Initial characterization of the proteins of keratinized epithelium of rat oral mucosa

Arch onrlfhol.Vol. 22,]pp.75 to 82.Pergamon Press1977Printedm GreatBritain.

INITIAL

CHARACTERIZATION OF THE PROTEINS OF KERATINIZED EPITHELIUM OF RAT ORAL MUCOSA BEVERLY A. DALE, I. B. STERN*

and J. A.

CLAGETT

Department of Periodontics, SM-44, University of Washington, School of Dentistry, Seattle, Washington 98195. U.S.A.

Smnma~-Rat cheek and palate epithelium, representing the least and most keratinized portions of the oral mucosa, were compared by biochemical and immunologic methods. Immunofluorescence using antibody to the epidermal fibrous proteins showed a positive reaction in the keratohyalin granules in cheek, palate and epidermis. A mild reaction was detectable in the stratum corneum of cheek and palate in contrast to the intense reaction of the epidermal stratum corneum, suggesting that antigenic sites in the oral epithelium differ or are blocked in situ. Proteins were extracted from the stratum corneum by detergent, urea and reducing agents and were compared to those of stratum corneum of newborn rat epidermis by sodium dodecyl sulphate (NaDS) polyacrylamide gel electrophoresis. Amino acid compositions of partially purified proteins of the stratum corneum of cheek and palate epithelium were very similar and to the fibrous proteins of epidermis. All these oral proteins cross-reacted with antibody to epidermal fibrous protein. The third major protein from rat epidermis which had a high content of arginine and histidine was not detected in extracts of the oral stratum corneum.

INTRODUCTION

In rodents, the entire oral mucosa is keratinized but differences in ultrastructural features and extent of keratinization have been described by Alvares and Meyer (1971), Chen and Meyer (1971) and Osmanski and Meyer (1967). The epithelium of the cheek is the least and that of the palate the most keratinized. Where the ora I epithelium is completely keratinized (i.e., palate), it has many ultrastructural features in common with the stratum comeum of epidermis, including a thickened cell membrane, flattened cells devoid of organelles but filled with fibrils embedded in a darker staining matrix (Gibbins, 1962; Hayward, Hamilton and Hackemann, 1973; Osmanski and Meyer, 1967). In contrast, Albright and Listgarten (1962), Osmanski and Meyer (1967) and Squier (1968) reported that rodent cheek stratum comeum cells contain filament:< which are distinguishable against a lighter background. These ultrastructural studies, as well as observations by Kempson (1974), also reveal differences between rodent palate and cheek epithelium in the granular layer, subjacent to the stratum corneum. In the palate, the granular layer contains keratohyalin granules which *are small and numerous and associated with both tonofilaments and ribosomes, as they are in epidermis. During keratinization, the amorphous material cd keratohyalin seems to spread along the tonofilaments and the two components become intimately associated. It has been suggested that kera*Present address: Tufts University, School of Dental Medicine, One Kneeland Street, Boston, Massachusetts 02111, U.S.A.

tohyalin may contribute to the interfilamentous matrix of the stratum corneum and/or to the fibrous component itself (Brody, 1959a, b; Fukuyama and Epstein, 1967; Odland, 1964). In contrast, the granules are larger and less numerous in the cheek and often composed of two distinct components. They are associated with ribosomes but not with tonofilaments. The biochemical bases for ultrastructural differences in keratinizing tissues have not been established. The fibrous and nonfibrous proteins of the keratin complex in oral epithelium have not been investigated biochemically; nor have they been compared to those of the epidermis. MATERIALS

Epithelial

AND METHODS

samples

Cheek and palate mucosa were removed from the molar region of adult male Sprague-Dawley, Berkeley strain rats after ether anaesthesia and decapitation. The tissues were rinsed in Earle’s balanced salt solution (EBSS) and treated with trypsin (Difco 1:250) in EBSS at 4°C for 18 h. Cheek was incubated in 0.25 per cent trypsin, palate in 0.125 per cent, and skin in 0.10 per cent. The epithelium was separated from the connective tissue using tissue forceps (Stem and Sekeri-Pataryas, 1972). Extraction

of proteins

The minced epithelium was stirred in 1 per cent NaDS in 0.1 M tris-HCl, pH 8.5 with 2Opg/ml phenylmethylsulphonyl fluoride (PMSF) for 1 h at room temperature. Intact pieces were subsequently

Beverly A. Dale, I. B. Stern and J. A. Clagett

76

extracted for 4-6 h with 1 per cent NaDS containing 8 M urea, 0.1 M trisHC1, pH 8.5, 0.1 M 2-mercaptoethanol, 1 mM dithiothreitol (UTME) with 20~g/ml PMSF. Extracted material was separated from the insoluble residue by centrifugation at 20,000 g x 15 min. The residue was resuspended in NaDS-UTME. All samples were dialyzed vs. distilled water containing 20 pg/ml PMSF for 6 h at room temperature, then 48 h at 4°C to remove NaDS. Precipitates formed during dialysis were removed by centrifugation at 20,000 g x 30 min, then resuspended in a small volume of UTME. All samples were stored at -20°C. Protein concentration was assayed by the method of Bramhall et al. (1969). Immunologic procedures Antiserum to the light chain of the fibrous protein of newborn rat epidermis was previously prepared in the goat (Huang et al., 1975). This antiserum is specific for epidermis; it does not react with rat dermis, liver, or kidney (Dale et al., 1976). Affinity-purified antibody was labelled with fluorescein isothiocyanate for direct immunofluorescence studies on cryostatsectioned adult rat cheek, dorsal and ventral tongue and palate, as well as newborn palate and skin (Dale et n2., 1976). Gel electrophoresis Separation of proteins by NaDS polyacrylamide gel electrophoresis was performed on 7.5 per cent gels as previously described (Dale and Stern, 1975a). Electrophoresis was carried out at 9 mA/tube for 3; to 4h. Preparation of samples for amino acid analysis Proteins of the NaDS-UTME extracts were separated by electrophoresis for 6 h on 18 duplicate NaDS gels. One gel was stained and the others were frozen on dry-ice and stored at -20°C. Areas with the major protein bands were identified by comparison with the stained gels. These regions were excised and the protein eluted by incubation over-night at 37°C in 5 ml 0.005 M NaHCO, buffer, pH 7, containing 0.05 per cent NaDS, 1 mM dithiothreitol and 10 pg/ml PMSF. The supematants were centrifuged to remove gel particles and dialyzed vs. H,O containing Bio-Rad AG l-X2 (acetate form) and 10 pg/ml PMSF at room temperature to remove NaDS. After further dialysis vs. H,O in the cold, the samples were divided into 4 and 1 ml portions and lyophilized. The 1 ml portion was redissolved in a small volume of 4 M urea, rerun on NaDS gels to determine purity, and tested for cross-reaction with antiserum to epidermal fibrous protein by rocket immunoelectrophoresis (Laurell, 1966). The 4ml portion was hydrolyzed for 24 h at 110°C in 6 M HCI and analyzed for amino acid composition using a Beckman 120C autoanalyzer. RESULTS

Epithelinl morphology Plate Figure 1 shows sections of trypsin oral epithelium and the NaDS-separated layers. After trypsinization, the basal layer intact although some separation of the widening of the intercell;lar space was

separated cornified was fairly cells and evident in

Table 1. Extraction

1 hr NaDS NaDS-UTME Residue *Total protein 5.01 mg; epidermis

of epithelial

Cheek

Palate

42% 50% 8%

39% 30% 31%

proteins* Epidermis

for cheek = 4.85 mg; = 31.1 mg.

34% 32% 34% palate =

both the basal and lower spinous layers. After one hour stirring in 1 per cent NaDS, the nucleated cells have been removed and only the stratum corneum remains intact, although some comified cells are removed by this treatment. ImmunoJuorescence The antibody used is directed against an epidermal fibrous protein. As expected, the fluorescence reaction was most intense in the stratum comeum and in the region of the keratohyalin granules of the newborn skin (Plate Fig. 2a). The fluorescence was well blocked in sections preincubated with non-fluorescent antiserum (Fig. 2b). In contrast, some fluorescence was detectable in the stratum comeum of oral epithelium, but the reaction was not intense as in the epidermis. The fluorescent reaction in the palate and cheek (Plate Fig. 2c and e) was primarily localized in the region of the keratohyalin granules. Pre-incubation with non-fluorescent antiserum completely blocks the reaction in the stratum corneum but is slightly less effective in blocking the reaction of the keratohyalin granules (Fig. 2d and f). Similar results were obtained with newborn palate as well as adult dorsal and ventral tongue. Epithelial protein extraction The total yield of extracted epithelial protein averaged 2 g per adult rat for cheek and palate and 4.4 g per newborn rat epidermis. The NaDS extract contained material from the lower cell layers and some material from the cornified layer but the NaDSUTME extract contained material from the cornified layer only. Table 1 shows distribution of extracted protein. Approximately one-third of the total protein

Fig. 3. Immunodiffusion of extracts of oral mncosa and epidermis vs. antiserum to epidermal fibrous protein (centre well). A. 1. partially purified fibrous protein; 2. palate NaDS extract; 3. palate, NaDS-UTME extract; 4. cheek, NaDS extract; 5. cheek, NaDS-UTME extract. B. 1. uartiallv_ _ nurified fibrous urotein: 2. eoidermis. NaDS A extract; 3. epidermis, NaDS-UTME extract.

Oral keratin was extracted by 1 per cent NaDS. However, when further extraction with NaDSUTME was performed, protein from the cheek was much more completely solubilized than from palate or epidermis. ImmunodifSusion The NaDS and NaDS-UTME extracts were tested for cross-reaction with antiserum to the epidermal fibrous protein by Ouchterlony double diffusion analysis. Preciptation lines could be seen for all extracts (Text Fig. 3A and B), including lines of identity between the purified epidermal fibrous protein and the NaDS extracts of epidermis, cheek and palate. Multiple bands were present with epidermis and palate as well as a line of identity between the purified epidermal fibrous protein and the NaDSUTME extract of epidermis. The newborn epidermis was originally used as a source of antigen in order to avoid contamination by hair proteins or possible alterations of the epiderma1 fibrous proteins by depilatory procedures. For comparison with the newborn epidermis, the stratum comeum of ad& epidermis was isolated and extracted following hot-wax treatment to remove hair. The resulting adult extract yielded a strong precipitant band with the antibody to newborn fibrous protein in Ouchterlony analysis with a small spur indicating loss of some antigenic sites during maturation or extraction procedures.

h

9

C

8T%?z Distance

from

30 Molecular

40 50607080 weight (daltons

NaDS polyacrylamide gel electrophoresis The extracts and residues from the three epithelia were subjected to NaDS polyacrylamide gel electrophoresis. The 1 per cent NaDS extract contained fastmigrating, poorly-resolved protein bands suggesting proteolysis before or during extraction. These extracts were not used for subsequent analysis. In contrast, the NaDSUTME extracts and residues showed sharp, well-resolved bands. Comparison of the gels of the NaDS-UTME (stratum corneum) extracts revealed that the major proteins in cheek and palate differ, but some of the oral proteins are similar to those of the epidermis (Text Fig. 4). Molecular weights of major bands, (a)(h), determined by comparison with standards on NaDS gels were 59,000, 5O,ooO, 42,000 in cheek; 67,000, 62,000, 53,000 in palate; and 67,000, 61,000 and 50,000 in epidermis. Identical results were obtained with NaDS-UTME extracts of NH,Cl separated epithelia; thus, differences in the molecular weights observed are not due to differential effects of trypsin on the three epithelia.

Amino-acid analysis of major protein bands The major proteins of the NaDSUTME extracts were separated by preparative electrophoresis on NaDS polyacrylamide gels. The samples were assayed for purity and reaction with the antiserum to epiderma1 fibrous protein. Amino acid analysis was performed on the remaining material (Table 2). In general, the composition of epidermal fractions A, B, C was similar to the purified proteins of similar molecular weight. Fractions A and B were characterized by high aspartic acid (asp), serine (ser), glutamic acid (glu), glycine (gly) content and fraction C was characterized by relatively high histidine (his), arginine (arg) and alanine (ala) content as well as high ser, glu and gly. The protein fractions eluted from NaDS gels differed somewhat from the chromatographically purified proteins. The major differences between epiderma1 fractions A and B and the purified fibrous proteins were greater yields of gly and ser and lesser yields of cysteine (cys) and methionine (met). The major differences between epidermal fraction C and the purified 50,000 molecular weight protein were increased yields of the nonpolar amino acids, valine (val), isoleucine (isol), leucine (leu), tyrosine (tyr), phenylalanine (phe), and lesser yields of his and arg. Fractions D-H from oral stratum comeum were very similar to each other and to the epidermal fractions A and B. None of these oral samples had the relatively high amounts of his and arg found in epidermal fraction C. Immunoelectrophoresis of stratum corneum prowin fractions

origin (mm) l-

io12.5

77

x 10w31

Fig. 4. Densitometric scans of NaDS polyacrylamide gels of stratum corneum extracts. A. epidermis; B. cheek; C. palate. Stained gels were scanned ising a Helena Instruments Quik Scan densitometer with a 545 pm filter. Major bands are indicated (a)-(h). Band (g) is a doublet.

Protein fractions isolated by elution from NaDS gels were tested for cross-reaction with the antibody to epidermal fibrous protein (61,OOOMW chain) by rocket immunoelectrophoresis. Epidermal fractions A, B and C all gave positive reactions. Cheek and palate fractions D-H all gave very weak positive reactions as might be expected from an immunologically related but non-identical protein.

Beverly A. Dale, I. B. Stern and J. A. Clagett

78

Table 2. Amino acid composition

Approx.

half cys lys his arg asp thr ser glu pro gly ala val met isol leu tyr phe try

m.w.$

Purified epidermal proteins? Fibrous protein 3rd light heavy band 67.000 61,000 50,000

0.8 7.1 0.8 7.4 8.9 2.8 9.5 14.3 2.0 11.5 4.0 3.2 4.9 3.6 5.9 2.2 4.8 1.5

0.8 4.6 0.7 5.8 8.2 3.2 11.9 17.9 0.6 15.1 2.8 1.7 2.8 2.8 7.7 5.5 3.6 2.3

0 1.0 8.2 16.0 3.7 5.6 14.7 21.2 trace 15.3 11.9 trace 0 1.7 0.5 0.4 trace

of epithelial

protein

fractions*

Preparative SDS gel fractions$ Epidermis Cheek Palate A B C D E F G H 67,000 61,000 50,000 59,000 50,000 42,000 67,000 53,OCMl 62,000 0 4.8 2.2 5.2 7.9 4.0 15.0 12.2 2.9 21.0 5.4 3.6 1.0 3.2 5.6 2.8 3.1

0 5.0 2.0 4.4 9.0 4.4 16.7 9.9 2.2 22.0 5.3 3.4 0.2 3.0 6.9 2.8 2.9

0 2.5 5.5 9.4 5.5 4.8 15.3 16.9 3.4 17.7 8.4 2.0 0.5 2.4 3.2 1.7 1.1

0 5.7 3.0 3.7 7.1 3.9 14.8 12.7 3.8 20.3 7.1 3.6 0.7 2.9 5.5 2.4 2.8

0 4.0 2.6 3.1 7.4 4.6 17.4 14.9 3.2 18.3 7.5 3.4 0.9 2.9 5.5 2.1 2.2

0 5.3 2.7 3.3 8.4 4.7 14.8 13.4 3.5 17.7 7.4 4.1 0.9 3.0 3.1 2.2 2.5

0 4.1 2.8 3.7 7.9 4.4 15.9 13.7 3.0 19.5 7.1 3.7 1.1 3.0 6.0 1.9 2.3

0 5.4 2.3 4.9 8.8 4.5 13.3 13.2 3.7 16.5 6.4 4.1 1.2 3.2 7.4 2.5 2.4

* Residues per 100 residues. t Fibrous protein amino acid composition Huang et ctl. (1975). 3rd band purified by DE-52 and CM-52 chromatography, manuscript in preparation. $ Fractions eluted from 18 to 25 duplicate NaDS-polyacrylamide gels with 5 ml 0.005 M NaHCO, buffer, pH 7, containing 0.05 per cent NaDS, 1 mM dithiothreitol and lOpg/ml PMSF. Supernatants were dialyzed vs. H,O containing BioRad AG l-x2 (acetate form) and 10 pg/ml PMSF at 24°C to remove NaDS. After further dialysis vs. H,O in the cold, the samples were divided into 4ml and 1 ml portion and lyophilized. The 4ml portion was hydrolyzed for 24 h at 110°C in 6N HCl and analyzed in a Beckman 12OC autoanalyzer. 0 Molecular weights determined by comparisons with protein standards on NaDS polyacrylamide gels.

DISCUSSION

with the antibody

in immunoelectrophoresis and in microscopy. All the extracted oral proteins have amino acid compositions similar to the fibrous proteins of epidermal keratin. The immunofluorescence study shows that the proteins of the oral stratum corneum are cross-reactive with those of epidermis. But the difference in intensity of fluorescence suggests that they differ in the type or availability of antigenic sites. The differences could be due to differences in primary. secondary, or tertiary structure of the proteins, or due to differences in the type or extent of post-translational modification of the fibrous protein. The keratohyalin granules of all epithelia tested bound the fluorescent antibody suggesting that these granules play a role in keratinization in both oral epithelium and epidermis. Perhaps some common antigenic determinants are localized in keratohyalin but their fate differs during keratinization giving rise to proteins in the oral stratum corneum which differ immunologically from those of epidermis. Brody (1959a), Fukuyama and Epstein (1957) and Odland (1964) have suggested keratohyalin granules as a site for post-translational modification of fibrous protein. Recent immunologic evidence consistent with post-translation modification during keratinization has been presented in studies of foetal rat

situ using fluorescence

We have applied to oral epithelium extraction procedures normally used for the extraction of epidermal keratins (Baden, Bonar and Katz, 1968; Baden and Goldsmith, 1972; Huang et al., 1975; Steinert, 1975). When alterations in techniques were introduced. they were employed on epidermis as a control. Extraction resulted in proteins similar to epidermal keratin in molecular weight and amino acid composition (Baden et al., 1968; Baden and Goldsmith, 1972; Bauer, 1972; Huang et al., 1975; Steinert, 1975; Steinert and Idler. 1975). In addition, these proteins gave a positive reaction with antibody to epidermal keratin. These results, taken together with the morphologic similarity of stratum corneum of oral mucosa and epidermis, suggest that we have extracted oral keratin. Our findings suggest both differences and similarities in the oral and epidermal proteins. Although three major proteins were extracted from cheek, palate and epidermis, the molecular weights of those from the more highly keratinized tissues, palate, skin, as well as tongue (Baratz, 1974) were higher than proteins extracted from cheek epithelium. The extracts gave a reaction of identity with epidermal fibrous protein in double diffusion analysis, but reacted weakly

79

Oral keratin epidermis (Dale et al., 1976), in which it was shown that more antigenic sites are present at 20 days gestation (the time of appearance of stratum corneum) than at 19 days, but the molecular weights of the cross-reacting species are similar at the two times. In contrast, in bovine hoof keratin, Steinert (1975), found no significant differences between the fibrous proteins of the living cell layers and those of the cornified layer except for the formation of disulphide bonds. The major diflerence we found between the proteins of the skin and oral stratum corneum is the apparent absence of the protein rich in histidine and arginine from oral stratum comeum extracts. The function of this protein in epidermal stratum corneum is unknown, but its absence from oral stratum corneum suggests that it may define a functional difference between oral and skin epithelium. One functional difference may be the barrier to water penetration which Adams (1974) found to be less effective in oral mucosa than in skin; on the other hand, the barrier to solute migration, as measured by penetration of horseradish peroxidase or lanthanum nitrate, is similar in nonkeratinized and keratinized oral epithelia and in skin (Elias and Friend, 1975; Squier, 1973). The amino acid composition of the epidermal protein with high histidine and arginine content closely resembles the “histidine-protein” studied by Hoober and Bernstein (1966) as well as that of a keratohyalin preparation (Dale and Stern, 1975b; Sibrack, Gray and Bernstein, 1974). It would be interesting to determine if this protein can be detected in oral keratohyalin granules. 1i high molecular weight protein (110,000) relatively rich in histidine has been identified in both tongue and skin epithelia by Baratz et al. (1975) who pulse-labelled with C3H]-histidine. In our study, the epidermal protein which as a relatively high histidine content has a molecular weight of about 50,000. Differences in age of animals, source of protein and extraction procedures could explain this difference. The fact that we did not detect this protein in oral stratum corneum extracts does not imply that it is absent from the lower layers of the epithelia, merely that it i:; not a major extractable constituent of the stratum comeurn as it is in both newborn and adult epidermis. Keratin is a complex biochemical product of differentiation in epithelia. Ultrastructural observations of mammalian epidermal stratum corneum suggest that keratin is composed of fibrous proteins embedded in a matrix (Brady, 1959a, b). Morphologically, it seems that keratohyalm granules could give rise to the interfilamentous matrix in the stratum corneum. This does not appear to be the case in cheek in which keratohyalin granules are not associated with tonofilaments. We have presented evidence in epidermis that components of the KHG remain in the stratum corneum (Dale and Stern, 1975b). One of these components is the 50,OOOMW band, which has a high histidine content. This protein may be a matrix protein. However, we have not isolated any protein component of either cheek or palate which is an obvious candidate for matrix substance. Thus, it may be the nonfilamentous ma.terial of the stratum corneum which differs significantly from one epithelium to another and contributes to their differing properties and func-

tions, while the fibrous proteins are the components which are basically similar in all keratinized epithelia.

Acknowledgements-This study was supported by USPHS Grant DE-02600, Center for Research in Oral Biology. We wish to thank Ms. Marie Doman for assistance with histologic preparations and Mr. Gaylord Cooper and Ms. Su Yu Ling for assistance with amino-acid analysis.

REFERENCES Adams D. 1974. Penetration of water through human and rabbit oral mucosa in vitro. Archs oral Bik 19. 865-872. Albriaht J. T. and Listaarten M. A. 1962. Observations of the fine structure OF the hamster cheek pouch epithelium. Archs oral Biol. 7. 613-620. Alvares 0. and Meyer J. 1971. Variable features and regional differences in oral epithelium. In: Current Concepts qf the Histology of Oral Mucosa (Edited by Squier C. A. and Meyer J.) Chap. 5, pp. 97-113, Thomas, Springfield, Ill. Baden H. P., Bonar L. and Katz E. 1968. Fibrous proteins of epidermis. J. invest. Derm. 50, 301-307. Baden H. P. and Goldsmith L. A. 1972. The structural protein of epidermis. J. invest. Derm. 59. 66-76. Baratz R. S. 1974. Protein profiles of differentiating rat tongue epithelium. Anat. Rec. 178. 304. Abstract. Baratz R. S., Telser A. and Farbman A. I. 1975. Molecular weights of histidine and cysteine-rich proteins in fetal keratinizing epithelia. J. Ce?/ Biol. 15a. -Abstract. Bauer F. W. 1972. Studies of isolated keratin fractions from mammalian epidermis. Dermatologica 144. 217-228. Bramhall S., Noack N., Wu M., Loewenberg J. R. 1969. A simple calorimetric method for determination of protein. Andyt. Biochem. 31, 146148. Brody I. 1959a. An ultrastructural study on the role of keratohyalin granules in the keratinization process. J. Ultrastruct. Res. 3, 84-104. Brody I. 1959b. The keratinization of epidermal cells of normal guinea pig skin as revealed by electron microscopy. J. Ultrustruct. Res. 2, 482-511. Chen S-Y. and Meyer J. 1971. Regional differences in tonofilaments and keratohyalin granules. In: Current Concepts of the Histology of Oral Mucosa (Edited by Squier C. A. and Meyer J.) Chap. 6, pp. 114-128, Thomas, Springfield, Ill. Dale B. A. and Stern I. B. 1975a. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of proteins of newborn rat skin. I. Cell strata and nuclear proteins. J. invest. Derm. 65, 22G222. Dale B. A. and Stern I. B. 1975b. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of proteins of newborn rat skin. II. Keratohyalin and stratum corneum proteins. J. invest. Derm. 65. 223-227. Dale B. A., Stern I. B., Rabin M. and Huang L-Y. 1976. The identification of fibrous proteins in fetal rat epidermis by electrophoretic and immunologic techniques. J. inoest. Derm. 66, 23G235. Elias P. M. and Friend D. S. 1975. The permeability barrier in mammalian epidermis. J. Cell Biol. 65, 18@191. Fukuyama K. and Epstein W. L. 1967. Ultrastructural autoradiographic studies of keratohyalin granules formation. J. invest. Derm. 49. 595dO4. Gibbins J. R. 1962. An electron microscopic study of the normal epithelium of the palate of the albino rat. Archs oral Bid. 7. 287-295. Hayward A. F., Hamilton A. I. and Hackemann M. M. A. 1973. Histological and ultrastructural observations on the keratinizing epithelia of the palate of the rat. Archs oral Bid. 18. 1041 1057.

80

Beverly

A. Dale, I. B. Stern and J. A. Clagett

Hoober J. K. and Bernstein I. A. 1966. Protein synthesis related to epidermal differentiation. Proc. natn. Acad. sci., U.S.A. 56. 594601. Huang L-Y., Stern I. B., Clagett J. A. and Chi E-Y. 1975. Two polypeptide chain constituents of the major proteins of the cornified layer of newborn rat epidermis. Biochemistry 14. 3573-3580. Kempson S. A. 1974. Ultrastructural observations on the keratohyalin granules of the rat oral epithelium. Archs oral Biol. 19. 101 l-1024. Laurel1 C. B. 1966. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15. 45-52. Odland G. F. 1964. Tonofilaments and keratohyalin. In: The Epidermis (Edited by Montagna W. and Lobitz W. C.1 no. 237-249. Academic Press. New York. Osmanski C. P. and Meyer J. 1967. Differences in the fine structure of the mucosa of mouse cheek and palate. J. inuest. Derm. 48. 309-317. Shimizu T., Fukuyama K. and Epstein W. L. 1974. Partial purification of proteins isolated from mammalian cornified cells. Biochim. biophys. Acta 359. 389400. I

.

.

Sibrack L. A., Gray R. H. and Bernstein I. A. 1974. Localization of the histidine-rich protein in keratohyalin: A morphologic and macromolecular marker in epidermal differentiation. J. invest. Derm. 62. 394405. Squier C. A. 1968. Ultrastructural observations on the keratinization process in rat buccal epithelium. Archs oral Biol. 13. 1445-1451. Squier C. A. 1973. The permeability of keratinized and nonkeratinized oral epithelium to horseradish peroxidase. J. Ultrastruct. Res. 43, 160-177. Stern I. B. and Sekeri-Pataryas K. H. 1972. The uptake of [‘%Z]leucine and [‘4C]histidine by cell suspensions of isolated strata of neonatal rat epidermis. J. invest. Derm. 59, 251-259. Steinert P. M. 1975. The extraction and characterization of bovine epidermal %-keratin. Biochem. J. 149, 3948. Steinert P. M. and Idler W. W. 1975. The polypeptide composition of bovine epidermal %-keratin. Biochem. J. 151, 603-614.

Oral keratin

Fig. 1. Oral epithelium and stratum corneum. 5 pm paraffin-embedded sections. Haematoxylin and eosin. ‘x 400 A. Adult rat buccal epithelium after treatment with 0.25 per cent trypsin for 16 h at 4°C. B. Adult rat palatal epithelium after treatment with 0.125 per cent trypsin for 16 h at 4°C. C-E. Stratum corneum after incubation of trypsinized epithelium in 1 per cent NaDS for 1 h at room temperature. C. Adult cheek. D. Adult palate. E. Newborn skin.

81

82

Beverly A. Dale, I. B. Stern and J. A. Clagett

Fig. 2. Direct immunofluorescence with antibody to epidermal fibrous protein. Sections incubated with normal goat serum then with fluorescent antiserum; controls incubated with nonfluorescent antiserum then with fluorescent antiserum. Blocked controls were photographed under the same conditions for equal or longer exposure times. A. Newborn rat skin. x 1100. B. Newborn rat skin blocked control. x 1100. C. Adult rat palate. ‘,’ 450. D. Adult rat palate blocked control. x 450. E. Adult rat cheek. Y 450. F. Adult rat cheek blocked control. x 450