RESUMEN
RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/sangre , Trastornos de las Plaquetas Sanguíneas/genética , Familia , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Heterocigoto , Homocigoto , Adolescente , Adulto , Plaquetas/metabolismo , Dominio Catalítico/genética , Niño , Femenino , Tamización de Portadores Genéticos/métodos , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Agregación Plaquetaria , Portugal , Adulto JovenRESUMEN
DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293 T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250 kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factores de Intercambio de Guanina Nucleótido/genética , Empalme Alternativo , Animales , Línea Celular Tumoral , Exones , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/inmunología , Células HEK293 , Humanos , Sueros Inmunes/química , Sueros Inmunes/metabolismo , Interleucina-4/inmunología , Células Jurkat , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Ratones , Ratones Noqueados , Isoformas de Proteínas , ARN Mensajero/genética , TranscriptomaRESUMEN
CONTEXT: Osteoporosis (OP) is a progressive systemic bone disease. Dual-energy X-ray absorptiometry (DXA) is routinely employed and is considered the gold standard method for the diagnosis of OP. OBJECTIVE: We aimed to investigate the potential use of combined information from multiple bone turnover markers (BTMs) as a clinical diagnostic tool for OP. MATERIALS AND METHODS: A total of 9053 Chinese postmenopausal women (2464 primary OP patients and 6589 healthy controls) were recruited. Serum levels of six common BTMs, including BAP, BSP, CTX, OPG, OST and sRANKL were assayed. Models based on support vector machine (SVM) were constructed to explore the efficiency of different combinations of multiple BTMs for OP diagnosis. RESULTS: Increasing the number of BTMs used in generating the models increased the predictive power of the SVM models for determining the disease status of study subjects. The highest kappa coefficient for the model with one BTM (BAP) compared to DXA was 0.7783. The full model incorporating all six BTMs resulted in a high kappa coefficient of 0.9786. CONCLUSION: Our findings showed that although single BTMs were not sufficient for OP diagnosis, appropriate combinations of multiple BTMs incorporated into the SVM models showed almost perfect agreement with the DXA.
Asunto(s)
Biomarcadores/sangre , Remodelación Ósea/genética , Osteoporosis/sangre , Absorciometría de Fotón , Anciano , Densidad Ósea/genética , China/epidemiología , Enzimas de Restricción del ADN/sangre , Femenino , Factores de Intercambio de Guanina Nucleótido/sangre , Humanos , Persona de Mediana Edad , Osteoporosis/diagnóstico por imagen , Osteoporosis/patología , Ligando RANK/sangre , Factores de Intercambio de Guanina Nucleótido Rho/sangre , Máquina de Vectores de SoporteRESUMEN
The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbß3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.
Asunto(s)
Plaquetas/metabolismo , Factores de Intercambio de Guanina Nucleótido/sangre , Trastornos Hemorrágicos/sangre , Trastornos Hemorrágicos/genética , Mutación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trastornos de las Plaquetas Sanguíneas/sangre , Trastornos de las Plaquetas Sanguíneas/genética , Plaquetas/patología , Niño , Preescolar , Femenino , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Masculino , LinajeRESUMEN
Dedicator of cytokinesis 2 (DOCK2) is a CDM family protein containing DOCK homology region (DHR)-1 and DHR-2, Src-homology 3 (SH3) domain, and C-terminal polybasic amino acid cluster. The CDM family consists of 11 mammalian members and is classified into four subfamilies, the DOCK-A, -B, -C, and -D. DOCK2 is a member of DOCK-A subfamily and an atypical guanine exchange factor regulating the loading of GTP to activate Rac. It is primarily found in peripheral blood, spleen, and thymus and mainly expressed in lymphocytes and macrophages of various organs. DOCK2 is also expressed in microglial in brain and is induced in neointima smooth muscle following vascular injury. Functionally, DOCK2 is involved in cell motility, polarity, adhesion, proliferation, and apoptosis. It is essential for lymphocyte migration and activation as well as neutrophil chemotaxis. DOCK2 also regulates the differentiation of natural killer T cells, type 2 T helper cells, and plasmacytoid dendritic cells. In addition, it is important for the growth of B cell lymphoma and prostate cancer cells. Deletion of DOCK2 enables long-term cardiac allograft survival. Moreover, DOCK2 is associated with the Alzheimer Disease, HIV development, and the early-onset of invasive infections. Recently, we found that DOCK2 plays a critical role in SMC phenotypic modulation and vascular remodeling. In this review, we will briefly summarize recent advancement of DOCK2 function. J. Cell. Physiol. 232: 1931-1940, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Sistema Inmunológico/metabolismo , Transducción de Señal , Animales , Enfermedad , Modelos Animales de Enfermedad , Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Sistema Inmunológico/inmunología , Unión ProteicaRESUMEN
Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.
Asunto(s)
Activación Plaquetaria/fisiología , Proteínas R-SNARE/sangre , Citoesqueleto de Actina/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/sangre , Animales , Plaquetas/fisiología , Plaquetas/ultraestructura , Gránulos Citoplasmáticos/fisiología , Exocitosis/fisiología , Factores de Intercambio de Guanina Nucleótido/sangre , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Selectina-P/sangre , Agregación Plaquetaria/fisiología , Proteínas R-SNARE/deficiencia , Proteínas R-SNARE/genéticaRESUMEN
Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.
Asunto(s)
Barrera Hematoencefálica/inmunología , Interleucina-9/inmunología , Accidente Cerebrovascular/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Complejo CD3/sangre , Estudios de Casos y Controles , Hipoxia de la Célula/fisiología , Células Cultivadas , Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Expresión Génica , Glucosa/metabolismo , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Interleucina-9/sangre , Interleucina-9/genética , Interleucina-9/farmacología , Masculino , Ratones , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Proteínas Nucleares/sangre , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Índice de Severidad de la Enfermedad , Accidente Cerebrovascular/patología , Subgrupos de Linfocitos T/inmunología , Proteínas de Uniones Estrechas/metabolismo , Transactivadores/sangre , Transactivadores/genética , Adulto JovenRESUMEN
OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.
Asunto(s)
Plaquetas/metabolismo , Factores de Intercambio de Guanina Nucleótido/deficiencia , Hemostasis , Activación Plaquetaria , Trombosis/prevención & control , Animales , Modelos Animales de Enfermedad , Genotipo , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones Transgénicos , Mutación , Fenotipo , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Trombosis/sangre , Trombosis/genética , Factores de Tiempo , Proteínas de Unión al GTP rap1/sangreRESUMEN
This postmarketing surveillance study assessed the safety and effectiveness of daily teriparatide treatment in patients with osteoporosis in a Japanese clinical setting. In this prospective, multicenter, observational study, patients with osteoporosis at high risk for fracture received subcutaneous injections of teriparatide (20 µg/day) for a maximum of 24 months. For this interim report, data from 1,671 patients were eligible for analysis at the cutoff date. The mean age was 75.3 years; 93% of patients (1,552/1,671 patients) were women. There were 117 adverse drug reactions (ADRs) reported in 101 of 1,671 patients (6.04%); the most common reported ADRs were nausea, dizziness, headache, and palpitations. No clinically significant safety issues were identified, although 5 serious ADRs were reported in 4/1,671 (0.24 %) patients. At 12 months, 71.9% of patients remained on teriparatide treatment. From 1 month, there were rapid increases in the biomarkers of bone formation P1NP and, to a lesser extent, BAP. In contrast, increases in the biomarkers of bone resorption, serum NTX, urinary NTX, and TRACP5b, were smaller. After 12 months of treatment, there was an increase in bone mineral density at the lumbar spine, femoral neck, and total hip, and a decrease in the Visual Analog Scale score for back pain. The incidence of new vertebral and nonvertebral fractures was 1.21% and 3.18%, respectively. In conclusion, the favorable safety profile and effectiveness of teriparatide observed in this population of Japanese patients with osteoporosis were accompanied by relatively high persistence with treatment, which is a key factor in the success of osteoporosis treatment.
Asunto(s)
Conservadores de la Densidad Ósea/administración & dosificación , Densidad Ósea/efectos de los fármacos , Resorción Ósea/tratamiento farmacológico , Osteoporosis/tratamiento farmacológico , Teriparatido/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/orina , Conservadores de la Densidad Ósea/efectos adversos , Resorción Ósea/sangre , Resorción Ósea/orina , Femenino , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/orina , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/sangre , Osteoporosis/orina , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/orina , Procolágeno/sangre , Procolágeno/orina , Estudios Prospectivos , Teriparatido/efectos adversosRESUMEN
BACKGROUND: Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene is thought to be a key regulator of E6AP. METHODS AND RESULTS: Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. CONCLUSIONS: Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.
Asunto(s)
Amish/genética , Síndrome de Angelman/genética , Factores de Intercambio de Guanina Nucleótido/genética , Mutación , Adolescente , Adulto , Proteínas de Ciclo Celular/química , Línea Celular , Niño , Preescolar , ADN/análisis , ADN/genética , Análisis Mutacional de ADN , Femenino , Fibroblastos/química , Fibroblastos/metabolismo , Estudio de Asociación del Genoma Completo , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Lactante , Masculino , Modelos Moleculares , Proteínas Nucleares/química , Linaje , Ubiquitina-Proteína LigasasRESUMEN
Two major pathways contribute to Ras-proximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbß3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI(-/-) blood, even in the presence of exogenous adenosine diphosphate and thromboxane A(2). In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI(-/-) mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Clopidogrel , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/genética , Hemostasis , Cinética , Masculino , Mesenterio/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/irrigación sanguínea , Inhibidores de Agregación Plaquetaria/uso terapéutico , Transducción de Señal/efectos de los fármacos , Trombosis/tratamiento farmacológico , Trombosis/genética , Ticlopidina/análogos & derivados , Ticlopidina/uso terapéuticoRESUMEN
OBJECTIVE: Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). METHODS: ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). RESULTS: Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. CONCLUSION: These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes.
Asunto(s)
Adenosina Trifosfato/sangre , Eritrocitos/metabolismo , Factores de Intercambio de Guanina Nucleótido/sangre , Proteína Quinasa C/sangre , Adenina/análogos & derivados , Adenina/farmacología , Cilostazol , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Activación Enzimática/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Humanos , Iloprost/farmacología , Técnicas In Vitro , Isoproterenol/farmacología , Modelos Biológicos , Naftalenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Rolipram/farmacología , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Tetrazoles/farmacología , Tionucleótidos/farmacologíaRESUMEN
Second messenger-mediated inside-out activation of integrin alphaIIbbeta3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent alphaIIbbeta3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for alphaIIbbeta3 activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for alphaIIbbeta3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Galphai-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Galphai signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and alphaIIbbeta3 activation in mouse platelets activated through the PAR4 receptor.
Asunto(s)
Plaquetas/metabolismo , Factores de Intercambio de Guanina Nucleótido/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteína Quinasa C/sangre , Adenosina Difosfato/sangre , Animales , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Degranulación de la Célula/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/sangre , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Cinética , Ratones , Ratones Noqueados , Modelos Biológicos , Oligopéptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Receptores de Trombina/sangre , Receptores de Trombina/efectos de los fármacos , Transducción de Señal , Proteínas de Unión al GTP rap1/sangreRESUMEN
Microarray searches have revealed potential genetic biomarkers in a wide variety of human diseases. Identification of biomarkers for disease status is particularly important in chronic neurodegenerative diseases where brain tissue cannot be sampled. A previous study identified 12 genes from microarray analysis as associated with Huntington's disease, although the relationships had not been validated. We used new machine learning approaches to reanalyse those microarray data and to rank the identified potential genetic biomarkers. We then performed quantitative reverse transcription-polymerase chain reaction analysis on a subset of the candidate genes in blood samples from an independent cohort of 23 Huntington's disease patients and 23 healthy controls. Our highest ranked genes did not overlap with the 12 previously identified, but two were significantly up-regulated in the Huntington's disease group: ARFGEF2 and GOLGA8G. Little is known about the latter, but the former warrants further analysis as it is known to be associated with intracellular vesicular trafficking, disturbances of which characterize Huntington's disease.
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Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Enfermedad de Huntington/genética , Adulto , Inteligencia Artificial , Estudios de Casos y Controles , Femenino , Factores de Intercambio de Guanina Nucleótido/sangre , Humanos , Enfermedad de Huntington/sangre , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (PREX1) was reported to be overexpressed in some cancers and involved in cancer development, but its expression and significance in gastric cancer remain unclear. AIM: To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer, especially to evaluate the potential mechanism of PREX1 in gastric cancer. METHODS: Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer. The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal. The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor (TGF) ß1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics. Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFß1 on the expression of PREX1. Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFß1 pathway. Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells. RESULTS: PREX1 was overexpressed in the gastric tumors, and the expression levels were positively associated with the development of gastric cancer. Also, the high expression of PREX1 revealed poor prognosis, especially for those advanced and specific intestinal gastric cancer patients. PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFß1-related mediators. Furthermore, TGFß1 could induce the expression of PREX1 at both the protein and mRNA level. Also, PREX1 could activate the TGFß1 pathway. The induced PREX1 could increase the migration and invasion activity of gastric cancer cells. CONCLUSION: PREX1 is overexpressed in gastric cancer, and the high level of PREX1 predicts poor prognosis. PREX1 is closely associated with TGFß signaling and promotes the metastasis of gastric cancer cells.
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Regulación Neoplásica de la Expresión Génica/genética , Factores de Intercambio de Guanina Nucleótido/sangre , Fosfatos de Fosfatidilinositol/sangre , Neoplasias Gástricas/genética , Factor de Crecimiento Transformador beta1/sangre , Adulto , Biología Computacional , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , ARN Mensajero/sangre , Transducción de Señal/genética , Neoplasias Gástricas/sangre , Neoplasias Gástricas/mortalidadRESUMEN
BACKGROUND: Behçet's disease (BD) is a multisystem inflammatory disorder characterized by recurrent oral ulcers, genital ulcers and ocular inflammation, as well as skin, joint, vascular, pulmonary, central nervous system (CNS) and gastrointestinal tract manifestations. The etiopathogenesis of BD has not yet been identified; but it has generally been accepted that several environmental factors may induce an inflammatory attack in genetically susceptible individuals. In this study, we aimed to identify antigens that could elicit high-titer IgG responses by the serological analysis of recombinant expression of cDNA libraries method (SEREX). METHODS: We screened a human testis cDNA library with pooled sera obtained from 4 BD patients by SEREX. Antigens that were identified with the initial analysis were selected for seroreactivity analysis of a larger group of BD patients (n=78) and controls (n=66) by serological immunoscreening. RESULTS: We observed seroreactivity against 6 antigens using the pooled sera. These included rabaptin 5 (RABPT5), PTEN-induced putative kinase 1 (PINK1), switch associated protein 70 (SWAP70), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), ankyrin repeat domain 20 family, member A1 (ANKRD20A1), and an unknown antigen. Eleven out of 82 (13.4%) BD patients were found to have antibodies elicited against PINK1 antigen, when none of the control sera showed reactivity (p=0.001). There was no significant difference in the frequency of other defined antigens between the patient and control groups. However, among BD clinical sub-groups, anti-SWAP70 antibodies were found to associate with vascular involvement. DISCUSSION: In this study, antibodies against PINK1 were found to specifically associate with BD while SWAP70 antibody was associated with clinical sub-groups of BD. Although variations in both genetic background and environmental factors may affect the outcome of serological responses, our results suggest that serological screening can be used to identify antigens that elicit antibody responses associated with BD.
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Autoanticuerpos/sangre , Síndrome de Behçet/enzimología , Proteínas Quinasas/inmunología , Adulto , Proteínas Reguladoras de la Apoptosis , Síndrome de Behçet/inmunología , Proteínas de Unión al ADN/sangre , Femenino , Biblioteca de Genes , Factores de Intercambio de Guanina Nucleótido/sangre , Humanos , Masculino , Antígenos de Histocompatibilidad Menor , Proteínas Nucleares/sangre , Proteínas/análisis , Proteínas de Unión al ARN , Turquía , Proteínas de Transporte Vesicular/sangreRESUMEN
Objective: The aim of the study was to determine the expression profile of long noncoding RNAs (lncRNAs) in CD4+ T cells from COPD patients and explore the clinical value of the lncRNAs. Methods: First, microarray analysis was performed. Differentially expressed lncRNAs were validated by quantitative real-time reverse transcription-PCR (qRT-PCR) in samples from 56 patients with acute exacerbations of COPD (AECOPD), 56 patients with stable COPD, and 35 healthy controls. Meanwhile, the clinical value was tested by receiver operating characteristic curve analysis. The functions of lncRNAs were analyzed by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database. The potential target genes that might be regulated by NR-026690 and ENST00000447867 were identified by the lncRNA-mRNA network and competing endogenous RNA network. The transcriptional expression level of rap guanine nucleotide exchange factor 3 (RAPGEF3) was tested by qRT-PCR. The correlation of the expression between NR-026690, ENST00000447867, and RAPGEF3 was analyzed by Spearman's correlation test. Results: We found that the relative expression levels of ENST00000447867 and NR-026690 in the CD4+ T cells of AECOPD patients were significantly higher than in the stable COPD patients and control subjects by microarray and qRT-PCR validation. The transcriptional expression level of RAPGEF3 in the CD4+ T cells was significantly higher in the AECOPD group compared to the control group (P<0.01) and the stable COPD group (P<0.05). RAPGEF3 expression was positively associated with NR-026690 (r=0.4925, P<0.01) and ENST00000447867 (r=0.4065, P<0.01). Conclusion: NR-026690 and ENST00000447867 might be potential biomarkers for COPD. They might affect RAPGEF3 as miRNA sponges to regulate COPD development.
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Linfocitos T CD4-Positivos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , ARN Largo no Codificante/sangre , Anciano , Estudios de Casos y Controles , Citocinas/sangre , Citocinas/genética , Bases de Datos Genéticas , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Marcadores Genéticos , Factores de Intercambio de Guanina Nucleótido/sangre , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Regulación hacia ArribaRESUMEN
Friedreich's ataxia (FA) is a neurodegenerative disease with no approved therapy that is the result of frataxin deficiency. The identification of human FA blood biomarkers related to disease severity and neuro-pathomechanism could support clinical trials of drug efficacy. To try to identify human biomarkers of neuro-pathomechanistic relevance, we compared the overlapping gene expression changes of primary blood and skin cells of FA patients with changes in the Dorsal Root Ganglion (DRG) of the KIKO FA mouse model. As DRG is the primary site of neurodegeneration in FA, our goal was to identify which changes in blood and skin of FA patients provide a 'window' into the FA neuropathomechanism inside the nervous system. In addition, gene expression in frataxin-deficient neuroglial cells and FA mouse hearts were compared for a total of 5 data sets. The overlap of these changes strongly supports mitochondrial changes, apoptosis and alterations of selenium metabolism. Consistent biomarkers were observed, including three genes of mitochondrial stress (MTIF2, ENO2), apoptosis (DDIT3/CHOP), oxidative stress (PREX1), and selenometabolism (SEPW1). These results prompted our investigation of the GPX1 activity as a marker of selenium and oxidative stress, in which we observed a significant change in FA patients. We believe these lead biomarkers that could be assayed in FA patient blood as indicators of disease severity and progression, and also support the involvement of mitochondria, apoptosis and selenium in the neurodegenerative process.
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Biomarcadores/sangre , Ataxia de Friedreich/sangre , Ganglios Espinales/metabolismo , Estrés Oxidativo/genética , Animales , Antioxidantes/metabolismo , Apoptosis/genética , Modelos Animales de Enfermedad , Factores Eucarióticos de Iniciación/sangre , Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Ganglios Espinales/patología , Regulación de la Expresión Génica/genética , Factores de Intercambio de Guanina Nucleótido/sangre , Humanos , Proteínas de Unión a Hierro/genética , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/sangre , Miocardio/metabolismo , Selenio/metabolismo , Factor de Transcripción CHOP/sangre , FrataxinaRESUMEN
Multiplexed biosensors hold great promise for early diagnosis of diseases where the detection of multiple biomarkers is required. Hyper Immunoglobulin E syndromes (HIES) are rare primary immunodeficiency disorders associated with mutations either in the signal transducer and activator of transcription 3 (STAT3), dedicator of cytokinesis 8 DOCK8) or phosphoglucomutase 3 (PGM3) genes. Yet, the diagnosis of HIES is challenged by the complexity of the existing laboratory assays. Here, we report for the first time the development of a multiplexed electrochemical immunosensor for the simultaneous detection of DOCK8, STAT3 and PGM3 proteins. The immunosensor was constructed on carbon array electrodes that were first modified by electrodeposition of gold nanoparticles (AuNPs). The array electrodes were then used to immobilize specific antibodies for the three proteins after the functionalization of the electrodes with cysteamine/glutaraldehyde linkers. The simultaneous detection of the DOCK8, PGM3 and STAT3 proteins was successfully realized by the immunosensor with respective limits of detections of 3.1, 2.2 and 3.5â¯pg/ml. The immunosensor has shown good sensitivity as well as selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and Duchenne Muscular Dystrophy (DMD). Moreover, the immunosensor was successfully applied in human serum samples showing capability to distinguish the HIES from the control samples.