RESUMEN
PURPOSE: Heterozygous defects in the highly conserved PAX6 homeobox gene are associated with aniridia, an inherited human disorder affecting several ocular structures, including the adult cornea. This work establishes the pattern of Pax-6 gene expression in the surface epithelia of the late embryonic and adult eye. METHODS: Chick embryo sections and wholemounts, as well as adult mouse and monkey tissues, were analyzed by in situ hybridization and immunohistochemistry with probes specific to Pax-6. Western immunoblots were used to detect Pax-6 protein, and mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: In days 5 and 6 chick embryos, Pax-6 protein is found in the nuclei of all cells within the corneal epithelium and in the future conjunctiva. Although not detected in the cornea by in situ hybridization, Pax-6 mRNA is, in fact, present at levels comparable to those observed in the retina. In the mature mouse, Pax-6 protein was expressed in all cells of the corneal epithelium, the limbus, and the entire conjunctiva. Similar results were obtained for the monkey cornea. CONCLUSIONS: These data indicate that in addition to its role in the embryo, Pax-6 is expressed strongly in surface epithelia of the adult cornea and conjunctiva. In cells of these tissues, the gene may function by regulating structural or secretory specializations. Pax-6 might play a direct role in the maintenance and proliferation of corneal stem cells, a vital process that appears to be defective in aniridia.
Asunto(s)
Conjuntiva/metabolismo , Córnea/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Homeodominio , Factores de Transcripción/biosíntesis , Animales , Western Blotting , Embrión de Pollo , Conjuntiva/embriología , Córnea/embriología , Cartilla de ADN/química , Proteínas de Unión al ADN/genética , Epitelio/metabolismo , Proteínas del Ojo , Expresión Génica , Técnicas para Inmunoenzimas , Hibridación in Situ , Macaca fascicularis , Masculino , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteínas Represoras , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
PURPOSE: This study was conducted to examine the effect of retinal ischemia-reperfusion injury on protein tyrosine phosphorylation, the production of angiogenic growth factors, and the activation of signal proteins in tyrosine kinase pathways. METHODS: Ischemia-reperfusion injury was induced in rats by compression of the optic nerve for 2 hours. The rats were killed, and the retinas were collected at 0, 1, 6, 24, 48, 96, or 168 hours of reperfusion. Tyrosine phosphorylation of proteins in the retina was examined by Western blot analysis and immunohistochemistry. Angiogenic growth factors and their receptors, such as basic fibroblast growth factor (bFGF) and Flg, vascular endothelial growth factor (VEGF) and Flk-1, platelet-derived growth factor (PDGF)-B chain and PDGF-beta receptor, and five intracellular signal proteins (phosphatidylinositol 3-kinase [PI3K], phospholipase C gamma [PLC gamma], C-Src, SHC, and mitogen-activated protein kinase [MAPK]) were examined by Western blot analysis. RESULTS: Protein tyrosine phosphorylation increased after ischemia-reperfusion injury, reaching a peak at 48 hours of reperfusion. Increased staining of tyrosine-phosphorylated proteins in the inner retina were evident on immunohistochemical examination. The amount of bFGF decreased after injury, but the amounts of VEGF and PDGF-B chain increased. Tyrosine phosphorylation of PLC gamma, SHC, and MAPK was increased at 48 hours of reperfusion, and tyrosine phosphorylation of PDGF-beta receptor and PI3K was increased at 168 hours of reperfusion. CONCLUSIONS: Ischemia-reperfusion injury in the rat retina leads to activation of the tyrosine kinase pathway, increasing the amounts of angiogenic growth factors. The resultant activation of signal proteins PLC gamma, SHC, MAPK, PI3K, and PDGF-beta receptor may play an important role in ischemia-induced retinal changes such as cell proliferation.
Asunto(s)
Proteínas del Ojo/metabolismo , Daño por Reperfusión/metabolismo , Retina/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Sustancias de Crecimiento/metabolismo , Técnicas para Inmunoenzimas , Isquemia/metabolismo , Masculino , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento/metabolismo , Vasos Retinianos/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Genistein (4',5,7-trihydroxyisoflavone) is a potent anti-angiogenic compound. We investigated the inhibition of phosphotyrosine as a putative signaling mechanism utilized by the drug in modulating basic fibroblast growth factor (bFGF)-mediated vascular endothelial cell proliferation. The studies included the effect of genistein on DNA synthesis, cell viability, phosphotyrosine induction and characterization of the FGF receptor (FGFR). DNA synthesis was attenuated significantly by genistein in a concentration- and time course-dependent manner with relatively low cytotoxicity during a 16-24 hr exposure (IC50 = 12.5 microM; LC50 = 300 microM). Ligand-stimulated cells exhibited significant increases in phosphotyrosine, affecting FGFR and several tyrosine kinase substrates, ranging in size from M(r) 28 to 200 kDa. Inhibition of phosphotyrosine induction as shown by western blots occurred only at high concentrations of the drug (> 500 microM). These results were supported by results obtained using fluorescence immunocytochemistry. FGFR was shown to be FGF-R1 beta 2, a dimer of approximately 85 and 62 kDa, which was prevented from being autophosphorylated when relatively high concentrations of the drug were applied. Low dose (< 20 microM) inhibition of DNA synthesis by genistein did not correlate with the high concentration required for phosphotyrosine inhibition. The data suggest that although cell stimulation results in phosphotyrosine induction, inhibition of phosphotyrosine is not required for inhibition of DNA synthesis. Furthermore, in endothelial cells, inhibition of DNA synthesis by genistein is not mediated primarily by the inhibition of protein tyrosine kinase activity.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Isoflavonas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Tirosina/análogos & derivados , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genisteína , Fosfotirosina , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Tirosina/biosíntesisRESUMEN
The GC-Ion Trap MS is recently one of the most efficient instrumental analysis recommended for understanding the chemistry of volatile organic compounds, not only in water but even in the food chain and other environmental media (air and soil). Results of the experiment conducted on water samples from Kuguri and Yatsutani sampling stations showed considerably higher levels of organic enrichment (COD = 10 mg/L and 11 mg/L respectively). Total concentrations of Pb (0.072 mg/L and 0.093 mg/L) and Cd (0.004 mg/L and 0.011 mg/L) on the other hand, invariably exceeded the maximum allowable concentrations for human health and the living environment (Pb = 0.005 mg/L; Cd = 0.001 mg/L respectively). And the toxicity levels for these contaminants at LC50 showed critical impact on rainbow trout (hypersensitive species) at 0.14 mg/L for Pb and 0.007 mg/L for Cd in 96 hours respectively. Although these major contaminants including phenol and 3-, 4-cresol, showed relatively higher toxicity impact in the experimental media, it would remain contentious to justify any associated potential dangers without regular routine water monitoring, at least for a period of one year. Nevertheless, the data could serve as a benchmark through which other phenomena can easily be investigated.
Asunto(s)
Metales Pesados/análisis , Compuestos Orgánicos/análisis , Contaminantes Químicos del Agua/análisis , Animales , Agua Dulce/química , Cromatografía de Gases y Espectrometría de Masas , Humanos , Metales Pesados/toxicidad , Oncorhynchus mykiss/metabolismo , Compuestos Orgánicos/toxicidad , Volatilización , Contaminantes Químicos del Agua/toxicidadRESUMEN
The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKC alpha), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO2 = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO2 = 3%) in humidified, 5% CO2, 37 degrees C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M(r) approximately 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKC alpha, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases.