RESUMEN
Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GalNAc). In this work, we determined a value of 1.0 x 10(-2) M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.
Asunto(s)
Glicopéptidos/química , Glicoproteínas/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Amaranthus , Secuencia de Aminoácidos , Animales , Electroforesis Capilar , Femenino , Glicopéptidos/metabolismo , Glicosilación , Masculino , Ratones , Ratones Endogámicos BALB C , Microsomas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Espectrometría de Fluorescencia , Timo/metabolismoRESUMEN
The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5 -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE). There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.
Asunto(s)
ATPasas Transportadoras de Calcio/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Empalme Alternativo , Animales , Secuencia de Bases , ATPasas Transportadoras de Calcio/biosíntesis , Células Cultivadas , Clonación Molecular , Componentes del Gen , Regulación de la Expresión Génica , Cardiopatías/genética , Cardiopatías/metabolismo , Humanos , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Hormonas Tiroideas/farmacologíaRESUMEN
Regional Symposium on Emergency Preparedness and Chemical Disasters: a Challenge for the XXI Century
Simposio Regional sobre Preparativos para Emergencias y Desastres Químicos: un Reto para el Siglo XXI. Organización Panamericana de la Salud; 30 oct. -1 nov. 1996
Asunto(s)
Liberación de Peligros Químicos , Desarrollo de Personal , Educación , Investigación , Cooperación Internacional , Estructura de los ServiciosRESUMEN
La ausencia de surfactantes pulmonares trae como consecuencia el incremento de la tensión superficial a lo largo del epitelio alveolar, provocando un colapso alveolar y la lisis de las células epiteliales. Este proceso culmina con la aparición de un síndrome de insuficiencia respiratoria, que es la causa principal de morbimortalidad en niños prematuros. Recientemente, la aplicación de mezclas de agentes surfactantes con fines terapéuticos ha constituido un gran apoyo para la terapia respiratoria, ya que permite una evolución más rápida de los niños que padecen este síndrome. Por todo esto, resulta de gran importancia el conocimiento más detallado de la función, el metabolismo y la regulación de la expresión genética de las proteíinas surfactantes, para el diseño de nuevas y mejores estrategias terapéuticas para combatir este síndrome