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1.
Nat Genet ; 25(4): 397-401, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10932181

RESUMEN

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.


Asunto(s)
Proteínas de Homeodominio/genética , Microftalmía/genética , Factores de Transcripción/genética , Adulto , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Análisis Mutacional de ADN , Exones , Salud de la Familia , Resultado Fatal , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Genes Homeobox/genética , Humanos , Lactante , Intrones , Masculino , Persona de Mediana Edad , Mutación , Linaje , Retina/crecimiento & desarrollo , Retina/metabolismo
2.
Cell Tissue Res ; 293(3): 375-87, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9716727

RESUMEN

An in vitro system has been established to study the migration of human melanoma cells through a monolayer of endothelial cells. Endothelial cells were cultured to confluence on Matrigel before the seeding of melanoma cells. Laser scanning confocal microscopy showed that, prior to migration, melanoma cells appeared round and showed cortical F-actin staining. The initial stage of transmigration was characterized by numerous membrane blebs protruding from basolateral surfaces of the melanoma cells, and contact regions showed an abundance of filaments arising in the underlying endothelial cells. Later, pseudopods from the melanoma cells inserted into contact regions between endothelial cells. Eventually, the melanoma cells intercalated with the endothelial cells. At this stage, many endothelial filament bundles terminated at contacts between the endothelial cells and the transmigrating melanoma cell, suggesting active interactions between the two cell types. Upon contact with the Matrigel, melanoma cells began to spread beneath the endothelium, displaying a fibroblastic morphology with prominent stress fibers. To reestablish the monolayer, adjacent endothelial cells extended processes over the melanoma cell. Tumor necrosis factor alpha did not affect the transmigration of melanoma cells from cell lines isolated from several stages of metastasis. However, tumor necrosis factor did promote the transmigration of melanoma cells derived from a non-metastatic lesion. These results thus define cell attachment and cell penetration of the monolayer as two distinct steps in transmigration and suggest that tumor necrosis factor may enhance the metastatic potential of tumor cells.


Asunto(s)
Movimiento Celular/fisiología , Citoesqueleto/ultraestructura , Endotelio Vascular/ultraestructura , Melanoma/ultraestructura , Células Neoplásicas Circulantes , Actinas/metabolismo , Tamaño de la Célula/fisiología , Medios de Cultivo , Citoesqueleto/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Cinética , Melanoma/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/ultraestructura , Fijación del Tejido , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología
3.
Arterioscler Thromb Vasc Biol ; 17(11): 2891-6, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9409272

RESUMEN

Most vascular endothelial cells at the edge of experimentally induced wounds have their centrosomes oriented toward the wound in the direction of cell migration. The finding that the centrosomes in endothelial cells of non-wounded aorta and vena cava are also oriented toward the heart suggested the hypothesis that endothelial cells are normally migrating in this direction. To test this hypothesis, endothelial cells in a segment of the rat abdominal aorta were labeled with a relatively nontoxic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and the position of the labeled cells was determined 3 and 6 weeks later. The results obtained showed that in 6 of the 9 rat aortas examined at 3 weeks and 15 of the 20 rat aortas examined at 6 weeks, DiI-labeled endothelial cells had migrated various distances up to 5000 microns toward the heart. In contrast, no migration of endothelial cells was detected at the opposite end of the labeled segment, in the direction away from the heart. These results demonstrate that vascular endothelial cells in the abdominal aorta of the rat are not stationary but are migrating toward the heart. The significance of the migration of endothelial cells toward the heart is presently unknown; however, it would be interesting to explore whether or not the impairment of this migration may contribute to disease processes in which the ability to maintain an intact and normally functioning endothelial cell lining is compromised as in atherosclerosis.


Asunto(s)
Endotelio Vascular/citología , Corazón , Animales , Aorta Abdominal/citología , Movimiento Celular , Polaridad Celular , Centrosoma/ultraestructura , Hemorreología , Microscopía Confocal , Microscopía Fluorescente , Ratas
4.
Artery ; 22(5): 251-65, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9209698

RESUMEN

Centrosomes are preferentially oriented toward the heart in endothelial cells (ECs) of the pig aorta and pig and rabbit inferior vena cava (IVC). In the rabbit aorta this preferential orientation of the centrosome toward the heart decreases with age. To determine if this is also true in the rat, a species which is more amenable to experimental manipulation than the pig or the rabbit, we determined the position of centrosomes relative to the nucleus in ECs lining the aorta and IVC using whole mounts of vessels that were immunofluorescently stained with sera specific for centrosomes. In both the thoracic and abdominal aorta of the rat the majority of the ECs (60%) had centrosomes on the heart side of the nucleus, 25% had centrosomes on the side of the nucleus away from the heart and 15% had centrosomes in a central position in the cell. Similar results were obtained in the IVC of the rat where these values were, 58%, 31% and 11% respectively. A comparable preferential orientation of centrosomes toward the heart was also seen in the ECs of thoracic and abdominal aortas and IVCs of weanling and young adult rats and this did not decrease with age as it does in the rabbit aorta. When segments of the rat aorta were placed in organ culture, the percentage of ECs with preferentially oriented centrosomes decreased by 48 hrs, even though the cells remained elongated in shape. We have recently demonstrated that ECs in the rat aorta are normally migrating in the direction of the heart and thus in the direction in which the centrosomes in rat aortic ECs are preferentially oriented. This correlation is consistent with the general hypothesis that the centrosome position defines the direction of migration in monolayers of cells.


Asunto(s)
Aorta/citología , Centrosoma/ultraestructura , Endotelio Vascular/citología , Vena Cava Inferior/citología , Análisis de Varianza , Animales , Técnica del Anticuerpo Fluorescente , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley
5.
Cell Motil Cytoskeleton ; 36(2): 149-63, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9015203

RESUMEN

To determine events that lead to the formation of intercellular contacts, we examined the spatial and temporal distribution of NCAM, cadherins, and F-actin in TM4 cells by immunofluorescence and laser scanning confocal microscopy. TM4 cells exhibited epithelioid characteristics and formed large overlapping lamella-like cell-cell contacts that contained a high concentration of NCAM. NCAM-rich lamellae formed from smaller NCAM patches at the ends of filopodia-like contacts between adjacent cells. Cadherins, as visualized by a pan-cadherin antibody, were present in a pattern distinctly different from that of NCAM. Although in filopodia-like contacts, both cadherins and NCAM were often concentrated at filopodial tips, in the larger lamella-like contacts that developed later, cadherins were located in an irregular punctate pattern only at the distal and more apical margins of the slanted NCAM-rich contact regions. Patterns of NCAM and microfilament (MF) bundle distribution were distinctly different, suggesting that the ends of these MF bundles were not physically linked to NCAM. By contrast, cadherins were concentrated at the ends of MF bundles at all stages of contact formation examined. Interestingly, this association of cadherins with MF bundles was mostly seen at the edge of the overlapping processes. In the lower cell process, MF bundles at the contact site were often arranged in random fashion, indicating an asymmetric distribution of MF in the junctional region. However, N-cadherin was enriched only at sites where MF bundles from both the upper and lower cell processes were aligned and terminated at the junctional membrane. Thus the organization of the actin cytoskeleton at cell-cell contact sites is influenced by the differential localization of different cadherins. These data also suggest that different mechanisms are involved in the accumulation of NCAM and cadherins in cell-cell contact regions.


Asunto(s)
Citoesqueleto de Actina/fisiología , Cadherinas/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Células de Sertoli/fisiología , Animales , Comunicación Celular , Línea Celular , Masculino , Ratones , Vinculina/metabolismo
6.
Cell Motil Cytoskeleton ; 36(3): 228-35, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9067618

RESUMEN

Locomoting cells exhibit a polarity whereby certain organelles, like the centrosome, and cytoskeletal structures, like stress fibers, are preferentially oriented in the direction of migration. To determine if this was also true in endothelial cells (ECs) of the rat aorta that are migrating toward the heart, whole mounts of abdominal and thoracic aorta were double stained with rhodamine phalloidin to label stress fibers and sera that labels centrosomes. Our results show that in 66% of the ECs of the abdominal aorta where stress fibers were present, 47% had stress fibers on the heart side of the nucleus, 21% had stress fibers on the side of the nucleus away from the heart, and 32% had stress across the cell. Similarly, in 50% of the ECs of the thoracic aorta where stress fibers were present, these values were 56, 19, and 25%, respectively. The results also showed that the centrosome was preferentially located toward the heart in the majority (61%) of the ECs with stress fibers as well as in ECs without stress fibers. Since in both, the same percentage of ECs had centrosome preferentially oriented toward the heart, these results imply that while the centrosome may determine the position of the stress fibers, the stress fibers do not appear to determine the position of the centrosome. Nevertheless, both centrosomes and stress fibers in aortic ECs are preferentially oriented in the direction of migration, where they may be involved in defining the direction and providing the force of locomotion, respectively.


Asunto(s)
Citoesqueleto de Actina/fisiología , Centrosoma/fisiología , Endotelio Vascular/citología , Animales , Aorta Abdominal , Aorta Torácica , Núcleo Celular , Técnica del Anticuerpo Fluorescente Indirecta , Ratas , Ratas Sprague-Dawley
7.
Cell Motil Cytoskeleton ; 38(4): 351-64, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9415377

RESUMEN

Transmigration of cancer cells through the vascular endothelium (diapedesis) is a key event in tumor metastasis. To investigate mechanisms involved in diapedesis, we used laser scanning confocal microscopy to examine the distribution of cadherins of WM239 melanoma cells as they migrated through a monolayer of activated human umbilical vein endothelial cells (EC) cultured on matrigel. Cadherins, including VE-cadherin, but not N-cadherin, were enriched in contacts between EC, whereas N-cadherin, but not VE-cadherin, was found in contacts between melanoma cells. During the early stages of diapedesis, EC located below the attached melanoma cells decreased in height and VE-cadherin disappeared from the EC contact located underneath the melanoma cell. Transendothelial migration began with small melanoma cell processes penetrating the VE-cadherin-negative regions between the EC. Subsequently, melanoma cells became intercalated between EC. Despite the absence of both VE-cadherin and N-cadherin, other members of the cadherin family were present in the heterotypic contacts between EC and melanoma cells. EC surrounding the intercalated melanoma cell subsequently extended processes and spread over the melanoma cell to re-form the endothelial monolayer. Interestingly, the leading margins of these EC processes contained high levels of N-cadherin, but not VE-cadherin. VE-cadherin-rich cell-cell contacts, however, reformed between advancing endothelial processes when they met above the melanoma cell. As the melanoma cells came into contact with the underlying matrigel, they spread out and adopted a fibroblast-like morphology. Addition of anti-N-cadherin antibodies to the assay resulted in a delay in the transendothelial migration of melanoma cells. Together, these results suggest that EC actively participate in diapedesis by disassembling and reassembling VE-cadherin-rich adherens junctions, and that N-cadherin plays an important role in the transmigration of melanoma cells and the reclosure of the endothelium.


Asunto(s)
Cadherinas/fisiología , Movimiento Celular , Endotelio Vascular/citología , Anticuerpos Monoclonales/inmunología , Antígenos CD , Adhesión Celular , Comunicación Celular , Humanos , Uniones Intercelulares , Melanoma , Células Tumorales Cultivadas
8.
Exp Eye Res ; 65(5): 681-9, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9367648

RESUMEN

Expression of SPARC (Secreted Protein, Acidic, Rich in Cysteine), a counteradhesive, calcium-binding extracellular matrix (ECM) glycoprotein, is associated with several morphogenetic events during early development. In this study, changes in the spatiotemporal distribution of SPARC transcripts and the protein during chicken retinal development were documented by in situ hybridization and indirect immunofluorescence microscopy. SPARC transcripts were first detected within the proliferating neural ectoderm at embryonic day 4. 5 (E4.5), followed short thereafter (E5) by appearance of SPARC. SPARC was enriched within the inner plexiform layer (IPL) by E10 and within the outer plexiform layer (OPL) by E14, several days after these layers became morphologically distinct. Significant levels of SPARC transcripts were first observed within the ganglion cell layer (GCL) at E17 prior to accumulation of SPARC within the nerve fiber layer, seen first at E20. SPARC protein was first detected within the developing retinal pigment epithelium (RPE) at E10 and increased significantly at RPE cells ceased to proliferate and continued differentiating. Of special note was the restriction of SPARC to the basal-half of the RPE cells. SPARC transcripts were similarly distributed in the adult retina, but at lower levels than in the period just prior to hatching. In the adult retina SPARC was retained in the nerve fiber layer and present in the inner nuclear layer (INL) and outer nuclear layer (ONL), but lost from the IPL and OPL. These changes in expression pattern with time indicate that SPARC is developmentally regulated and therefore may have important function(s) in both morphological development of the retina and functioning of the mature eye.


Asunto(s)
Pollos/metabolismo , Osteonectina/metabolismo , Retina/embriología , Secuencia de Aminoácidos , Animales , Northern Blotting , Embrión de Pollo , Secuencia Conservada , Inmunohistoquímica , Hibridación in Situ , Microscopía Fluorescente , Datos de Secuencia Molecular , Morfogénesis , Osteonectina/análisis , Osteonectina/genética , Epitelio Pigmentado Ocular/química , Epitelio Pigmentado Ocular/embriología , Epitelio Pigmentado Ocular/metabolismo , ARN Mensajero/análisis , Retina/química , Retina/metabolismo
9.
Nat Genet ; 12(4): 376-84, 1996 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-8630490

RESUMEN

Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off


Asunto(s)
ADN/genética , Anomalías del Ojo/genética , Genes Homeobox , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/genética , División Celular , Mapeo Cromosómico , Cartilla de ADN/genética , Anomalías del Ojo/patología , Femenino , Expresión Génica , Homocigoto , Masculino , Ratones , Datos de Secuencia Molecular , Retina/anomalías , Retina/patología , Células Madre/patología
10.
Exp Eye Res ; 62(1): 55-61, 1996 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-8674513

RESUMEN

The retinal pigment epithelial cells, which form a monolayer between the choroid and photoreceptor cell layer of the eye, generally do not divide after birth. There is, however, a gradual loss of retinal pigment epithelial cells die and the mechanism by which the integrity of retinal pigment epithelium is maintained after cell death has not been examined. Confocal laser scanning microscopy of whole mounts of retinal pigment epithelium of 12 to 16 day old chick embryos showed that among the great majority of retinal pigment epithelial cells which were regular in size and hexagonal in shape, single, scattered, irregularly shaped, dying cells are present. The distribution of the dying retinal pigment epithelial cells, their morphology and the presence of apoptotic bodies, including pyknotic and fragmented nuclei, above such defects suggests that the death occurs by apoptosis. The defects created by the dying or dead cells were repaired by spreading of the surrounding normal retinal pigment epithelial cells and a series of stages in the repair of the defects could be identified. During the repair of the defects, fine microfilament bundles running parallel to the edge of the defect in each of the surrounding retinal pigment epithelial cells could be detected by confocal laser scanning microscopy giving a 'spider-web' appearance to the region around the defect. Since induction of proliferation in retinal pigment epithelial cells during healing of the defect requires cell migration, we would not expect the spreading of retinal pigment epithelial cells into the single cell defects to trigger cell proliferation. The death of single cells and the spreading of adjacent ones in the absence of cell proliferation would however explain at least in part the increase in the average size of retinal pigment epithelial cells with age.


Asunto(s)
Epitelio Pigmentado Ocular/fisiología , Regeneración , Actinas/análisis , Animales , Muerte Celular/fisiología , Tamaño de la Célula/fisiología , Embrión de Pollo , Citoesqueleto/ultraestructura , Microscopía Confocal , Microscopía Fluorescente , Epitelio Pigmentado Ocular/citología
11.
Biochem Cell Biol ; 74(3): 373-81, 1996.
Artículo en Inglés | MEDLINE | ID: mdl-8883843

RESUMEN

The neural cell adhesion molecule NCAM is a member of the immunoglobulin (Ig) superfamily. NCAM can undergo homophilic binding and heterophilic interactions with cell surface components and is often concentrated at sites of intercellular contact. To investigate the molecular basis of this biased surface distribution, we examined L cell transfectants expressing wild-type or mutant forms of chick NCAM-140 by laser scanning confocal microscopy. Mutant NCAMs that lacked Ig-like domains 1, 2, 4, or 5 were preferentially localized in contact regions. However, the relative concentration of these mutant NCAMs in contact sites was substantially reduced compared with wild-type NCAM. In contrast, NCAM redistribution to intercellular contacts was abolished in cells expressing mutant NCAMs that either lacked Ig-like domain 3 or contained mutations in the homophilic binding site in this domain. In heterotypic contacts between PC12 cells and L cell transfectants, colocalization of rat NCAM and chick NCAM was again dependent on the integrity of the homophilic binding site of the NCAM expressed on L cells. These results provide evidence that homophilic binding is the main mechanism by which NCAM becomes redistributed to intercellular contacts. They also implicate a role for other Ig-like domains in the accumulation of NCAM at cell-cell contacts.


Asunto(s)
Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Sitios de Unión , Comunicación Celular , Células Cultivadas , Embrión de Pollo , Células L , Ratones , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Células PC12 , Ratas , Transfección
12.
Biochem Cell Biol ; 73(9-10): 709-22, 1995.
Artículo en Inglés | MEDLINE | ID: mdl-8714692

RESUMEN

Several systems of microfilaments (MF) associated with adherens-type junctions between adjacent retinal pigment epithelial (RPE) cells and between these cells and the substratum play an important role in maintaining the integrity and organization of the RPE. They include prominent, contractile circumferential MF bundles that are associated with the zonula adherens (ZA) junctions. In chick RPE, these junctions are assembled from smaller subunits thus giving greater structural flexibility to the junctional region. Because the separation of the junctions requires trypsin and low calcium, both calcium-dependent and -independent mechanisms are involved in keeping adjacent RPE cells attached to one another. Another system of MF bundles that crosses the cell at the level of ZA junctions can be induced to form by stretching the epithelium. The MF bundles forming this system are oriented in the direction in which the RPE is stretched, thereby preventing the overextension of the cell in any one direction. The system may be useful as an indicator of the direction in which tension is experienced by RPE during development of the eye, in animal models of disease and during repair of experimentally induced wounds. Numerous single-cell wounds resulting from death of RPE cells by apoptosis at various stages of repair are normally present in developing chick and adult mammalian RPE. These wounds are repaired by the spreading of adjacent RPE cells and by the contraction of MF bundles oriented parallel to the wound edge, which develop during this time. As a result of the spreading in the absence of cell proliferation, the RPE cells increase in diameter with age. Experimentally induced wounds made by removing 5-10 RPE cells are repaired by a similar mechanism within 24 h. In repair of larger wounds, over 125 microns in width, the MF bundles oriented parallel to the wound edge characteristic of spreading cells are later replaced by stress fibers (SFs) that run perpendicularly to the wound edge and interact with the substratum at focal contacts (FCs) as RPE cells start to migrate. Cell proliferation is induced in cells along the wound edge only when the wounds are wide enough to require cell migration. In the presence of antibodies to beta-1-integrins, a component of FCs, cell spreading is not prevented but both cell migration and cell proliferation are inhibited. Thus, only the organization of the cytoskeleton characteristic of migrating RPE cells that have SFs that interact with the substratum at FCs, is associated with the induction of cell proliferation.


Asunto(s)
Citoesqueleto de Actina/ultraestructura , Desmosomas/fisiología , Epitelio Pigmentado Ocular/fisiología , Retina/fisiología , Cicatrización de Heridas/fisiología , Animales , División Celular , Movimiento Celular , Embrión de Pollo , Epitelio Pigmentado Ocular/lesiones , Epitelio Pigmentado Ocular/ultraestructura , Retina/lesiones , Retina/ultraestructura
13.
Neuron ; 13(2): 377-93, 1994 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-7914735

RESUMEN

Few potential regulatory proteins of vertebrate retinal development have been identified. We describe a 39 kDa murine polypeptide (Chx10) with a homeodomain 82% identical to that of the nematode protein ceh-10. In the developing mouse, the Chx10 transcript is expressed throughout the anterior optic vesicle and all neuroblasts of the optic cup. In the mature retina, the Chx10 protein is restricted to the inner nuclear layer, in which its expression decreases from the outer to the inner margin. Chx10 transcripts are also detected in regions of the developing thalamus, hindbrain, and ventral spinal cord. The data suggest that Chx10 plays critical roles in the formation of the neuroretina and in the development and maintenance of the inner nuclear layer.


Asunto(s)
Genes Homeobox , Proteínas de Homeodominio , Retina/embriología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Mapeo Encefálico , Clonación Molecular , Expresión Génica , Genes , Secuencias Hélice-Asa-Hélice , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Morfogénesis , Neuronas Motoras/fisiología , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido
14.
Invest Ophthalmol Vis Sci ; 34(9): 2761-8, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-7688361

RESUMEN

PURPOSE: To investigate the effect that antibodies against the beta 1 subunit of integrin, a cell-surface extracellular matrix receptor, would exert on chick embryo retinal pigment epithelial (RPE) cells maintained in organ culture after mechanical wounding of the epithelium. METHODS: RPE cells maintained in organ culture in the presence of antibodies against the beta 1 subunit of integrin were observed to quantify their spreading and migration. Antibodies against proliferating cell nuclear antigen (PCNA) were used to assess cell proliferation under the experimental conditions. RESULTS: In the presence of monoclonal antibodies against the beta 1 subunit of integrin, cell migration is inhibited whereas the initial cell spreading response still occurs. This implies that the RPE cells along the wound edge use different mechanisms in interacting with the substratum in spreading and in migration. Moreover, the RPE cells along the wound edge of cultures in which migration is inhibited do not express PCNA. Higher concentrations of the anti-integrin antibodies, however, are required to inhibit cell proliferation than to inhibit cell migration. CONCLUSIONS: These results suggest that specific cell-substratum interactions may be involved in the initiation of a proliferative response to wound healing in this model system.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Integrinas , Epitelio Pigmentado Ocular/citología , Cicatrización de Heridas/efectos de los fármacos , Animales , Autoantígenos/metabolismo , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Embrión de Pollo , Medios de Cultivo , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Integrina beta1 , Proteínas Nucleares/metabolismo , Técnicas de Cultivo de Órganos , Epitelio Pigmentado Ocular/metabolismo , Antígeno Nuclear de Célula en Proliferación
15.
Exp Gerontol ; 28(1): 69-75, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8436206

RESUMEN

The position of centrosomes in endothelial cells (EC) lining the aorta was examined in rabbits at different ages, using en face preparations and immunofluorescent staining with a serum that specifically labels centrioles. The results obtained show that in young rabbits (4 h-6 weeks) the great majority of the EC (61%) had centrosomes on the heart side of the nucleus, whereas in older rabbits (6-156 weeks) only 41% of the EC had centrosomes oriented toward the heart. The results suggest that the orientation of structures normally associated with centrosomes such as the microtubule organizing centers and the Golgi apparatus also change with age. The change in the orientation of centrosomes and associated structures along the longitudinal axis of the cell with age could affect the function and behaviour of EC and their ability to respond to injury.


Asunto(s)
Envejecimiento , Aorta/ultraestructura , Endotelio Vascular/ultraestructura , Orgánulos/ultraestructura , Animales , Polaridad Celular , Centriolos/ultraestructura , Microscopía Fluorescente , Conejos
16.
Biochem Cell Biol ; 70(10-11): 1135-41, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1297337

RESUMEN

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Citoesqueleto/ultraestructura , Endotelio Vascular/ultraestructura , Cicatrización de Heridas/fisiología , Animales , Aorta Torácica/lesiones , Cateterismo , Movimiento Celular/fisiología , Endotelio Vascular/lesiones , Femenino , Técnica del Anticuerpo Fluorescente , Conejos
17.
Neuron ; 8(6): 1171-84, 1992 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-1610568

RESUMEN

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (15/16 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.


Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular , ADN/genética , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso , Disco Óptico/metabolismo , Células Fotorreceptoras/metabolismo , Enfermedades de la Retina/etiología , Adulto , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Humanos , Proteínas de Filamentos Intermediarios/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Neuropéptidos/fisiología , Periferinas , Segmento Externo de la Célula en Bastón/metabolismo
18.
Glia ; 6(2): 149-53, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1398896

RESUMEN

Immuno-electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP-vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP-vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell.


Asunto(s)
Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Vimentina/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Astrocitos/química , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/análisis , Ratones , Ratones Endogámicos C3H , Microscopía Inmunoelectrónica , Vimentina/análisis
19.
Exp Cell Res ; 195(2): 307-14, 1991 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-1676970

RESUMEN

Although 12-day-old chick embryo retinal pigment epithelial (RPE) cells in situ do not express the proliferating cell nuclear antigen (PCNA) which is known to function as an auxiliary protein of DNA polymerase delta, they do so when cultured on glass. Conversely, PCNA was not expressed by RPE cells of the same age maintained in organ culture. If, however, the organ cultures were wounded, allowing the RPE cells to spread and migrate over the exposed basal lamina, the nuclei of cells along the wound edge were stained for PCNA. The time required for cells to express PCNA was longer in organ culture than in tissue culture. This time lag in the expression of PCNA was independent of the time in culture prior to wounding and occurred regardless of whether or not the continuity of the epithelial sheet was reestablished. In organ culture, the staining did not persist as long as in tissue culture. We found that only in wounds exceeding 125 +/- 48 microns did the RPE cells along the wound edge express PCNA. This suggests that a certain degree of either spreading or migration is required for PCNA expression in the wounded region.


Asunto(s)
Proteínas Nucleares/biosíntesis , Epitelio Pigmentado Ocular/metabolismo , Cicatrización de Heridas , Animales , División Celular , Embrión de Pollo , Técnicas de Cultivo , Técnicas de Cultivo de Órganos , Antígeno Nuclear de Célula en Proliferación
20.
Can J Ophthalmol ; 25(3): 159-63, 1990 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-2163281

RESUMEN

We report the light and electron microscopic findings for two lesions from two patients who died of complications of familial adenomatous polyposis. In the first case microscopy of a small (100 to 200 mu), uniformly dark lesion (the commonest type seen in this condition) showed enlarged retinal pigment epithelial cells with an increased number of pigment granules. This is consistent with the term "hypertrophy of the retinal pigment epithelium", currently used to describe these lesions. In the second case we sectioned a larger (1000 to 1500 mu), oval, grey lesion from the posterior pole. The pigment epithelium was normal, but between it and outer retina was an unusual choristoma consisting largely of myelinated axons and astrocytes.


Asunto(s)
Poliposis Adenomatosa del Colon/complicaciones , Retina/ultraestructura , Enfermedades de la Retina/patología , Adulto , Fondo de Ojo , Humanos , Masculino , Persona de Mediana Edad , Células Fotorreceptoras/patología , Epitelio Pigmentado Ocular/ultraestructura , Enfermedades de la Retina/congénito , Enfermedades de la Retina/etiología
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