carbocyanines and Retinitis-Pigmentosa

Progressive dynamic, relative quantitative changes were compared in glycans associated with retinal proteins of wild type (wt) and retinal degeneration 1 (rd1) mice during neonatal development and degeneration of retinae.. Proteins extracted from retinae of postnatal days 2 (PN2), PN7, and PN14 wt and rd1 mice were labeled with Cy3-fluorescent dye. Glycome of these proteins was quantified relatively by lectin microarray technique. Net fluorescence emitted by individual complexes formed between 45 lectins and Cy3-labeled proteins was measured by evanescent-field fluorescence-assisted microarray reader.. GlcNAcβ1-oligomer and high-mannose/Manα1-6Man were major glycans associated with the proteins of PN2, PN7, and PN14 wt and rd1 mice retinae. Gal/GalNAc/Man3-core-bi-/tri-antennary-complex, Sia2-3Galβ1-4GlcNAc, and high-mannose glycans were conjugated mainly to proteins from PN7 rd1 and PN14 wt retinae, respectively. With increasing neonatal age, mannosylated, GlcNAcβ, and sialylated (minor component) glycans were increased, and fucosylated GlcNAc/Galβ glycans were decreased significantly in wt retinal proteins. This trend was less evident in PN14 rd1 retinal proteins. Mouse retina was almost devoid of Siaα2-6 (except WGA bound Sia), Fucα1-2, and Gal/GalNAc-containing glycans. STL reacting GlcNAc oligomers were high in PN2 rd1 retinae.. Quantitative dynamic, relative variation in high-mannose and GlcNAc glycans, Siaα2-3Galβ1-4GlcNAc associated with proteins from PN2, PN7, and PN14 wt and rd1 mice retinae suggested that these glycans participate in retinal development and degeneration, and may be used as markers for retinal electrophysiologic integrity during transplantation/therapy studies; Siaα2-3Galβ1-4GlcNAc-specific Agrocybe cylindracea lectin and other lectins may be used to enrich/purify retinal ribbon synapse glycoproteins and other glycoproteins including rhodopsin. Further investigations are required.

Topics: Animals; Animals, Newborn; Carbocyanines; Disease Models, Animal; Eye Proteins; Fluorescent Dyes; Glycomics; Glycoproteins; Lectins; Mice; Polysaccharides; Protein Array Analysis; Retina; Retinitis Pigmentosa

Cell replacement has the potential to be applied as a therapeutic strategy in retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) for which no adequate pharmacological and surgical treatments are currently available. Although controversial, the use of ciliary epithelium (CE)-derived cells is supported by evidence showing their differentiation into retinal phenotypes. This study examines the differentiation potential of porcine CE-derived cells in vitro and their survival, migration, morphological characteristics, and immunohistochemical phenotype in vivo, upon transplantation into the subretinal space of normal pigs.. Cells were isolated from the CE of postnatal pigs and were grown in a suspension sphere culture. Differentiation was assessed in vitro after exposure to laminin and the addition of serum. For transplantation, CE-derived spheres were dissociated, labeled with CM-DiI vital dye, and the cells were injected subretinally into one eye of eight week-old allorecipients. The eyes were examined at eight days and at two and four weeks after transplantation.. Cells positive for neuronal and retinal pigment epithelium (RPE) markers were detected by immunohistochemistry in differentiation cultures. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) revealed upregulation of neuronal markers after in vitro differentiation. CM-DiI dye-labeled CE-derived cells dissociated from primary spheres survived for up to four weeks after transplantation in vivo. Some of the surviving cells migrated distantly from the injection site. Large clusters of transplanted cells integrated into the RPE layer and multilayered RPE-like structures positive for RPE65 were often observed. Grafted cells were also identified in the neuroretina where 5%-10% were positive for recoverin, protein kinase C alpha (PKCα), and calbindin.. The efficient conversion to an RPE-like phenotype suggests that CE-derived cells could be a potential source of RPE for cell replacement. Our data also suggest that the ability of these cells to acquire neuronal phenotypes is influenced by the environment. Thus, pre-differentiated or (re)programmed CE-derived cells may be more amenable for retinal repair.

Topics: Animals; Animals, Newborn; Biomarkers; Carbocyanines; Carrier Proteins; Cell Differentiation; Cell Transplantation; Cells, Cultured; cis-trans-Isomerases; Epithelial Cells; Eye Proteins; Fluorescent Dyes; Immunohistochemistry; Injections, Intraocular; Laminin; Macular Degeneration; Neurons; Retinal Pigment Epithelium; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Swine; Transplantation, Homologous

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.

Topics: Actins; Ataxia; Benzimidazoles; Calcium; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line, Tumor; Cytoskeleton; DNA, Mitochondrial; Fluorescent Dyes; Fura-2; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ionophores; Membrane Potentials; Microscopy, Fluorescence; Mitochondrial Myopathies; Muscle Weakness; Mutation; Osteosarcoma; Phalloidine; Retinitis Pigmentosa; Rhodamines; Thapsigargin