RESUMEN
Antimetastatic effect of Metformin has been documented in epithelial ovarian cancer (EOC). Presently, we investigated the regulatory mechanism of Metformin in EOC metastasis. First, Girdin was significantly enhanced in EOC tumorous tissues and cell lines. Seconded, knockdown of Girdin significantly suppressed EOC cell viability, migration, and invasion, while upregulation of Girdin produced the opposite effects in vitro and facilitated lung metastasis in EOC cell xenograft in vivo. In addition, we confirmed that the inhibitory effect of Metformin on Girdin expression. Mechanistically, the oncogenic effects of Girdin could be reversed by LY294002 (an AKT pathway inhibitor) and Metformin. These results suggested that Metformin attenuated EOC metastasis through Girdin and targeting Girdin may be a promising therapeutic strategy for EOC in the future.
Asunto(s)
Carcinoma Epitelial de Ovario/metabolismo , Proteínas de Microfilamentos/genética , Metástasis de la Neoplasia/tratamiento farmacológico , Proteínas de Transporte Vesicular/genética , Adulto , Animales , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Transición Epitelial-Mesenquimal , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metformina/metabolismo , Metformina/farmacología , Ratones Desnudos , Proteínas de Microfilamentos/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transcriptoma/genética , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Flavonoides/farmacología , Isoflurano/antagonistas & inhibidores , Isoflurano/toxicidad , Complejo Mayor de Histocompatibilidad/efectos de los fármacos , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/prevención & control , Polifenoles/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Hipocampo/efectos de los fármacos , Ratones , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.
Asunto(s)
Trastorno Depresivo Mayor/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Serotonina/metabolismo , Conducta Social , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Animales , Autopsia , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Femenino , Fluoxetina/farmacología , Humanos , Macaca mulatta , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/fisiología , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/fisiología , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Caracteres SexualesRESUMEN
Multiple sclerosis (MS) is a neurologic autoimmune disorder featured by chronic inflammation of the central nervous system, demyelination and axonal damage. Recently, the term "oxinflammation" has been proposed to depict the vicious circle of chronic inflammation and oxidative stress (OS). OS promotes demyelination and neurodegeneration directly, by oxidation of lipids, proteins, and DNA but also indirectly, by inducing a dysregulation of the immunity and favoring the state of pro-inflammatory response. Many of the actors of this delicately tuned network are controlled by Keap1/Nrf2/ARE signaling pathway, a principal regulator of antioxidant and phase II detoxification genes. This pathway also has a pivotal role in inflammation, and therefore possesses a great potential in the treatment of MS. The aim of this review is to provide the newest insights in the preclinical and clinical evidence of Nrf2 induction in the regeneration of the antioxidant response and attenuation of inflammation in MS. Preclinical studies have indicated that activators of this pathway, such as epigallocatechin gallate (EGCG), curcumin, melatonin, resveratrol, and sulforaphane might be a promising therapeutic option in amelioration of MS symptoms, nevertheless, the efficacy and safety of these compounds have to be confirmed in future clinical trials.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/efectos de los fármacos , Esclerosis Múltiple/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacos , Animales , HumanosRESUMEN
Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.
Asunto(s)
Relojes Circadianos/fisiología , Colágeno/metabolismo , Homeostasis/fisiología , Vías Secretoras/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/efectos de los fármacos , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carbazoles/farmacología , Colágeno/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Matriz Extracelular/metabolismo , Ratones Transgénicos , Pirrolidinas/farmacología , Canales de Translocación SEC/efectos de los fármacos , Canales de Translocación SEC/metabolismo , Vías Secretoras/genética , Sulfonamidas/farmacología , Tiofenos/farmacología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células A549 , Factor 1 de Ribosilacion-ADP/metabolismo , Antibacterianos/farmacología , Antígenos de Neoplasias/efectos de los fármacos , Autofagia/fisiología , Brefeldino A/farmacología , Disentería Bacilar/tratamiento farmacológico , Disentería Bacilar/metabolismo , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HT29 , Células HeLa , Humanos , Shigella flexneri/efectos de los fármacos , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
OBJECTIVE: Giant cell arteritis (GCA) is pathologically characterized by dysfunctional angiogenesis and inflammatory cell infiltration. Acute-phase serum amyloid A (A-SAA) is an acute-phase reactant, but is also produced at sites of inflammation and may contribute to vascular inflammation in atherosclerosis. This study was undertaken to examine the effect of A-SAA on proinflammatory pathways and angiogenesis in GCA, using a novel ex vivo temporal artery tissue explant model. METHODS: Serum A-SAA levels were measured by enzyme-linked immunosorbent assay (ELISA). Temporal artery explants and peripheral blood mononuclear cell (PBMC) cultures were established from patients with GCA. Temporal artery explant morphology, viability, and spontaneous release of proinflammatory mediators following 24-hour culture were assessed by hematoxylin and eosin, calcein viability staining, and ELISA. Temporal artery explants and PBMC cultures were stimulated with A-SAA (10 µg/ml), and interleukin-6 (IL-6), IL-8, vascular endothelial growth factor, Ang2, and matrix metalloproteinase 2 (MMP-2)/MMP-9 were quantified by ELISA and gelatin zymography. The effect of conditioned medium from temporal artery explants on angiogenesis was assessed using endothelial cell Matrigel tube-formation assays. Temporal artery explants were also embedded in Matrigel, and myofibroblast outgrowth was assessed. RESULTS: Serum A-SAA levels were significantly higher in GCA patients versus healthy controls (P < 0.0001). Intact tissue morphology, cell viability, and spontaneous cytokine secretion were demonstrated in temporal artery explants. A-SAA treatment induced a significant increase in the levels of IL-6 and IL-8 from temporal artery explants (P < 0.05) and IL-8 from PBMCs (P < 0.05) compared to basal conditions. Conditioned medium from A-SAA-treated explants significantly induced angiogenic tube formation (P < 0.05 versus basal controls). Finally, A-SAA induced myofibroblast outgrowth and MMP-9 activation. CONCLUSION: Our findings demonstrate a functional role for A-SAA in regulating temporal artery inflammation, angiogenesis, and invasion, all key processes in the pathogenesis of GCA.
Asunto(s)
Arteritis de Células Gigantes/inmunología , Miofibroblastos/efectos de los fármacos , Neovascularización Patológica/inmunología , Proteína Amiloide A Sérica/farmacología , Arterias Temporales/efectos de los fármacos , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/farmacología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Arteritis de Células Gigantes/metabolismo , Humanos , Técnicas In Vitro , Inflamación/inmunología , Interleucina-6/inmunología , Interleucina-8/efectos de los fármacos , Interleucina-8/inmunología , Leucocitos Mononucleares , Masculino , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/inmunología , Persona de Mediana Edad , Proteína Amiloide A Sérica/inmunología , Arterias Temporales/inmunología , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/inmunología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/inmunologíaRESUMEN
El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.(AU)
In 2008, Brazils Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMFs ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.(AU)
Asunto(s)
Animales , Ratas , Apoptosis/genética , Regulación Enzimológica de la Expresión Génica/genética , Hemo/deficiencia , Degeneración Nerviosa/genética , Neuronas/metabolismo , Porfirias/complicaciones , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Colágeno Tipo XI/efectos de los fármacos , Colágeno Tipo XI/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo/biosíntesis , Heptanoatos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Células PC12 , Poli(ADP-Ribosa) Polimerasas , Porfirias/metabolismo , Porfirias/fisiopatología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.
In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.
Asunto(s)
Animales , Ratas , Apoptosis/genética , Regulación Enzimológica de la Expresión Génica/genética , Hemo/deficiencia , Degeneración Nerviosa/genética , Neuronas/metabolismo , Porfirias/complicaciones , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Colágeno Tipo XI/efectos de los fármacos , Colágeno Tipo XI/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo/biosíntesis , Heptanoatos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Poli(ADP-Ribosa) Polimerasas , Porfirias/metabolismo , Porfirias/fisiopatología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Anticuerpos Monoclonales de Origen Murino/análisis , Vasos Linfáticos/patología , Psoriasis/tratamiento farmacológico , Factores de Necrosis Tumoral/antagonistas & inhibidores , Factores de Crecimiento Endotelial Vascular/análisis , Proteínas de Transporte Vesicular/análisis , Anticuerpos Monoclonales de Origen Murino/efectos de los fármacos , Biopsia , Biomarcadores/análisis , Inmunohistoquímica , Inmunoglobulina G/uso terapéutico , Factores Inmunológicos/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Psoriasis/metabolismo , Psoriasis/patología , Valores de Referencia , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Estadísticas no Paramétricas , Piel/efectos de los fármacos , Piel/patología , Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/análisis , Vasos Linfáticos/patología , Psoriasis/tratamiento farmacológico , Inhibidores del Factor de Necrosis Tumoral , Factores de Crecimiento Endotelial Vascular/análisis , Proteínas de Transporte Vesicular/análisis , Adulto , Anticuerpos Monoclonales de Origen Murino/efectos de los fármacos , Biomarcadores/análisis , Biopsia , Etanercept , Femenino , Humanos , Inmunoglobulina G/uso terapéutico , Inmunohistoquímica , Factores Inmunológicos/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Psoriasis/metabolismo , Psoriasis/patología , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Valores de Referencia , Piel/efectos de los fármacos , Piel/patología , Estadísticas no Paramétricas , Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
HIV-1 release requires a direct interaction between the p6 domain of the Gag protein and Tsg101, a component of the cellular endosomal sorting complex required for transport I (ESCRT-I). Disruption of the binding between Gag and Tsg101 is highly detrimental to particle release, making this viral-host cell interaction a potential target for the development of novel anti-HIV-1 agents. An article in this issue reports on the application of a bacterial reverse two-hybrid strategy to identify a cyclic peptide that disrupts Gag-Tsg101 binding and suppresses HIV-1 particle release.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , VIH-1/efectos de los fármacos , VIH-1/fisiología , Péptidos Cíclicos/farmacología , Factores de Transcripción/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Humanos , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Synthetic activators of peroxisome proliferator-activated receptors (PPAR)-alpha and -gamma are capable of reducing macrophage foam cell cholesterol accumulation through the activation of genes involved in cholesterol homeostasis. Since conjugated linoleic acids (CLA) were also demonstrated to activate PPARalpha and PPARgamma in vivo and in vitro, we tested the hypothesis that CLA are also capable of reducing macrophage foam cell cholesterol accumulation. Thus, mouse RAW264.7 macrophage-derived foam cells were treated with CLA isomers, c9t11-CLA and t10c12-CLA, and linoleic acid (LA), as reference fatty acid, and analyzed for the concentrations of free and esterified cholesterol, cholesterol efflux and expression of genes involved in cholesterol homeostasis (CD36, ABCA1, LXRalpha, NPC-1, and NPC-2). Treatment with c9t11-CLA and t10c12-CLA, but not LA, lowered cholesterol accumulation, stimulated acceptor-dependent cholesterol efflux, and increased relative mRNA concentrations of CD36, ABCA1, LXRalpha, NPC-1, and NPC-2 (P < 0.05). In conclusion, the present study showed that CLA isomers reduce cholesterol accumulation in RAW264.7 macrophage-derived foam cells presumably by enhancing lipid acceptor-dependent cholesterol efflux.
Asunto(s)
Colesterol/metabolismo , Células Espumosas/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , PPAR gamma/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antígenos CD36/efectos de los fármacos , Antígenos CD36/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Espumosas/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isomerismo , Ácidos Linoleicos Conjugados/farmacología , Receptores X del Hígado , Ratones , Proteína Niemann-Pick C1 , Receptores Nucleares Huérfanos , PPAR gamma/efectos de los fármacos , Proteínas/efectos de los fármacos , Proteínas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The effect of alpha-tocopherol, the major vitamin E component, on the release of endogenous glutamate has been investigated using rat cerebrocortical nerve terminals. Results showed that alpha-tocopherol facilitated the Ca2+-dependent but not the Ca2+-independent glutamate release evoked by 4-aminopyridine (4AP). This release facilitation was insensitive to glutamate transporter inhibitor L-trans-PDC or DL-TBOA, and blocked by the exocytotic neurotransmitter release inhibitor tetanus neurotoxin, indicating that alpha-tocopherol affects specifically the physiological exocytotic vesicular release without affecting the non-vesicular release. Facilitation of glutamate exocytosis by alpha-tocopherol was not due to its increasing synaptosomal excitability, because alpha-tocopherol did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. Rather, examination of the effect of alpha-tocopherol on cytoplasmic free Ca2+ concentration revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca2+ influx. Consistent with this, the alpha-tocopherol-mediated facilitation of glutamate release was significantly reduced in synaptosomes pretreated with omega-CgTX MVIIC, a wide spectrum blocker of N- and P/Q-type Ca2+ channels. In addition, alpha-tocopherol modulation of glutamate release appeared to involve a protein kinase C (PKC) signalling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitor GF109203X effectively suppressed the facilitatory effect of alpha-tocopherol on 4AP- or ionomycin-evoked glutamate release. Furthermore, alpha-tocopherol increased the phosphorylation of MARCKS, the major presynapic substrate for PKC, and this effect was also significantly attenuated by PKC inhibition. Together, these results suggest that alpha-tocopherol exerts an increase in PKC activation, which subsequently enhances voltage-dependent Ca2+ influx and vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical glutamatergic terminals. This finding might provide important information regarding to the action of vitamin E in the central nervous system.
Asunto(s)
Corteza Cerebral/metabolismo , Exocitosis/fisiología , Ácido Glutámico/metabolismo , Terminales Presinápticos/metabolismo , alfa-Tocoferol/metabolismo , 4-Aminopiridina/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Corteza Cerebral/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Exocitosis/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/farmacología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Sinaptosomas , Toxina Tetánica/farmacología , Proteínas de Transporte Vesicular de Glutamato/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
BACKGROUND: Many studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD. RESULTS: Here, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone. CONCLUSION: Our findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.
Asunto(s)
Dopamina/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/efectos de los fármacos , Neuronas/efectos de los fármacos , Rotenona/toxicidad , Desacopladores/toxicidad , Línea Celular , Complejo I de Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Mitocondrias/metabolismo , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Proteómica/métodos , Nexinas de Clasificación , Proteína 3 de Membrana Asociada a Vesículas/efectos de los fármacos , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
PURPOSE: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. EXPERIMENTAL DESIGN: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. RESULTS: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. CONCLUSIONS: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.
Asunto(s)
Neoplasias del Colon/genética , Regulación hacia Abajo/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Progresión de la Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fosforilación , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Nexinas de Clasificación , Relación Estructura-Actividad , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/genética , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Palmitatos/farmacología , Palmitatos/toxicidad , Potasio/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
In the current report we review the results that lay grounds for the model of intracellular sodium-mediated dopamine-induced endocytosis of Na,K-ATPase. Under conditions of a high salt diet, dopamine activates PKCzeta, which phosphorylates NKA alpha1 Ser-18. The phosphorylation produces a conformational change of alpha1 NH2-terminus, which through interaction with other domains of alpha1 exposes PI3K- and AP-2-binding domains. PI3K bound to the NKA alpha1 induces the recruitment and activation of other proteins involved in endocytosis, and PI3K-generated 3-phosphoinositides affect the local cytoskeleton and modify the biophysical conditions of the membrane for development of clathrin-coated pits. Plasma membrane phosphorylated NKA is internalized to specialized intracellular compartments where the NKA will be dephosphorylated. The NKA internalization results in a reduced Na+ transport by proximal tubule epithelial cells.
Asunto(s)
Cardiotónicos/farmacología , Dopamina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Túbulos Renales Proximales/citología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Endocitosis/efectos de los fármacos , Humanos , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Botulinum neurotoxin serotype A (BoNT/A) has achieved a dichotomous status in modern medicine; it is both a versatile treatment for several neurological disorders and a lethal poison responsible for causing the neuroparalytic syndrome botulism. The extent of paralysis largely depends on the dosage of toxin received. The toxins block neurotransmitter release by delivering their Zn(2+)-dependent protease components to the presynaptic side of chemical synapses. These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE (soluble N-ethylmaleiamide-sensitive factor attachment protein receptor) proteins. Recently, the structural basis for the highly specific interaction between BoNT/A and its target SNARE, SNAP-25 (synaptosomal-associated protein of 25kDa), was elucidated. New details regarding the nature of the toxin-SNARE interactions could be exploited for novel inhibitor design.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Fármacos Neuromusculares/farmacología , Neurotransmisores/antagonistas & inhibidores , Proteínas de Transporte Vesicular/efectos de los fármacos , Toxinas Botulínicas Tipo A/química , Proteínas SNARE , Proteínas de Transporte Vesicular/químicaRESUMEN
Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.