RESUMEN
This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.
Asunto(s)
Ácido Gálico/farmacología , Folículo Ovárico/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Ovinos/fisiología , Animales , Cromatina , Cromonas/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Mitocondrias/metabolismo , Morfolinas/farmacología , Folículo Ovárico/crecimiento & desarrollo , Fosfatidilinositol 3-Quinasa/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Técnicas de Cultivo de Tejidos/veterinariaRESUMEN
The interaction of dendritic cells and macrophages with a variety of rigid noncellular particles triggers activation of the NLRP3 inflammasome and consequent secretion of interleukin 1ß (IL-1ß). Noncellular particles can also be generated in the context of helminth infection, since these large pathogens often shed their outermost structures during growth and/or molting. One such structure is the massive, mucin-based, soft, flexible laminated layer (LL), which protects the larval stages of cestodes of the genus Echinococcus We show that particles from the Echinococcus granulosus LL (pLL) trigger NLRP3- and caspase-1-dependent IL-1ß in lipopolysaccharide (LPS)-primed mouse bone marrow-derived dendritic cells (BMDC). This response can be elicited by pLL too large for phagocytosis and nonetheless requires actin dynamics, Syk, and phosphatidylinositol 3-kinase (PI3K). These three requirements had already been observed in our previous study on the alteration by pLL of CD86, CD40, IL-10, and IL-12 responses to LPS in BMDC; however, we now show that these alterations are independent of NLRP3 and caspase-1. In other words, an initial interaction with particles requiring actin dynamics, Syk, and PI3K, but not phagocytosis, elicits both NLRP3-dependent and NLRP3-independent responses. Intraperitoneal injection of pLL induced IL-1ß, suggesting that contact with LL materials induces IL-1ß in the E. granulosus infection setting. Our results extend our understanding of NLRP3 inflammasome activation by noncellular particulate materials both to helminth-derived materials and to flexible/soft materials.
Asunto(s)
Micropartículas Derivadas de Células/química , Células Dendríticas/efectos de los fármacos , Echinococcus granulosus/química , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Parásitos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Micropartículas Derivadas de Células/inmunología , Células Dendríticas/inmunología , Echinococcus granulosus/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Parásitos/genética , Indazoles/farmacología , Inflamasomas/efectos de los fármacos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/agonistas , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal , Estilbenos/farmacología , Sulfonamidas/farmacología , Wortmanina/farmacologíaRESUMEN
Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.
Asunto(s)
Mecanotransducción Celular/genética , Óxido Nítrico Sintasa/genética , Fosfatidilinositol 3-Quinasa/genética , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Wortmanina/farmacología , Inductores de la Angiogénesis/farmacología , Proteínas de Unión al ADN , Dioxigenasas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Mecanotransducción Celular/efectos de los fármacos , Oxigenasas de Función Mixta , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo III/genética , Fosfatidilinositol 3-Quinasa/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-akt/genética , Resistencia al Corte/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Estrés Mecánico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Red and processed meat consumption has been strongly related to increase the risk of colorectal cancer (CRC), although its impact is largely unknown. Hemin, an iron-containing porphyrin, is acknowledged as a putative factor of red and processed meat pro-carcinogenic effects. The aim of this study was to investigate the effects of high dietary hemin on the promotion/progression stages of 1,2-dimethylhydrazine (1,2-DMH)-induced colon carcinogenesis. Twenty-four Wistar male rats were given four subcutaneous 1,2-DMH injections and received either balanced diet or balanced diet supplemented with hemin 0.5â¯mmol/kg for 23 weeks. Colon specimens were analyzed for aberrant crypt foci (ACF) and tumor development. Dietary hemin significantly increased ACF number and fecal water cytotoxicity/genotoxicity in Caco-2 cells when compared to 1,2-DMH control group. However, tumor incidence, multiplicity and cell proliferation did not differ between 1,2-DMHâ¯+â¯hemin and 1,2-DMH control group. Gene expression analysis of 91 target-genes revealed that only three genes (Figf, Pik3r5 and Tgfbr2) were down-regulated in the tumors from hemin-fed rats compared to those from 1,2-DMH control group. Therefore, the findings of this study show that high hemin intake promotes mainly DNA damage and ACF development and but does not change the number nor incidence of colon tumors induced by 1,2-DMH in male rats.
Asunto(s)
Focos de Criptas Aberrantes/inducido químicamente , Neoplasias del Colon/inducido químicamente , Daño del ADN , Hemina/toxicidad , Lesiones Precancerosas/inducido químicamente , 1,2-Dimetilhidrazina , Alimentación Animal , Animales , Células CACO-2 , Cocarcinogénesis , Ensayo Cometa , Regulación hacia Abajo/efectos de los fármacos , Heces , Humanos , Masculino , Fosfatidilinositol 3-Quinasa/genética , Ratas , Ratas Wistar , Receptor Tipo II de Factor de Crecimiento Transformador beta/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Carne Roja , Factores de Tiempo , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Factor D de Crecimiento Endotelial Vascular/genéticaRESUMEN
Root hair curling is an early and essential morphological change required for the success of the symbiotic interaction between legumes and rhizobia. At this stage rhizobia grow as an infection thread within root hairs and are internalized into the plant cells by endocytosis, where the PI3K enzyme plays important roles. Previous observations show that stress conditions affect early stages of the symbiotic interaction, from 2 to 30 min post-inoculation, which we term as very early host responses, and affect symbiosis establishment. Herein, we demonstrated the relevance of the very early host responses for the symbiotic interaction. PI3K and the NADPH oxidase complex are found to have key roles in the microsymbiont recognition response, modulating the apoplastic and intracellular/endosomal ROS induction in root hairs. Interestingly, compared with soybean mutant plants that do not perceive the symbiont, we demonstrated that the very early symbiont perception under sublethal saline stress conditions induced root hair death. Together, these results highlight not only the importance of the very early host-responses on later stages of the symbiont interaction, but also suggest that they act as a mechanism for local control of nodulation capacity, prior to the abortion of the infection thread, preventing the allocation of resources/energy for nodule formation under unfavorable environmental conditions.
Asunto(s)
Bradyrhizobium/fisiología , Glycine max/enzimología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta , Simbiosis , Interacciones Huésped-Patógeno , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Glycine max/genética , Glycine max/microbiología , Glycine max/fisiologíaRESUMEN
Low levels of high-density lipoprotein-cholesterol (HDL-C) constitute an independent biomarker of cardiovascular morbi-mortality. However, recent advances have drastically modified the classical and limited view of HDL as a carrier of 'good cholesterol', and have revealed unexpected levels of complexity in the circulating HDL particle pool. HDL particles are indeed highly heterogeneous in structure, intravascular metabolism and biological activity. This review describes recent progress in our understanding of HDL subpopulations and their biological activities, and focuses on relationships between the structural, compositional and functional heterogeneity of HDL particles.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Antioxidantes/metabolismo , Enfermedades Cardiovasculares/metabolismo , HDL-Colesterol/metabolismo , Fibrinolíticos/metabolismo , Vasodilatadores/metabolismo , Animales , Antiinflamatorios no Esteroideos/clasificación , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/clasificación , Antioxidantes/farmacología , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Biomarcadores/metabolismo , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patología , HDL-Colesterol/clasificación , HDL-Colesterol/farmacología , Citoprotección , Fibrinolíticos/clasificación , Fibrinolíticos/farmacología , Regulación de la Expresión Génica , Humanos , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasodilatadores/clasificación , Vasodilatadores/farmacologíaRESUMEN
The aim of this study was to evaluate the effects of yerba maté (YM) extract on the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in vivo. The mice were introduced to either standard- or high-fat diet (HFD). After 8 weeks on an HFD, mice were randomly assigned to one of the two treatment conditions, water or yerba maté extract at 1.0 g/kg. After treatment, glucose blood level and hepatic insulin response were evaluated. Liver tissue was examined to determine the mRNA levels using the PI3K-AKT PCR array. The nuclear translocation of forkhead box O1 (FOXO1) was determined by an electrophoretic mobility-shift assay. Our data demonstrated that yerba maté extract significantly decreased the final body weight, glucose blood levels, and insulin resistance of mice. Molecular analysis demonstrated that an HFD downregulated Akt2, Irs1, Irs2, Pi3kca, Pi3kcg, and Pdk1; after yerba maté treatment, the levels of those genes returned to baseline. In addition, an HFD upregulated Pepck and G6pc and increased FOXO1 nuclear translocation. The intervention downregulated these genes by decreasing FOXO1 nuclear translocation. The results obtained demonstrate for the first time the specific action of yerba maté on the PI3K-AKT pathway, which contributed to the observed improvement in hepatic insulin signaling.
Asunto(s)
Ilex paraguariensis/química , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa , Regulación hacia Abajo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Fosfatidilinositol 3-Quinasa/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Cyclin D1 (CCND1) plays a significant role in G1-S transition of cell cycle, and phosphatase and a tensin homologue (PTEN) negatively regulate cell cycle through phosphatidylinositol 3-kinase (PI3K)/AKT signaling. CCND1 and PTEN genetic polymorphisms might induce susceptibility to the occurrence of esophageal squamous cell carcinoma (ESCC). Three hundred and four ESCC patients and 413 healthy controls from Anyang, China, were enrolled in this study. All genotyping at CCND1 (807 G/A) and PTEN (rs701848 T/C and rs2735343 C/G) were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Unconditional logistic regression model was used to analyze the correlation between the polymorphisms and the susceptibility to develop ESCC. Statistically significant differences were observed between cases and controls in distribution of genotypes or alleles at PTEN rs701848 T/C and rs2735343 C/G, with either haplotype TG or CG possessing notably higher proportion in cases than in the controls. However, such difference could not be found in the distribution of the polymorphisms at CCND1 807 G/A. In summary, the polymorphisms of PTEN rs701848 T/C and rs2735343 C/G might represent crucial modifying factors for development of ESCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Ciclina D1/genética , Neoplasias Esofágicas/genética , Fosfohidrolasa PTEN/genética , Estudios de Casos y Controles , Progresión de la Enfermedad , Carcinoma de Células Escamosas de Esófago , Femenino , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasa/genética , Polimorfismo de Longitud del Fragmento de Restricción/genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND/OBJECTIVES: Highly palatable food (HPF), which is enriched in simple sugars and saturated fat, contributes to obesity and insulin resistance in humans. These metabolic changes are associated with serious complications of the central nervous system, including an elevated risk of cognitive dysfunction. We, herein, treated rats with HPF and then examined the insulin-signaling pathway, in particular, the levels of phosphatidylinositol-3 kinase (PI3K), Akt, and insulin receptor substrate-1 (IRS-1) in the hippocampus and hypothalamus. METHODS: Adult Wistar rats fed with HPF (heated or not during preparation) for 4 months and then measured the levels of PI3K, Akt, and IRS-1 in the hippocampus and hypothalamus, by western blotting and quantitative real-time polymerase chain reaction. RESULTS: We observed changes in body weight, glucose intolerance, and lipidemia, confirming that peripheral metabolic alterations were induced using this model. Hippocampal PI3K and hypothalamic Akt were affected in rats that are submitted to chronic exposure to an HPF diet. Moreover, heated HPF caused differentiated alterations in the regulatory subunit of PI3K in the hippocampus. DISCUSSION: Our data suggest that this diet alters insulin signaling differentially in each brain region, and that hippocampal changes induced by this diet could contribute to the understanding of cognitive impairments that are dependent on the hippocampus.
Asunto(s)
Hipocampo/metabolismo , Hipotálamo/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Fosfatidilinositol 3-Quinasa/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Animales , Peso Corporal , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Dieta , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Masculino , Obesidad/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling. EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Expresión Génica , Hormona del Crecimiento/metabolismo , Hígado/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Factor de Crecimiento Epidérmico/genética , Hormona del Crecimiento/genética , Hígado/citología , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Serina/genética , Serina/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Treonina/genética , Treonina/metabolismoRESUMEN
INTRODUCTION: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia. OBJECTIVE: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations) in phosphatidylinositol-3-kinase (PI3K), Janus kinase 2 (JAK2), FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1), which are involved in leukemia and other cancers, in a population of Brazilian MDS patients. METHODS: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression. RESULTS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression. CONCLUSIONS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression.
Asunto(s)
Janus Quinasa 2/genética , Mutación/genética , Síndromes Mielodisplásicos/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasa/genética , Tirosina Quinasa 3 Similar a fms/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , NucleofosminaRESUMEN
Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam(3) -Cys-Ser-Lys(4) . These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478-511) and exposed to Pam(3) -Cys-Ser-Lys(4) in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.
Asunto(s)
Dexametasona/farmacología , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptor Toll-Like 2/inmunología , Línea Celular , Citocinas/biosíntesis , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Pulmón/metabolismo , FN-kappa B/biosíntesis , Péptidos/farmacología , Fosfatidilinositol 3-Quinasa/genética , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Factor de Transcripción AP-1/biosíntesis , Activación Transcripcional , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37°C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS-PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3-4 h at 37°C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20°C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37°C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.