Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
Acta Biomater ; 10(1): 183-93, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24055455

RESUMEN

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin ß-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.


Asunto(s)
Membrana Basal/metabolismo , Detergentes/farmacología , Andamios del Tejido/química , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Colágeno/metabolismo , ADN/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Glicosaminoglicanos/metabolismo , Humanos , Imagenología Tridimensional , Etiquetado Corte-Fin in Situ , Integrina beta1/metabolismo , Antígeno Ki-67/metabolismo , Microvasos/citología , Coloración y Etiquetado , Sus scrofa
2.
Biochem J ; 224(1): 117-27, 1984 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-6095814

RESUMEN

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.


Asunto(s)
Química Encefálica , Proteínas de Unión al Calcio/aislamiento & purificación , Cromatografía en Gel/métodos , Proteínas Quinasas/aislamiento & purificación , Aminoácidos/análisis , Animales , Encéfalo/enzimología , Calcio/farmacología , Bovinos , Cromatografía por Intercambio Iónico , Calor , Fragmentos de Péptidos/análisis , Fosfolípidos/farmacología , Monoéster Fosfórico Hidrolasas/análisis , Proteína Quinasa C , Proteínas Quinasas/metabolismo
3.
Biochem J ; 218(3): 863-70, 1984 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-6326748

RESUMEN

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.


Asunto(s)
Músculo Liso/enzimología , Proteínas Quinasas/aislamiento & purificación , Animales , Calcio , Proteínas de Unión a Calmodulina , Pollos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Molleja de las Aves/enzimología , Métodos , Quinasa de Cadena Ligera de Miosina , Fragmentos de Péptidos/análisis , Fosfoproteínas Fosfatasas/aislamiento & purificación , Especificidad por Sustrato
4.
Biochem Biophys Res Commun ; 115(3): 855-63, 1983 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-6312998

RESUMEN

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.


Asunto(s)
Anticuerpos Monoclonales , Molleja de las Aves/enzimología , Proteínas Quinasas/aislamiento & purificación , Animales , Complejo Antígeno-Anticuerpo , Pollos , AMP Cíclico/farmacología , Inmunoensayo , Peso Molecular , Quinasa de Cadena Ligera de Miosina , Fosforilación , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA