Eileen Masterson

A quantitative system for studying phagocytosis in pigment epithelium tissue culture

Abstract A quantitative assay for phagocytosis has been described using a heterologous system of chick pigment epithelial culture and radiolabeled bovine rod outer segment suspensions. Electron microscopy was used to verify results obtained in radiolabeled uptake studies. Freezing and mild shearing of the outer segment suspensions do not significantly inhibit phagocytosis. Outer segments radiolabeled with [ 3 H]choline and [ 14 C]mannose showed similar kinetics of phagocytosis. Maximum uptake was between 1 and 2 hr of incubation and approached 1% of the total applied radioactivity for mannose and 2% for choline.

Characterization of glucose transport by cultured chick pigmented epithelium

Abstract Glucose transport was studied in well-differentiated, confluent cultures of chick pigmented epithelial cells. Glucose permeation was stereospecific and was not influenced by energy poisons. Glucose, 3-O-methyl-glucose, and 2-deoxy- d -glucose all appeared to use the same carrier system. Countertransport of 3-O-methyl-glucose was demonstrated. The Km and Vmax values for 2-deoxy- d -glucose uptake were 2·70 m m and 22 nmol/min/mg protein. Hexose transport in these cultured cells appears to take place by a facilitated diffusion system.

Changes in retinoid binding levels during development of the chicken cornea

[3H]-retinol binding to cellular retinol-binding protein (CRBP) increased more than five-fold in developing chicken corneal epithelium at 14 days of incubation, the time when the initial increase in corneal transparency occurs in the developing embryo. In contrast, [3H]-retinoic acid binding to cellular retinoic acid-binding protein (CRABP) was highest at the earliest stages and decreased progressively to a very low level in the corneal epithelium near the time of hatching. Thus, there appear to be changing and differing requirements for retinol and retinoic acid during development of the chicken cornea.

Pentose shunt activity in developing chick retina and pigment epithelium: A switch in biochemical expression in cultured pigment epithelial cells

Abstract Pentose shunt activity in developing chick retina and pigment epithelium was studied by measuring the rate of 14 CO 2 evolution from glucose selectively labelled in the C-1 and C-6 positions. In the retina, shunt activity declines from appreciable levels at stages 29–31 to minimal activity in the 2-week-old hatched chick. Overall retinal metabolism also declines up to stage 45, but dramatically increases again after hatching. Developing chick pigment epithelium has minimal shunt activity at all stages studied. In contrast, cultured chick pigment epithelium has appreciable shunt activity which is constant over a period of several weeks in culture. This appears to be a switch in biochemical differentiation which could form the basis at least in part for subsequent changes in cell types observed in cultured pigment epithelial cells by Eguchi and Okada (1973) .

The pentose phosphate pathway in developing chick cornea.

Embryonic chick corneas at different stages of development were evaluated for activity of the pentose phosphate pathway. The appearance of activity was concurrent with the onset of corneal transperancy (stage 40). Highest values were found after complete transparency is achieved (stage 45 and after hatching). Phenazine methosulfate, an artificial electron acceptor, increased activity at all stages studied even before endogenous activity was measurable; however, no increase in glucose uptake was observed. Thus, the enzymes for the pathway are present at early stages (i.e., stage 38 and 40) although in latent form. The pathway probably functions in the developing cornea to generate NADPH rather than sugar moieties for macromolecular incorporation.

Metabolic inhibitors and glucose oxidation in developing chick cornea.

Abstract Stages 40 to 45 is a critical period of corneal deturgescence and development of transparency in the chick embryo. Glucose uptake, incorporation, and oxidation were studied in the chick cornea during this period and the effects of several inhibitors on these variables were tested. Corneal 14CO2 formation is constant between stages 38 and 45 but rises after hatching. Glucose uptake and incorporation increases between stage 38 and 40, is constant between stage 40 and 45, and increases again after hatching. Both DON, an inhibitor of proteoglycan synthesis, and actinomycin D had little effect on glucose uptake and incorporation by corneas at stages 40 and 45. Puromycin, on the other hand, significantly decreased glucose incorporation by the corneas at stages 40 and 45. The results indicate that much of the glucose taken up is utilized for protein synthesis which appears to be especially important during this period of corneal development.