RESUMEN
Gene and stem cell therapies hold promise for the treatment of a wide variety of inherited and acquired human diseases. Identification of genes involved in human disease and development of novel vectors and devices for delivering therapeutic genes to different tissues in vivo have resulted in significant progress in the area of gene therapy. Isolation of stem cells from organs formerly thought to have no regenerative potential, the demonstration of stem cell plasticity, and the creation of human embryonic stem cells clearly demonstrate the feasibility of human stem cell therapy. Much additional work remains to be done in the areas of vector development and stem cell biology before the full therapeutic potential of these approaches can be realized. Of equal importance, the ethical issues surrounding gene- and cell-based therapies must be confronted.
Asunto(s)
Terapia Genética , Investigación/tendencias , Células Madre , Animales , Ética , Terapia Genética/tendencias , Humanos , Trasplante de Células MadreAsunto(s)
Puente de Arteria Coronaria , Enfermedad Coronaria/diagnóstico por imagen , Ecocardiografía , Prueba de Esfuerzo/efectos adversos , Paro Cardíaco/etiología , Cateterismo Cardíaco , Enfermedad Coronaria/fisiopatología , Enfermedad Coronaria/cirugía , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In cultured hepatoma HepG2 cells, serum retinol-binding protein (RBP) is secreted more rapidly in the presence of retinol than in its absence (Tosetti, F., Ferrari, N., Pfeffer, U., Brigati, C., and Vidali, G. (1992) Exp. Cell Res. 200, 467-472). In the presence of millimolar concentration of DTT, HepG2 cells synthesize fully reduced RBP within the endoplasmic reticulum (ER) which, upon removal of DTT, forms disulfide bonds post-translationally. Secretion of this post-translationally folded RBP is also dependent on the presence of retinol. Using nonreducing gel electrophoresis, we resolved disulfide-bonded RBP folding intermediates. In addition, two other intracellular folding intermediates, compact I and II, which co-migrate with mature RBP were resolved by their different sensitivity to DTT-induced unfolding. Retinol, as well as retinoic acid, stabilized both compact I and II RBP intermediates to DTT-induced unfolding, suggesting that RBP assumes different conformations in the ER in the presence and absence of a ligand. However, only RBP synthesized in the presence of retinol is rapidly secreted, indicating that the ER export quality control system recognizes RBP containing retinol, but not retinoic acid, as fully folded and competent for export. Folding of RBP so that it is stabilized to DTT reduction is not a sufficient condition for ER exit.
Asunto(s)
Ditiotreitol/química , Pliegue de Proteína , Proteínas de Unión al Retinol/química , Vitamina A/química , Disulfuros/química , Humanos , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
HepG2 cells in the presence of DTT synthesize fully reduced serum retinol-binding protein (RBP) within the endoplasmic reticulum (ER). Upon removal of DTT, RBP forms disulfide bonds and a folding intermediate, compact II, accumulates within the ER. Compact II RBP co-migrates on nonreducing gel electrophoresis with the secreted form of RBP but is differentiated from secreted RBP by its sensitivity to DTT-induced unfolding (see accompanying article; Kaji, E. H., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22188-22194). Here, we have reconstituted DTT-induced unfolding of compact II RBP in a broken cell system and demonstrate that ER-associated factors enhance the unfolding of RBP by DTT. Protein disulfide isomerase is likely to be one such factor since it enhances the rate of RBP unfolding by DTT in vitro; protein disulfide isomerase-induced unfolding requires the absence of retinoids, similar to the DTT-induced unfolding in vivo. ATP enhances the unfolding of RBP in the absence but not in the presence of retinol, both in intact and broken cells. Thus, protein disulfide isomerase and other ATP-dependent factors can unfold partly folded (or misfolded) RBP in the ER, suggesting how improperly folded proteins might be correctly refolded in vivo.
Asunto(s)
Ditiotreitol/química , Retículo Endoplásmico/metabolismo , Pliegue de Proteína , Proteínas de Unión al Retinol/química , Adenosina Trifosfato/metabolismo , Catálisis , Citosol/metabolismo , Humanos , Isomerasas/metabolismo , Cinética , Oxidación-Reducción , Proteína Disulfuro Isomerasas , Células Tumorales CultivadasRESUMEN
A calcitonin receptor complementary DNA (cDNA) was cloned by expression of a cDNA library from a porcine kidney epithelial cell line in COS cells. The 482-amino acid receptor has high affinity for salmon calcitonin (dissociation constant Kd approximately 6 nM) and is functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP). The receptor shows no sequence similarity to other reported G protein-coupled receptors but is homologous to the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor, indicating that the receptors for these hormones, which regulate calcium homeostasis, represent a new family of G protein-coupled receptors.
Asunto(s)
Calcitonina/metabolismo , Receptores de Superficie Celular/genética , Adenilil Ciclasas/fisiología , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , AMP Cíclico/fisiología , ADN/genética , Expresión Génica , Riñón/fisiología , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores de Calcitonina , PorcinosRESUMEN
By screening a cDNA library derived from the A10 rat vascular smooth muscle cell line for functional expression in COS cells, we have isolated a high-affinity receptor for endothelin 1 (Kd = 476 pM) and endothelin 2. The affinity of the cloned endothelin receptor for endothelin 3 is greater than 100 times less in A10 cells and in a CHO cell line stably transformed by the endothelin receptor cDNA. The 426-amino acid receptor polypeptide has seven putative hydrophobic transmembrane domains and is presumed to be a member of the family of guanine nucleotide-binding regulatory (G) protein-coupled receptors. Microinjection of in vitro transcripts of the cloned cDNA into CHO cells confers a transient increase in intracellular calcium in response to endothelin 1, indicating that the receptor is functional and couples to the appropriate G protein(s). RNA analysis reveals high expression in rat lung and heart, tissues known to exhibit binding to iodinated endothelin 1.