RESUMEN
Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.
Asunto(s)
Genes Recesivos , Histamina N-Metiltransferasa/genética , Discapacidad Intelectual/genética , Mutación Missense , Adolescente , Adulto , Secuencia de Aminoácidos , Dominio Catalítico , Niño , Preescolar , Simulación por Computador , Análisis Mutacional de ADN , Exoma , Femenino , Histamina N-Metiltransferasa/metabolismo , Humanos , Lactante , Discapacidad Intelectual/enzimología , Irak , Masculino , Datos de Secuencia Molecular , Linaje , Alineación de Secuencia , Turquía , Población Blanca/genéticaRESUMEN
In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.
Asunto(s)
Cromosomas Humanos Par 16/genética , Proteínas F-Box/genética , Genes Recesivos , Discapacidad Intelectual/genética , Eliminación de Secuencia , Proteínas Supresoras de Tumor/genética , Western Blotting , Mapeo Cromosómico , Consanguinidad , Femenino , Mutación del Sistema de Lectura/genética , Homocigoto , Humanos , Técnicas para Inmunoenzimas , Discapacidad Intelectual/patología , Masculino , Pakistán , Linaje , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A novel method for pumping very small volumes of liquid by using surface acoustic waves is employed to create a microfluidic flow chamber on a chip. It holds a volume of only a few mul and its planar design provides complete architectural freedom. This allows for the reconstruction of even complex flow scenarios (e.g. curvatures, bifurcations and stenosis). Addition of polymer walls to the planar fluidic track enables cell culturing on the chip surface and the investigation of cell-cell adhesion dynamics under flow. We demonstrate the flexibility of the system for application in many areas of microfluidic investigations including blood clotting phenomena under various flow conditions and the investigation of different stages of cell adhesion.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Adhesión Celular , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Humanos , Tamaño de la Partícula , Polímeros/química , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
The calcium-sensing receptor (CaSR) provides a fundamental mechanism for diverse cells to detect and respond to modulations in the ionic and nutrient compositions of their extracellular milieu. The roles for this receptor are largely unknown in the intestinal tract, where epithelial cells are normally exposed to large variations in extracellular solutes. Here, we show that colonic CaSR signaling stimulates the degradation of cyclic nucleotides by phosphodiesterases and describe the ability of receptor activation to reverse the fluid and electrolyte secretory actions of cAMP- and cGMP-generating secretagogues, including cholera toxin and heat stable Escherichia coli enterotoxin STa. Our results suggest a paradigm for regulation of intestinal fluid transport where fine tuning is accomplished by the counterbalancing effects of solute activation of the CaSR on neuronal and hormonal secretagogue actions. The reversal of cholera toxin- and STa endotoxin-induced fluid secretion by a small-molecule CaSR agonist suggests that these compounds may provide a unique therapy for secretory diarrheas.
Asunto(s)
Secreciones Intestinales/metabolismo , Nucleótidos Cíclicos/metabolismo , Receptores Sensibles al Calcio/metabolismo , Compuestos de Anilina/farmacología , Animales , Toxinas Bacterianas/farmacología , Calcio/química , Calcio/metabolismo , Cationes Bivalentes/química , Secreciones Intestinales/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Fenetilaminas , Hidrolasas Diéster Fosfóricas/metabolismo , Propilaminas , Ratas , Receptores Sensibles al Calcio/agonistas , Receptores Sensibles al Calcio/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
The hepatic glucose transporter, GLUT2, facilitates bidirectional glucose transport across the hepatocyte plasma membrane under insulin regulation. We studied the interactions of IR and GLUT2 proteins to determine whether they are physically coupled in a receptor-transporter complex. By comparing endosome and plasma membrane IR and GLUT2 ratios before and after feeding, it was determined that IR and GLUT2 are internalized in a fixed ratio. When solubilized hepatocytes were immunoprecipitated with antibodies against either IR or GLUT2, both proteins co-precipitated. The association of IR and GLUT2 was further assessed by confocal microscopy. Sections of fed liver were incubated with fluorescein-tagged anti-GLUT2 or Texas Red-tagged anti-IR. Colocalization was observed both at the plasma membrane and in the cytosol. Fluorescence-resonance energy transfer studies further confirmed this association. We conclude that IR and GLUT2 form a receptor-transporter complex in hepatocytes, which forms a mechanism of insulin-mediated hepatic glucose regulation.