Changes in RNA and protein synthesis in the neural retina during lens regeneration in newts

Since neural retina stimulates regeneration of a lens from the dorsal iris in newts, RNA and protein synthesis in the neural retina was investigated during this process. Incorporation of 3H-uridine and 3H-leucine using liquid scintillation counting was employed to compare RNA and protein synthesis in the neural retina from sham-operated control eyes with that in eyes during lens regeneration. An initial increase in 3H-uridine uptake was seen one to three days after lentectomy. This was followed by greater incorporation of 3H-leucine, indicating increased protein synthesis between 5 to 15 days after lens removal. A decrease in 3H-uridine uptake was also seen at 5 to 12 days after lentectomy. After 20 days both the RNA and protein synthesis returned to the normal level. Since the increase in protein synthesis is preceded by an increase in RNA synthesis, the two processes might be related. The results indicate significant changes in the synthesis of macromolecules by the neural retina following lentectomy. These may be indirectly related to the production of the neural retinal factor with stimulates lens differentiation.     

A lens fibre differentiation factor from calf neural retina

During growth of the eye lens, epithelial cells differentiate into fibre cells under the influence of neural retina. The fibre differentiation factor (FDF) was partially characterized from calf retina-conditioned medium, using lens epithelial explants from young rats, to provide a bioassay for differentiation. FDF was associated with large-protein aggregates, the smallest of which eluted at approximately 500-600 kD on Sephacryl S-300 columns and migrated as a single protein band near 600 kD on gradient gels. This protein resolved into nine major peptides on SDS-polyacrylamide gels, ranging between 23 and 27 kD. Eight of these peptides were present oa four doublets, but did not appear to contain specific carbohydrate residues. The approximately 500-600 kD complex could be slightly disrupted by trypsin or heat treatment to release a less stable 90 kD component. Fractionation of FDF invariably led to loss of activity, possibly due to gradual dissociation into less active and/or less stable components. A working hypothesis suggested by these findings is that FDF is associated with a small group of peptides, each contributing an essential function to the process of fibre differentiation.     

Peroxidases in the neural retina and pigment epithelium.

Peroxidase activity, assayed with 2 mM-H₂O₂ and suitable hydrogen donors (either p-phenyl-enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle-associated in the native state, but is readily extractable by brief sonication or freeze-thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium. Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both.     

Proliferation of the cells of iris and pigment epithelium of the retina in the early periods after removal of the lens and iris in Spanish newts]

It has been shown by means of autoradiography that following the simultaneous removal of lens and retina in the eyes of adult ribbed newts (Pleurodeles waltlii) the proliferative processes related to the regeneration of retina, rather than lens, are most active at the early stages of eye restoration. During the lens regeneration in the absence of retina, the proliferation of the cells of pars iridica of the dorsal iris zone, a source of lens regeneration, is delayed, possibly due to the increase of the duration of mitotic cycle of these cells.     

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

Ligatin from embryonic chick neural retina inhibits retinal cell adhesion

Ligatin, a filamentous cell-surface protein purified from embryonic chick neural retina, has been found to inhibit the reassociation of dissociated retinal cells. This inhibition was demonstrated using two methods, a single cell disappearance assay and an improved monolayer collection assay utilizing microtiter plates. Monomeric ligatin at approximately 20 μg/ml inhibited rates of adhesion, but polymeric ligatin and tryptic fragments of ligatin were ineffective. Ligatin's inhibitory effect is suggested to be mediated through binding to retinal cell surfaces since preincubation of dissociated retinal cells with monomeric ligatin inhibited the cells' adhesiveness and removed the inhibitory activity from the culture media. Ligatin homologues prepared from mammalian tissues were ineffective in inhibiting retinal cell adhesion, suggesting a tissue and/or species specificity. Similarities in physicochemical and biological properties suggest that ligatin may be the inhibitor of adhesion previously described by Merrell et al.[Merrell, R., Gottlieb, D. I., and Glaser, L. (1975). J. Biol. Chem., 250, 4825].     

Lactate dehydrogenase isoenzymes during regeneration of the lens and retina in adult tritons

The range of lactate dehydrogenase (LDG) isozymes has been studied at the consecutive stages of retina regeneration from pigmented epithelium cells and lens regeneration from iris margin in adult crested newts. It was shown that the spectra of LDG isozymes peculiar to pigment epithelium cells and iris and characterized by the predominance of slowly migrating forms are replaced in the lens and retina regenerates by spectra characterized by the predominance of rapidly migrating isozymes which are peculiar to definitive lens and retina.     

Macrophage invasion and phagocytic activity during lens regeneration from the iris epithelium in newts

Following removal of the lens through the cornea, early stages of lens regeneration from the dorsal iris of the adult newt, Notophthalmus viridescens, were studied using light and electron microscopic observations on sectioned, plastic-embedded irises. Specimens were fixed in Karnovsky's fixative every 2 days from 0 to 12 and 15 days after lentectomy. Infiltration of the iris epithelium by macrophages and their phagocytosis of melanosomes and small fragments of iris epithelial cells were observed. These macrophages were characterized by coarse nuclear chromatin, numerous mitochondria, free ribosomes, granular endoplasmic reticulum, Golgi complexes, vesicles, lysosomes, and phagosomes containing ingested melanosomes. Lamellipodia of varying length projected from their surface. Most of the cells lying on or close to the posterior surface of the iris could be identified as macrophages by these criteria. During this period, there was enlargement of the intercellular spaces within the iris epithelium. The iris epithelial cells near the margin of the pupil elongated, lost their melanin pigment and some associated cytoplasm, and acquired abundant free polyribosomes to form a lens vesicle of depigmented cells.