receptor signaling in enhancement of lymphangiogenesis during proliferative inflammation. METHODS AND RESULTS: Lymphangiogenesis estimated by podoplanin/vascular endothelial growth factor (VEGF) receptor-3/LYVE-1 expression was upregulated during proliferative inflammation seen around and into subcutaneous Matrigel plugs containing fibroblast growth factor-2 (125 ng/site). A COX-2 inhibitor (celecoxib) significantly reduced lymphangiogenesis in a dose-dependent manner, whereas topical PGE2 enhanced lymphangiogenesis. Topical injection of fluorescein isothiocyanate-dextran into the Matrigel revealed that lymphatic flow from the Matrigels was COX-2 dependent. Lymphangiogenesis was suppressed in the granulation tissues of mice lacking either EP3 or EP4, suggesting that these molecules are receptors in response to endogenous PGE2. An EP3-selective agonist (ONO-AE-248) increased the expression of VEGF-C and VEGF-D in cultured macrophages, whereas an EP4-selective agonist (ONO-AE1-329) increased VEGF-C expression in cultured macrophages and increased VEGF-D expression in cultured fibroblasts. CONCLUSIONS: Our findings suggest that COX-2 and EP3/EP4 signaling contributes to lymphangiogenesis in proliferative inflammation, possibly via induction of VEGF-C and VEGF-D, and may become a therapeutic target for controlling lymphangiogenesis.