928 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Estimation of Keratin The area covered by orange-red cells compared to the area covered by blue cells was independently estimated by two observers at two dif- ferent time intervals and the results were averaged. Variation in esti- mation of keratin by this method was 10-15%. No quantitative method for the determination of keratin is known. RESULTS The accumulation of a band of amorphous material in a 21-day culture of adult skin epidermal cells is shown in Fig. 1. The material is intensely eosinophilic, and reacts with -SH and -SS- group reagents when stained by the method of Barnett and Seligman (11). Stains for DNA (Feulgen) and mucopolysaccharides (PAS) were negative orange G stain for keratin (12) was strongly positive. Figure 1. Accumulation of a keratin-like protein in epidermal cells in cell structure. Cells were grown from an cxplant of abdominal skin obtained at autopsy. Magnification 240 )

RETINOIC ACiD AND EPITHELIAL CELLS 929 The amorphous material is completely insoluble in dilute acid (0.1N HC1) and dilute base (0.1N NaOH). Also, the amorphous ma- terial is not hydrolyzed by the proteolytic enzymes trypsin, chymotryp- sin, or pepsin (Table I). Table I Insolubility Properties and Resistance to Proteolytic Enzy•nes of Keratin-Likc Proteins Micrograms Protein in Supernatant Reagent 0 hr 24 hr HC1 0 10 NaOH 0 10 Pepsin (10 t•g/mg) 0 10 Trypsin (10/•g/mg) 0 10 Chymotrypsin (10 t•g/mg) 0 10 In Fig. 2, the effect of increasing concentration of retinoic acid on the growth of skin epidermal cells is shown for a 2-week time interval. As shown in this figure, a concentration of retinoic acid of 0.01-0.05 rng/ml produces a marked stimulation of the outgrowth from foreskin similar stimulation was observed with skin obtained from the abdomen. In Table II, the effect of retinoic acid on the rate of growth is shown. ioo 9o o so fi 7o • so o • $o • 4o m 3o tJ •o • Io 0 0.1 0.5 1.0 50.0 Retinolc Acid (/Jg/ml) 5.0 I0.0 Figure 2. Effect of retinoic acid on the outgrowth of human posteml)ryonic epidermal cells. The growth is from a 14-day culture of adult human skin obtained at autopsy