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Biomedical subjects

M O Hall

Publications and source records attributed to M O Hall.

At least 37 records · Page 2Linked to original sources
PubMed1986

Rat retinal pigment epithelial cells show specificity of phagocytosis in vitro.

The retinal pigment epithelial (RPE) cell of the eye normally phagocytozes only retinal rod outer segments (ROS). The specificity of this phagocytic process was examined by incubating RPE cells with a variety of particle types. Confluent RPE cell cultures were incubated for 3 h at 37 degrees C in the presence of rat ROS, rat red blood cells (RBC), algae, bacteria, or yeast. Other cell cultures were incubated with equal numbers of ROS and one other particle type. Quantitative scanning electron microscopy was used to determine the numbers and morphology of particles bound to RPE cells, while double immunofluorescence labeling (Chaitin, M. H., and M. O. Hall, 1983, Invest. Ophthalmol. Vis. Sci., 24:812-820) was used to quantitate particle binding and ingestion. Both assays demonstrated phagocytosis to be a highly specific process. RPE cells bound 40-250 X more ROS than RBC, 30 X more ROS than algae, and 5 X more ROS than bacteria or yeast. Ingestion was more specific than binding; RPE cells ingested 970 X more ROS than RBC, 140 X more ROS than bacteria, and 35 X more ROS than yeast. The phagocytic preference for ROS was maintained in competition experiments with other particle types. Serum was found to be essential for phagocytosis. This study demonstrates that both the binding and ingestion phases of phagocytosis are highly specific processes.

Animals↗
PubMed1986

RPE cell surface proteins in normal and dystrophic rats.

Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

Animals↗
PubMed1985

An improved method for isolation and culture of rat retinal pigment epithelial cells.

Retinal pigment epithelial cells from normal, Long Evans (LE) and retinal dystrophic (RCS) rats can be grown in vitro (Edwards, 1977). An improved technique is described which permits a more rapid isolation of RPE cells, and routinely gives high cell yields (30,000-40,000/eye), excellent cell viability (95%), and high plating efficiencies (95-100%). Whole eyes are treated with hyaluronidase and collagenase followed by trypsin. These enzymes degrade components of the extracellular matrix, releasing sheets of RPE from adherent attachments to the retina and choroid. Trypsin was then used to dissociate the sheets into single cells. RPE cells are grown to confluence in primary culture. This technique permits RPE cell isolation from both normal and retinal dystrophic (RCS) rats, 8-15 days of age. Normal cells isolated by this technique consistently show excellent phagocytosis in vitro.

Animals↗
PubMed1984

Identification of some plasma membrane proteins of cultured rat pigment epithelial cells by labeling with 125I.

Cultured rat retinal pigment epithelial (PE) cells were labeled with 125I by lactoperoxidase (LPO)-catalyzed radioiodination. Examination of 125I-labeled cells by electron microscopic autoradiography suggested that 125I was incorporated into proteins at both the apical and basal surfaces of the PE cells, and into the extracellular matrix (ECM). Analysis of labeled cells by SDS-polyacrylamide gel electrophoresis and autoradiography showed that 125I was incorporated into at least 15 polypeptides. In order to determine which of these labeled proteins were derived from the plasma membrane. 125I-labeled cells were subjected to differential detergent extraction. Triton X-100 (2% v/v), which removed the cell plasma membrane, solubilized only three of the labeled proteins (152 000, 138 000, 123 000 daltons). SDS (0.25% w/v) completely removed cells from the tissue culture dish and solubilized all but four of the labeled proteins (225 000, 173 000, 87 000 and 72 100 daltons). When 125I-labeled PE cells were mechanically disrupted, and the resulting cell fractions separated by sucrose density gradient centrifugation, labeled proteins separated into two subcellular fractions. One fraction was especially enriched in the 152 000, 138 000 and 123 000 dalton labeled proteins, in addition to the plasma membrane marker enzymes Na+, K+-ATPase, and alkaline phosphodiesterase I. The second fraction was enriched in the 225 000, 196 000 and 173 000 dalton labeled proteins, the ECM proteins laminin and fibronectin, and the 43 000 actin band . It is proposed that 125I-labeled proteins in the former cell fraction are truly plasma membrane proteins, while those found in the latter cell fraction are a mixture of 125I-labeled ECM and basal plasma membrane proteins. The 152 000, 138 000 and 123 000 dalton labeled proteins of the plasma membrane fraction are glycoproteins and become firmly anchored to the Triton X-100 insoluble cytoskeleton when labeled cells are treated with concanavalin A.

Animals↗
PubMed1984

Actin in the photoreceptor connecting cilium: immunocytochemical localization to the site of outer segment disk formation.

Actin has been localized in Rana pipiens retinas that were fixed and embedded in aldehyde cross-linked BSA. Thin sections were reacted sequentially with (a) affinity-purified antiactin antibodies induced in rabbits; (b) biotinyl-sheep anti-rabbit antibodies; and (c) avidin-ferritin conjugates. As expected, antiactin labeling density was high in the apical pigment epithelial cell processes and in the calycal processes of photoreceptors. Actin was also localized in a new site. The connecting cilium that joins the inner and outer segments of both rods and cones was heavily labeled by antiactin at its outer segment (OS), or distal, end. In this region of the cilium, the plasma membrane evaginates to form new OS disks and these basal disks were labeled in some instances. Below the new disks in rods, the cytoplasm of liplike expansions of the distal cilium was also heavily labeled. The plasma membrane and interior of the connecting cilium and the remainder of the OS were unlabeled. These findings suggest that actin may participate in the vectorial transport of opsin and other intrinsic membrane proteins that are incorporated into newly forming OS disks. The results also implicate actin in the membrane expansion involved with OS disk formation.

Actins↗
PubMed1983

Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells.

A new procedure for assaying the phagocytosis of rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Using an ROS antiserum and a double immunofluorescent labeling procedure, ROS attached to the external surfaces of these cells can be distinguished from those that have already been ingested. We have used this procedure to study the phagocytosis of ROS by PE cells isolated from normal rats and rats with inherited retinal dystrophy (RCS rats). With this approach we have been able to show that the attachment of ROS to the external surfaces of dystrophic PE cells does take place to a normal extent. However, only a small number of these bound ROS are subsequently ingested, demonstrating that the ingestion phase of phagocytosis is defective. After a 4-hr incubation during which ROS are continuously present, normal rat PE cells ingest about 80% of the ROS that have bound to the cell surfaces. In contrast, after this time period, less than 20% of the ROS bound to the dystrophic PE cells have been ingested. These results, as well as the results of pulse-chase experiments in which ROS are rinsed away after two hours and the incubation continued without further addition of ROS, have demonstrated that normal PE cells rapidly ingest most of the bound ROS, whereas the dystrophic PE cells show no such rapid ingestion. Both cell types, however, are able to slowly ingest additional bound ROS with time.

Animals↗
PubMed1983

The distribution of actin in cultured normal and dystrophic rat pigment epithelial cells during the phagocytosis of rod outer segments.

In the previous article the authors reported that the ingestion phase of phagocytosis is defective in cultured dystrophic rat pigment epithelial (PE) cells. When these cells are challenged with isolated rod outer segments (ROS), attachment of ROS to the PE cell surfaces occurs to a normal extent. However, only a small number of these bound ROS are subsequently ingested. This raised the possibility that the contractile protein actin might not function normally in the dystrophic rat PE cells, since actin is intimately involved in the ingestion mechanism in other phagocytic cells. Utilizing actin antibodies and the technique of indirect immunofluorescence, we have studied the distribution of actin in cultured normal and dystrophic rat PE cells. Results show that the arrangement of actin fibers in the dystrophic cells appears normal both before and during the attachment of ROS to the cell surfaces. With the additional use of an ROS antiserum to label externally bound ROS, it is also possible to show that actin is involved with the ingestion of ROS by both normal and dystrophic PE cells. Thus, it appears that actin can function normally in dystrophic PE cells, but that the ingestion mechanism becomes activated at only a few sites of ROS attachment. The results of a scanning electron microscope study support this conclusion and also show the presence of a saucer-shaped elaboration of the PE cell plasma membrane beneath attached ROS. These may correspond to the actin feltworks seen with immunofluorescence microscopy at sites of ROS attachment.

Actins↗
PubMed1980

Experimental tractional retinal detachment in rabbits. Clinical picture and histopathologic features.

A simple reproducible model for the creation of tractional retinal detachments in rabbits is presented. This model comprises retinal membranes composed of different cell types derived from ocular tissues, and eliminates the need for lengthy and expensive tissue culturing procedures. The injected cell bolus rapidly migrates to the optic disc and forms surface retinal membranes. These lead to tractional retinal detachment confined to the area of the vascular medullary rays. The rapidity and severity of the clinical process seems to be directly dependent upon the number of cells injected. Good visibility of the posterior pole is maintained throughout the course of the development of detachment. This model is acceptable for testing agents to suppress intraocular cellular proliferation. It can also serve to teach surgical techniques for the management of vitreo-retinal membranes without endangering human eyes.

Animals↗
PubMed1979

Donor eye program.

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Academic Medical Centers↗
PubMed1978

Phagocytosis of light- and dark-adapted rod outer segments by cultured pigment epithelium.

Pigment epithelial cells in culture retain their ability to phagocytize rod outer segments. These cells phagocytize rod outer segments isolated from light-adapted rats, or from dark-adapted rats killed after the time at which the lights would normally be turned on. However, they phagocytize for fewer rod outer segments prepared in the dark from the retinas of rats killed before the onset of the normal light cycle. Phagocytosis of dark rod outer segments is variable, but that of light outer segments is reproducible. It is postulated that the effect of light is to synchronize the chemical events that occur at the surface of the rods to prepare them for phagocytosis. These processes also occur in the dark, but more slowly and irregularly than in the light.

Animals↗