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The molecular cues that control patterning of the heart tube during early cardiogenesis are largely unknown. The present study has explored the embryonic stem (ES) cell differentiation system to determine if this in vitro model could be useful in studying the process of regional specification of cardiac muscle cells at the earliest possible stages. As assessed by polymerase chain reaction, ribonuclease protection, in situ hybridization, and immunohistochemical analyses, ES cell differentiation into embryoid bodies is characterized by the transcriptional and translational activation of the ventricular regulatory (phosphorylatable) myosin light chain gene, demonstrating that ventricular specification occurs during ES cell cardiogenesis. The finding of a ventricular-specific marker in an in vitro system in the absence of an intact heart tube provides evidence for cardiac regional specification independent of positional cues or physiologic stimuli. The temporal expression of the myogenic regulatory factors, myogenin and MyoD, suggests activation of the skeletal muscle program following cardiac myogenesis in vitro, indicating temporal fidelity to the progression of in vivo myogenesis. These data establish the mouse embryonic stem cell system as a model for cardiac chamber specification and suggest a promising approach in the study of regional specification in genetically engineered cardiac muscle cells.

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The structures of five neurotrophic molecules have so far been published. Nerve growth factor, fibroblast growth factor and purpurin, have been identified as nerve-cell survival molecules. More recently, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor have been cloned and sequenced. As all these proteins stimulate the survival of ciliary or sensory neurons, a new cell survival assay is required if novel neurotrophic molecules are to be discovered. P19 teratoma cells differentiate to nerve-like cells in the presence of 5 x 10(-7) M retinoic acid (RA). But when P19 cells are plated in N2 synthetic medium without being exposed to RA, they die within 48 h. In an attempt to identify a molecule(s) that can substitute for RA in promoting P19 survival, we assayed serum-free growth-conditioned media for their ability to promote P19 survival. One cell line from the rat eye secreted a molecule that promoted the survival of P19 cells and some types of nerve cell. We identified this molecule as activin, better known for its role in hormone secretion.

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A secreted form of the amyloid beta protein precursor was isolated from the growth conditioned medium of the PC12 sympathetic nerve-like cell line. This protein is recognized by an antiserum that detects a protein of 140 kDa and a less abundant species of 115 kDa on NaDodSO4/acrylamide gels. The amyloid precursor proteins contain O-linked sugars and tyrosine sulfate and bind to the glycosaminoglycan heparin. These results suggest a role for extracellular sulfated glycoproteins in the pathogenesis of Alzheimer disease.

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Schwann cell movement and proliferation occur during peripheral nerve regeneration and remyelination. We asked whether soluble factors promoting these activities were present in fluid surrounding rat sciatic nerves regenerating across a 10-mm gap bridged by a silicone tube. In this model, regenerated and remyelinated axons extend across the gap by 28 days following nerve transection and tube implantation. Fluid conditioned by cells participating in nerve regeneration (RCF) was assayed for its ability to promote Schwann cell adhesion, migration and proliferation in vitro. RCFs collected at post-transectional days 1-28 were equally effective in promoting Schwann cell-substratum adhesion. In contrast, the motility-promoting activity of RCF was minimal at 1-2 days following nerve-transection, peaked at 7 days and remained elevated through 21 days. The RCF peak response was 87-fold greater than control. Schwann cell proliferative activity of RCF exhibited peaks of activity at 1 and 14 days post-transection. The biological potency of this fluid for each activity assayed in vitro correlated well with the behavior of Schwann cells chronicled during nerve repair in vivo. These findings suggest that soluble factors promoting Schwann cell adhesion, migration, and proliferation accumulate extracellularly during peripheral nerve regeneration and remyelination.

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The amyloid beta protein peptide is a major constituent of amyloid plaque cores in Alzheimer's disease and is apparently derived from a higher molecular weight precursor. It is now shown that the core protein of a heparan sulfate proteoglycan secreted from a nerve cell line (PC12) has an amino acid sequence and a size very similar to those of the amyloid beta protein precursor and that these molecules are antigenically related. This amyloid beta protein precursor-related protein is not found in the conditioned medium of a variant cell line (F3 PC12) that does not secrete heparan sulfate proteoglycan. The synaptic localization and metabolism of this class of proteoglycans are consistent with its potential involvement in central nervous system dysfunction.

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A cDNA for purpurin, a secreted 20,000 dalton neural retina cell adhesion and survival protein, has been sequenced and expressed in mammalian cells. Purpurin mRNA is found in both embryonic and adult retina, but not the brain, heart, or liver. The protein is highly concentrated in the neural retina between the pigmented epithelium and the outer segments of the photoreceptor cells; it is synthesized by photoreceptor cells. The predicted purpurin sequence contains 196 residues, has approximately 50% sequence homology with serum retinol binding protein, and is a member of the alpha-2 mu-globulin superfamily. Purpurin binds retinol and may play a major role in retinol transport across the interphotoreceptor cell matrix.

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A 20,000-D protein called purpurin has recently been isolated from the growth-conditioned medium of cultured embryonic chick neural retina cells (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 101:1071-1077). Purpurin is a constituent of adherons and promotes cell-adheron adhesion by interacting with a cell surface heparan sulfate proteoglycan. It also prolongs the survival of cultured neural retina cells. This paper shows that purpurin is a secretory protein that has sequence homology with a human protein synthesized in the liver that transports retinol in the blood, the serum retinol-binding protein (RBP). Purpurin binds [3H]retinol, and both purpurin and chick serum RBP stimulate the adhesion of neural retina cells, although the serum protein is less active than purpurin. Purpurin and the serum RBP are, however, different molecules, for the serum protein is approximately 3,000 D larger than purpurin and has different silver-staining characteristics. Finally, purpurin supports the survival of dissociated ciliary ganglion cells, indicating that RBPs can act as ciliary neurotrophic factors.

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A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP.

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Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.

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Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).

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Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.

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Both the skeletal muscle myoblast cell line L6 and an adhesion-deficient variant of L6 released glycoprotein complexes, termed adherons, into their culture medium. The adherons from the variant, M3A, differed from those of L6 in a number of properties. M3A adherons were much less effective in promoting the cell-substratum and cell-cell adhesion of myoblasts than L6 particles. The adherons from the two cell lines also differed in their relative sedimentation velocities in sucrose gradients and had different chemical compositions. The M3A particle lacked chondroitin and contained relatively less collagen and fibronectin than the L6 adheron. Both L6 and M3A particles adhered to plastic surfaces and cells equally well in the absence of calcium ions. Neither cell-cell adhesion nor particle aggregation occurred in calcium-free medium. However, in the presence of calcium, the L6 adherons aggregated completely and M3A particles aggregated poorly. These data suggest that at least two sets of interactions are required for adheron-mediated adhesion: a calcium-independent binding of the adheron to the cell, and a calcium-dependent interaction between particles that is directly responsible for adhesion. The M3A variant is blocked at the calcium-dependent step, resulting in an adhesion deficiency.

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Complexes containing glycoproteins and glycosaminoglycans were released into the culture medium by both smooth and skeletal muscle cells. The particles, termed adherons, from three smooth muscle cell lines promoted cell-substratum adhesion of a clonal sympathetic nerve cell line, PC12. In contrast, the glycoprotein complexes from three skeletal muscle preparations inhibited the adhesion of PC12 cells to Petri dish surfaces. The skeletal muscle adherons all sedimented in calcium-free sucrose gradients with an S value of 16, while the smooth muscle particles had a sedimentation value of 12. Although both classes of adherons contained fibronectin and collagen, hyaluronic acid was present only in those from skeletal muscle. An antiserum prepared against skeletal muscle adherons blocked myoblast adhesion to skeletal muscle adherons but did not alter PC12 adhesion to smooth muscle adherons. These data suggest that the glycoprotein complexes released by muscle have some intrinsic specificity with respect to their ability to mediate cellular adhesion.

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Nerve growth factor (NGF), epidermal growth factor (EGF), adenosine 3',5'-cyclic monophosphate (cAMP), and cholera toxin all increase the specific activity of ornithine decarboxylase (ODC) in the PC12 nerve-like cell line. A phosphodiesterase inhibitor augments the effect of NGF on the induction of ODC in these cells, while it is only additive with the EGF response. These data suggest that cAMP has an intermediary role in the induction of ODC by NGF and that cAMP is not involved in the EGF response.

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A variant of the anchorage-dependent L6 skeletal muscle myoblast cell line which grows in suspension culture was selected. This cell line, designated M3A, is aneuploid and tumorigenic, while the parental L6 line is near diploid and nontumorigenic. L6 secretes high molecular weight collagen alpha-chains, and M3A secretes a collagen-like protein of Mr = 56,000, or approximately half the size of collagen alpha-chains. Finally, both L6 and M3A synthesize hyaluronic acid, heparan sulfate, chondroitin sulfate, and chondroitin. The latter glycosaminoglycan is not, however, released into the culture medium by M3A, nor is it found in the substrate-attached material synthesized by M3A. The changes in secreted and substrate-attached material synthesized by M3A are discussed in relation to its altered growth characteristics.

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An adhesion-deficient variant, designated M3A, was derived from the anchorage-dependent L6 skeletal muscle myoblast line (Schubert, D., and La Corbiere, M. (1980) J. Biol. Chem. 255, 11557-11563). To investigate the defect in the M3A variant, adhesion to various substrata was studied. M3A adhered rapidly to substrate-attached material (SAM) prepared from L6 cultures and serum, but adhered slowly to SAM derived from M3A itself. The role of collagen and fibronectin in the adhesion of M3A cells to L6- and M3A-derived SAMs was ruled out, but several experiments suggested that glycosaminoglycans play a rate-limiting role in the adhesion process. The adhesive interaction of the M3A cells with the different substrata is specific with respect to the glycosaminoglycans involved, since the type and concentration of purified glycosaminoglycans required to inhibit the interaction is unique to each surface. An alteration in glycosaminoglycan synthesis by M3A cells may account for the difference in the adhesive properties of SAM derived from L6 and from the M3A variant.

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Myogenic cells release into their culture medium a glycoprotein complex that mediates cellular adhesion. In the absence of calcium this complex has a sedimentation value of 16S; it aggregates in the presence of calcium. The 16S material both agglutinates and increases the rate of cell-substratum adhesion of a myoblast variant and inhibits the adhesion of a nerve-like cell line to culture dishes. It is also a hemagglutinin. The 16S particle is composed of glycosaminoglycans and several proteins, including fibronectin and collagen.

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Glucocorticoids stimulate tyrosine hydroxylase activity and catecholamine synthesis, while markedly inhibiting acetylcholine synthesis and storage in the a clone of sympathetic nerve-like cells. Nerve growth factor enhances the effect of glucocorticoids on tyrosine hydroxylase. The steroid effect is specific for glucocorticoids, since 11-desoxycortisol, testosterone, and estradiol-17 beta do not reproduce the effects of hydrocortisone and dexamethasone. Concomitant with the shift in neurotransmitter synthesis, there is an increase in the mean diameter of intracellular dense core vesicles. In contrast to glucocorticoids, insulin increases the specific activity of choline acetyltransferase through the interaction with typical insulin receptors. Insulin does not, however, alter the morphology of the cells, nor does it block the morphological response of the cells to nerve growth factor.

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