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2.
Graefes Arch Clin Exp Ophthalmol ; 239(8): 613-8, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11585319

RESUMEN

BACKGROUND: Posterior capsule opacification (PCO) is still one of the major complications following modern cataract surgery. Several attempts have been made to find an appropriate therapeutic concept to significantly lower the rate of PCO. Here, we wanted to focus on the antimetabolic strategy, reducing PCO by using mitomycin C, further characterizing the pathway of apoptosis in human lens epithelial cells (hLECs). METHODS: Human lens epithelial cells were obtained from anterior lens capsules during cataract surgery. The expression of Fas, TRAMP, TRAIL-R1-R4, Apo-3L and TRAIL mRNA was investigated by means of RT-PCR using specific primers. For investigations on bcl-2, bax, p53 and the active form of caspase 3, cell cultures of hLECs were pretreated with mitomycin C and processed for immunocytochemistry thereafter. RESULTS: We detected the expression of the receptors Fas, TRAMP, TRAIL-R2 and TRAIL-R3 in hLECs. We further obtained evidence of the upregulation of the intracellular apoptotic signalling cascade, represented by bcl-2 and bax, the transcription factor p53 and the active form of caspase 3, after pretreatment with mitomycin C. CONCLUSION: We demonstrated the presence of the apoptosis-receptor system in hLECs. Furthermore, we demonstrated the possibility of the induction of key proteins of the apoptotic signalling cascade in these cells by the antimetabolic drug mitomycin C. This could have important implications on the strategies regarding both the prevention and the treatment of PCO after cataract surgery.


Asunto(s)
Antimetabolitos/farmacología , Apoptosis/fisiología , Cristalino/efectos de los fármacos , Mitomicina/farmacología , Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/genética , Cartilla de ADN/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteínas de la Matriz Extracelular/genética , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Cristalino/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Miembro 25 de Receptores de Factores de Necrosis Tumoral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2 , Receptor fas/genética
3.
Invest Ophthalmol Vis Sci ; 42(1): 241-6, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11133875

RESUMEN

PURPOSE: The goal of this study is the characterization of the strong yellow fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid. METHODS: Naturally occurring melanin in the human retina and choroid was oxidized by exposing fixed and plastic-embedded sections of a human eye to light and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melanin was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy. RESULTS: Naturally occurring melanin oxidized in situ exhibited a lipofuscin-like yellow fluorescence. Oxidation of melanin in vitro degraded the melanin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluorescence. CONCLUSIONS: The results indicate that the strong yellow fluorescence of melanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materials. The data presented allow a reinterpretation of the results obtained from fluorescence investigations of melanin-containing tissue and suggest a link between melanin degradation and lipofuscin formation.


Asunto(s)
Melaninas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Coroides/efectos de los fármacos , Coroides/metabolismo , Fluorescencia , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Melaninas/química , Microscopía Fluorescente , Persona de Mediana Edad , Oxidación-Reducción , Epitelio Pigmentado Ocular/efectos de los fármacos , Espectrometría de Fluorescencia , Espectrofotometría , Factores de Tiempo
4.
Graefes Arch Clin Exp Ophthalmol ; 238(12): 979-84, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11196361

RESUMEN

BACKGROUND: Trophic factors [e.g. basic fibroblast growth factor (bFGF)] released by transplanted retinal pigment epithelial (RPE) cells are able to slow down the hereditary degeneration of the retina in the Royal College of Surgeons rat in sites distant from the site of transplantation where rod outer segment (ROS) phagocytic activity is not reconstituted by the transplants. METHODS: To investigate whether iris pigmented epithelial (IPE) cells are also able to generate this rescue by trophic factors, we transplanted IPE cells from Long-Evans rats into the choroid and subretinal space of 17 young RCS rats. The eyes were enucleated after 6 months and prepared for light microscopy. Six age-matched RCS rats served as controls. Light microscope sections from the whole choroid, healthy choriocapillaris, transplanted cells and the maximum thickness of the choroid, and outer nuclear layer parameters were analyzed by computer-assisted morphometry. RESULTS: In transplanted animals photoreceptor cells were rescued from degeneration although the majority of the transplanted IPE cells were located in the choroid. In the non-transplanted group photoreceptors were absent. CONCLUSIONS: Transplantation of IPE cells slows down degeneration of the photoreceptors in the RCS rat. This photoreceptor-sparing effect by the IPE cells was observed even when the transplants were predominantly located within the choroid. The beneficial effect observed may be related to trophic factors possibly secreted by the transplanted IPE cells.


Asunto(s)
Coroides/cirugía , Iris/citología , Células Fotorreceptoras de Vertebrados/fisiología , Epitelio Pigmentado Ocular/trasplante , Degeneración Retiniana/prevención & control , Animales , Supervivencia Celular/fisiología , Trasplante de Células , Procesamiento de Imagen Asistido por Computador , Células Fotorreceptoras de Vertebrados/patología , Epitelio Pigmentado Ocular/ultraestructura , Ratas , Ratas Mutantes , Degeneración Retiniana/genética , Degeneración Retiniana/patología
6.
Gastroenterology ; 113(1): 249-54, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9207285

RESUMEN

BACKGROUND & AIMS: Canalicular secretion is rate limiting in overall blood-to-bile transport of bile acids. Studies using transfected cells have implicated the canalicular ecto-adenosine triphosphatase (ecto-ATPase) in adenosine triphosphate (ATP)-dependent bile acid transport. However, the structural features of this ecto-ATPase are not those anticipated for an in-to-out ATP-dependent transporter. The aim of this study was to explore the possible existence of an ATP-dependent bile acid transport mechanism distinct from ecto-ATPase. METHODS: Bile acid transport activity and ecto-ATPase expression were analyzed in primary rat hepatocytes, rat hepatoma HTC cells, and specially adapted HTC (HTC-R) cells using plasma membrane vesicles and Northern blot, slot blot, ribonuclease protection assay, and Western blot analyses. RESULTS: Plasma membranes isolated from HTC-R cells exhibited ATP-dependent taurocholate transport, which was many-fold greater than that in HTC cells. Hepatocytes showed the highest transport rates. Protein and RNA analyses showed very low expression of ecto-ATPase in HTC and HTC-R cells compared with hepatocytes. There was no difference between the two cell types at both the RNA and protein level. CONCLUSIONS: These findings show the presence in HTC-R cells and, apparently in hepatocytes, of one or more proteins other than the ecto-ATPase that mediate ATP-dependent transport of bile acids.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Ácidos y Sales Biliares/metabolismo , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfatasas/fisiología , Animales , Canalículos Biliares/enzimología , Northern Blotting , Western Blotting , Neoplasias Hepáticas Experimentales , Ratas , Ácido Taurocólico/metabolismo , Células Tumorales Cultivadas
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