RESUMEN
To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.
Asunto(s)
Insulina/metabolismo , Vacunas Antiprotozoos/inmunología , Receptor de Insulina/metabolismo , Schistosoma japonicum/crecimiento & desarrollo , Esquistosomiasis Japónica/inmunología , Animales , Transporte Biológico/genética , China , Femenino , Glucosa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Filipinas , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Receptor de Insulina/genética , Schistosoma japonicum/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/prevención & control , Esquistosomiasis Japónica/terapiaRESUMEN
Autophagy is a cellular lysosomal degradation pathway essential for regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mammalian target of rapamycin (mTOR) signaling and often counteracts efficacy of certain cancer therapeutic agents. NVP-BEZ235 (BEZ235) is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against non-small cell lung cancer (NSCLC) in preclinical models. The current study focuses on evaluating the role of BEZ235 in regulating autophagy. BEZ235 was effective in inhibiting the growth of NSCLC cells including induction of apoptosis. It also potently induced the expression of type-II LC3, indicating induction of autophagy. When BEZ235 was used in combination with the lysosomal or autophagic inhibitor chloroquine (CQ), enhanced inhibitory effects on monolayer growth and colony formation of NSCLC cells was observed. In addition, enhanced induction of apoptosis was also detected in cells exposed to the combination of BEZ235 and CQ. Moreover, the combination of BEZ235 and CQ was more effective than each single agent alone in inhibiting the growth of NSCLC xenografts in nude mice. Thus, induction of autophagy by BEZ235 appears to be a survival mechanism that may counteract its anticancer effects. Based on these, we suggest a strategy to enhance BEZ235's anticancer efficacy by blockade of autophagy.
Asunto(s)
Autofagia/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Imidazoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Quinolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cloroquina/farmacología , Cloroquina/uso terapéutico , Sinergismo Farmacológico , Femenino , Humanos , Imidazoles/uso terapéutico , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolinas/uso terapéutico , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The purpose of the study is to develop a mouse ocular melanoma model with human uveal melanoma cells that forms hepatic micrometastases. Human uveal melanoma Mel290 cells were transfected with a lentiviral-enhanced green fluorescent protein (EGFP) expression vector. Proliferation assays were performed by comparing Mel290-EGFP and Mel290 cells. After stable expression of EGFP and proliferation was ascertained, 1 x 10 Mel290-EGFP cells were introduced into NU/NU mice by posterior compartment (PC) inoculation or tail vein injection. Control groups were inoculated or injected with Mel290 cells. Ocular and hepatic frozen sections were examined by fluorescence microscopy, and the number of hepatic micrometastases was determined. EGFP expression was observed at 24 h after transfection. At 72 h after transfection, more than 70% of Mel290 cells expressed EGFP. At 45 days (six passages), 90% of Mel290 cells stably expressed EGFP. Histologic examination showed that Mel290-EGFP cells formed hepatic micrometastases after either PC inoculation or tail vein injection. A significant difference in the number of hepatic micrometastases between PC inoculation and tail vein injection (P<0.01) was observed. Mel290-EGFP cells stably expressed green fluorescent protein in vitro at 45 days (six passages). These cells formed hepatic micrometastases in NU/NU mice after PC inoculation or tail vein injection, with significantly more micrometastases developing in the PC inoculation model than after tail vein injection.
Asunto(s)
Neoplasias Hepáticas/secundario , Melanoma/patología , Melanoma/secundario , Neoplasias de la Úvea/patología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
Beta-amyloid peptides (Abeta) are the major component of plaques in brains of Alzheimer's patients, and are they derived from the proteolytic processing of the beta-amyloid precursor protein (APP). The movement of APP between organelles is highly regulated, and it is tightly connected to its processing by secretases. We proposed previously that transport of APP within the cell is mediated in part through its sorting into Mint/X11-containing carriers. To test our hypothesis, we purified APP-containing vesicles from human neuroblastoma SH-SY5Y cells, and we showed that Mint2/3 are specifically enriched and that Mint3 and APP are present in the same vesicles. Increasing cellular APP levels increased the amounts of both APP and Mint3 in purified vesicles. Additional evidence supporting an obligate role for Mint3 in traffic of APP from the trans-Golgi network to the plasma membrane include the observations that depletion of Mint3 by small interference RNA (siRNA) or mutation of the Mint binding domain of APP changes the export route of APP from the basolateral to the endosomal/lysosomal sorting route. Finally, we show that increased expression of Mint3 decreased and siRNA-mediated knockdowns increased the secretion of the neurotoxic beta-amyloid peptide, Abeta(1-40). Together, our data implicate Mint3 activity as a critical determinant of post-Golgi APP traffic.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas Portadoras/metabolismo , Red trans-Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Péptidos beta-Amiloides/aislamiento & purificación , Encéfalo/metabolismo , Cadherinas/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Línea Celular Tumoral , Endosomas/metabolismo , Humanos , Mutación , Proteínas del Tejido Nervioso/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Fracciones Subcelulares/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructuraRESUMEN
In this study, we tested the hypothesis that the elongation 1A (eEF1A) family regulates the cell surface density of the M4 subtype of the muscarinic acetylcholine receptors (mAChR) following agonist-induced internalization. Here, we show that mouse brains lacking eEF1A2 have no detectable changes in M4 expression or localization. We, however, did discover that eEF1A1, the other eEF1A isoform, is expressed in adult neurons contrary to previous reports. This novel finding suggested that the lack of change in M4 expression and distribution in brains lacking eEF1A2 might be due to compensatory effects of eEF1A1. Supporting this theory, we demonstrate that the overexpression of either eEF1A1 or eEF1A2 inhibits M4 recovery to the cell surface after agonist-induced internalization in PC12 cells. Furthermore, eEF1A1 or eEF1A2 had no effect on the recovery of the M1 subtype in PC12 cells. These results demonstrate the novel ability of the eEF1A family to specifically regulate the M4 mAChR.
Asunto(s)
Factor 1 de Elongación Peptídica/fisiología , Receptor Muscarínico M4/metabolismo , Animales , Encéfalo/metabolismo , Endocitosis , Inmunohistoquímica , Ratones , Células PC12 , RatasRESUMEN
BACKGROUND: Genetic, epidemiologic, and biochemical evidence suggests that apolipoprotein E, low-density lipoprotein receptors, and lipid metabolism play important roles in sporadic Alzheimer disease (AD). OBJECTIVE: To identify novel candidate genes associated with sporadic AD. DESIGN: We performed an unbiased microarray screen for genes differentially expressed in lymphoblasts of patients with sporadic AD and prioritized 1 gene product for further characterization in AD brain. SETTING: Emory University, Atlanta, Ga. SUBJECTS: Cell lines were used from 14 patients with AD and 9 normal human control subjects. RESULTS: Six genes were differentially expressed in lymphoblasts of 2 independent groups of patients with probable AD and autopsy-proven AD. We hypothesized that 1 of the genes, termed low-density lipoprotein receptor relative with 11 binding repeats (LR11) (reduced 1.8- and 2.5-fold in AD lymphoblasts vs controls), might be associated with sporadic AD on the basis of its function as neuronal apolipoprotein E receptor. We found dramatic and consistent loss of immunocytochemical staining for LR11 in histologically normal-appearing neurons in AD brains. This reduction of LR11 protein was confirmed by quantitative Western blotting (P =.01). CONCLUSIONS: There is loss of the microarray-derived candidate, LR11, in neurons of AD brains. This study shows that microarray analysis of widely available lymphoblasts derived from patients with AD holds promise as a primary screen for candidate genes associated with AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Línea Celular Transformada , Femenino , Humanos , Proteínas Relacionadas con Receptor de LDL , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Linfocitos/metabolismo , Masculino , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Proteínas del Tejido Nervioso/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores de LDL/biosíntesis , Receptores de LDL/genéticaRESUMEN
Agonist-induced internalization followed by subsequent return to the cell surface regulates G-protein-coupled receptor (GPCR) activity. Because the cellular responsiveness to ligand depends on the balance between receptor degradation and recycling, it is crucial to identify the molecules involved in GPCR recovery to the cell surface. In this study, we identify mechanisms involved in the recycling of the M4 subtype of muscarinic acetylcholine receptor. M4 is highly expressed in the CNS, plays a role in locomotor activity, and is a novel therapeutic target for neurologic and psychiatric disorders. Previous studies show that, after cholinergic stimulation, M4 internalizes from the cell surface to endosomes in cell culture and the rat brain. Here, we show that, after activation, M4 traffics to transferrin receptor- and Rab11a-positive perinuclear endosomes. Expression of the constitutively GDP-bound, inactive mutant Rab11aS25N inhibits M4 trafficking to recycling endosomes. Expression of the C-terminal tail of myosin Vb, a Rab11a effector, enhances M4 accumulation in perinuclear endosomes. Both Rab11aS25N and the myosin Vb tail impair M4 recycling. The results demonstrate that GPCR recycling is mediated through a discrete pathway using both Rab11a and myosin Vb.