The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this ...study, we describe the synthesis of 5'-digoxigenin-labeled phosphorothioate oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Barr virus-transformed B cells. Comparison of digoxigenin-labeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosome-associated membrane protein indicate that oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have been studied: immunogold labeling in heavily fixed and resin-embedded cells and immunogold labeling in lightly fixed and cryoultramicrotomy processed cells. The results confirm the major lysosomal accumulation of digoxigenin-labeled oligonucleotides and demonstrate that the antigenic capacity of digoxigenin is not damaged by any of the procedures used. Therefore, the conjugation of the functionalized digoxigenin molecule at the 5' end of phosphorothioate oligonucleotides provides a new tool in the study of oligonucleotide uptake and intracellular distribution at both cellular and ultrastructural levels.
RESUMO La interacción entre la matriz extracelular y los componentes celulares es la principal responsable de la diferenciación celular y dei desarrollo tisular. Mediante técnicas inmunocitoquímicas ...para microscopía óptica y electrónica, hemos estudiado el receptor de membrana celular dei subgrupo β1 de la familia de las integrinas, la laminina (LN) y la fibronectina (FN) en membranas epirretinianas (MER) de pacientes sometidos a vitrectomía portadores de una vitreorretinopatía proliferativa (VRP) como complicación de un desprendimiento de retina, y en membranas vítreorretinianas fibrovasculares de la retinopatía diabética proliferativa (RDP). Los resultados obtenidos indican que las MER presentan el complejo β1 en disposición pericelular, unido a la' membrana plasmática. La LN y la FN se hallan en la matriz extracelular en estrecha relación con la integrina. El receptor parece ser observado más frecuentemente en las MER de hasta 2 meses de evolución cuando comparadas a las de mayor tiempo de evolución. Por otro lado, dicha integrina se encuentra localizada en los capilares de neoformación de las membranas fibrovasculares obtenidas de pacientes diabéticos. Estos hallazgos aportan nuevos datos para una mejor comprensión de los mecanismos fisiopatológicos de la interacción célula-sustrato en el proceso proliferativo intraocular.
Cell surface proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. To investigate the organization of these molecules at the cell surface, the ...distribution of two well-known proteoglycan ligands has been studied. These ligands, lipoprotein lipase and basic fibroblast growth factor, showed a characteristic binding pattern consisting of highly organized parallel arrays that crossed the upper surface of human skin fibroblasts. The proteoglycan nature of the binding sites was evident from their susceptibility to heparinases, and from ligand displacement by heparin. Parallel localization of the ligands and actin, and treatment of the cells with cytochalasin, showed that the binding proteoglycans are organized by the actin cytoskeleton. The ligands induced a different behaviour of the binding sites on incubation of the cells at 37 degrees C. Lipoprotein lipase produced a movement of the binding proteoglycans along the actin filaments towards the cell center. In contrast, after binding of basic fibroblast growth factor the binding proteoglycans remained spread over the cell surface and actin depolymerization was induced. Since an increasing number of ligands appear to depend on proteoglycans for their interactions with their high affinity receptors, distribution and movement of proteoglycans at the cell surface that is organized by the actin cytoskeleton could direct and enhance the encounters between the ligands and their specific receptors.
RESUMEN La Retinopatía Diabética Proliferativa (RDP) se caracteriza por una proliferación fibrovascular intraocular con la formación de membranas prerretinianas que predisponen a Desprendimientos de ...Retina (DR) traccionales y hemorragias en el vítreo. Aunque sean considerables los estudios morfológicos acerca de estas membranas, su fisiopatogénesis es apenas conocida. Recientemente se ha descrito la posible participación de factores humorales y de la matriz extracelular en la angiogénesis y en su formación. Con el objetivo de identificar la distribución de algunos de estos posibles factores, hemos realizado um estudio inmunocitoquímico para la detección de la Laminina (LN) y de la Fibronectina (FN), en una serie de 18 membranas fibrovasculares, obtenidas de ojos portadores de RDP y sometidos a vitrectomía. Ambas glucoproteínas presentam una localización vascular; la FN en disposición luminal, y la LN en disposición basal con respecto a las células endoteliales de los capilares. La inmunolocalización también ha confirmado la presencia de macrófagos, sugiriendo que el proceso proliferativo intraocular puede ser considerado como una forma modificada de formación cicatricial.
Lipoprotein lipase (LPL, E.C. 3.3.1.34) is the enzyme responsible for hydrolysis of triacylglycerols in plasma lipoproteins, making the fatty acids available for use by subjacent tissues. LPL is ...functional at the surface of endothelial cells, but it is not clear which cells synthesize the enzyme and what its distribution is within tissues and vessels. We have searched for specific cell expression of the LPL gene by in situ hybridization using a RNA probe and for the corresponding protein distribution by immunocytochemistry on cryosections of some LPL-producing tissues of guinea pigs. In white and brown adipose tissues, heart and skeletal muscle, and lactating mammary gland, there was positive hybridization for LPL mRNA over all members of the major cell types, indicating that mature and immature adipocytes, muscle cells, and mammary epithelial cells are main sources of LPL. In large vessels, LPL expression was detected in some smooth muscle cells in the media layer. There was no positive hybridization for LPL mRNA over endothelial cells in any of the tissues studied, but there was immunoreaction for LPL protein at endothelial surfaces of all blood vessels. In the kidney, there was strong immunofluorescence at the vascular endothelium, particularly in the glomeruli, but little or no LPL mRNA was detected in the surrounding cells. These observations suggest that in some tissues LPL is synthesized by parenchymal cells and spreads along the vascular mesh. Transfer to the vascular endothelium is, however, not the only route taken by LPL. In the mammary gland most of the enzyme protein appeared to be secreted, partly in association with milk fat droplets.
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