0022-202X/84/8204 -0381 $02.00/0 THE JOURNAL 0~' INVESTIGATIVE DERMATOLOGY, 82:381 -385, !984 Copyright © 1984 by The Williams & Wilkins Co.

Vol. 82, No.4 Printed in U.S.A.

New Findings on the Proteins of Sebaceous Glands*

MASAAKI !TO, M.D., MEGUMI SUZUKI, M.D., KATSUHIRO MOTOYOSHI, TOMOHIRO MARUYAMA, M.D., AND YosHIO SATO, M.D.

Department of Dermatology, Niigata University Sclwol of Medicine, Niigata, Japan

In order to understand the distribution and concentra­tion of proteins with -SH groups or S -S linkages in sebaceous cells during differentiation and holocrine se­cretion of sebaceous glands, skin specimens from the inner side of ears of New Zealand white rabbits were examined histochemically and ultrastructurally.

DACM (N -[7 -dimethylamino-4-methyl-3-coumarinyl] .maleimide) staining method showed that proteins con­taining -SH groups were present in the cells (cytoplasm and nuclei) in all layers from the peripheral to the ter­minally differentiated cells of sebaceous glands and that proteins containing S-S linkages were present in the terminally differentiated cells and their pyknotic nuclei but not in the peripheral and differentiating cells of sebaceous glands. Lipid droplets in all sebaceous cells contained neither -SH groups nor S-S linkages. Ultra­structurally, the terminally differentiated cells were very electron dense and seemed to be abruptly formed from the differentiating cells that were producing lipid droplets.

These findings indicate that the conversion of -SH groups to S-S linkages of proteins also occurs in seba­ceous glands as in epidermis and hair.

It is well known that the cells of sebaceous glands undergo holocrine secretion, which is characterized by complete cellular death. These sebaceous cells originate from the peripheral cells of the glands, produce lipid droplets in their cytoplasm, and move toward center of the glands in a coordinate fashion [1) . These differentiating cells become condensed and eventually die with breakdown of their cytoplasm, extruding their lipid into the sebaceous ducts, namely by holocrine secretion.

A large number of investigations have been performed on sebaceous glands morphologically, cytochemically, and bio­chemically. The process of holocrine secretion of sebaceous cells has been thought to be caused by an autolytic mechanism by lysosomal enzymes produced in their cytoplasm [2-4]. Cell autolysis, however, does not seem to be the only factor inducing holocrine secretion, because such a sudden change from the differentiating cells to the condensed cells does not seem to be explainable by the autolytic mechanism alone. Therefore, it seems necessary to examine this secretion mechanism from a different view, i.e., the concept of cell keratinization.

Recently, a new histochemical method, DACM (N-[7-di­methylamino-4-methyl-3-coumarinyl]maleimide) staining, has been developed and applied to the study of keratinization [5-

Manuscript received July 18, 1983; accepted for publication Novem­ber 21, 1983.

* This paper was presented in part at the Joint Meeting of The Society for Investigative Dermatology, Inc., and the European Society for Dermatological Research, April 27- May 1, 1983, Washington, D.C.

Reprint requests to: Yoshio Sa to, M.D., Department of Dermatology, Niigata University School of Medicine, Niigata 951, Japan.

Abbreviations: DACM: N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide DTT: dithiothreitol EDT A: disodium ethylenediaminetetraacetate NEM: N-ethylmaleimide TAS: 5 mM Tris acetate buffer containing 0.85% NaCl (pH 6.8)

10]. This method (Ogawa and Taneda method) is very useful to demonstrate the distribution and concentration of -SH groups or S-S linkages of proteins in tissue sections under a fluorescent microscope [5-8]. Since sebaceous glands are one of the appendages of the epidermis, the proteins containing sulfhydryl groups may be distributed in the sebaceous cells as well as in the cells of the epidermis [ 5,6], hair and hair follicle [9], and sweat glands (10] .

The purpose of this study is to investigate the distribution of these proteins in sebaceous glands during holocrine secretion using the DACM staining method combined with light and electron microscopic methods.

MATERIALS AND METHODS

Well-developed sebaceous follicles of the inside skin of ears of male New Zealand white rabbits were biopsied. The biopnied specimens were divided into 3 pieces. The first was immediately frozen with acetone­dry ice, kept in a deep freezer at -4o·c until use, and sectioned into a thickness of 3 !Lm in a cryostat at -3o·c. The second piece was fixed in 10% formalin solution, dehydrated in ethanol and xylene, and embedded in paraffin. Deparaffinized sections of 3·JLm thickness were made in a microtome. The remaining piece was cut into small pieces, double-fixed in 5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 4 hand in 1% Os04 in the same buffer for 1.5 h, block­stained in 1% uranyl acetate in 50% ethanol, dehydrated in graded ethanol solutions and propylene oxide, and embedded in Epon 812. Ultrathin sections were cut in an ultramicrotome (Sorval MT-1), double-stained with 1% uranyl acetate and Reynolds' lead citrate [11). and observed in JEM·T8 and JEM-100S ultramicroscopes.

DACM staining was performed on both frozen and deparaffinized sections according to the previously reported [5- 7,9] and modified method [8,10]. DACM was obtained from Wako Pure Chemical Indus· tries (Tokyo) and N-ethylmaleimide (NEM), disodium ethylenedia­minetetraacetate (EDTA), and dithiothreitol (DTT) from Sigma Chemical Company (St. Louis, Missouri) .

(I) Staining method for -SH groups: Sections were rinsed in cold 5 mM Tris acetate buffer containing 0.85% NaCl (pH 6.8) (TAS) at 4•C for 5 min and incubated in 0.01 mM DACMt in TAS at room temper· ature for 3 min. After the incubation, each section was washed in cold T AS, embedded in glycerine solution, and observed under a fluorescent microscope (model BH-RFL, Olympus Co., Japan). The BG-12 filter ()I 405 nm) was chosen as an exiting filter and B filter as an absorption filter with Y -455 filter as an absorption subfilter. B and Y -455 filters absorb the wavelengths below 500 nm and 455 nm, respectively.

(II) Blocking method for free -SH groups: After incubation in 0.15 M NEM in TAS at 37•c for 15 min for frozen sections and 45 min for deparaffinized ones, each section was washed in cold TAS.:j:

(III) Staining method for S-S linkages: After complete blocking of -SH groups by NEM, each section was washed in cold TAS and incubated in TAS containing 0.5 mM EDTA and 40 mM DTT at 37•c for 3 min to reduce S-S linkages to -SH groups. After the reduction, each section was stained with DACM-TAS solution and observed in the same manner as in (I).

After the -SH or S-S staining with DACM, the deparattinized sections were stained with hematoxylin-eosin to conftrm what struc­tures in the sections contained -SH groups or S-S linkages.

t DACM was stored at -2o·c as 4.0 mM of DACM-acetone solution, which was di luted to a concentration of 0.01 mM of DACM with fresh TAS in each experiment.

:j: Following procedure (II), sections were stained with DACM and observed as in (I). They showed no fluorescence. This confirmed a complete blocking of free -SH groups in those sections by NEM, indicating an accuracy of the S-S distribution shown in (III) .

381

382 ITO ET AL

(!)

---....::- ~~ ,- ...----- ---

FIG 1. -SH and S-8 distribution in the epidermis. Deparaffinized sections of rabbit ear skin. a, DACM staining for -SH groups. b, Hematoxylin-eosin staining in the same section. The membranes of the horny cells (arrowheads) and the cyto~lasm of the living cells (lc) except for their nuclei (long arrows) contam -SH. The nucleoh (small arrows) in the nuclei show a small amount of -SH. -SH fluorescence gradually becomes weaker toward the ~pper. horny ~ell~. c, DACM staining for S-8 linkages. d, Hematoxyhn-eosm stammg m ~he same section. 8-S is not distributed in the living cells (lc) including the1r nuclei (long arrows) but along the membranes of the horny cells (arrowheads). he= horny cells. a and b, X716; c and d, X608.

RESULTS

DACM Staining and Hematoxylin-Eosin Staining

There was no difference between frozen and deparaffinized sections in the distribution pattern and concentration of -SH groups and S-S linkages ~n t~e n?rmal skin, a~ previously reported [10]. The incubatiOn time m NEM solutiOn to block free -SH groups should be prolonged in deparaffinized sections (for 45 min) compared with frozen sections (for 15 min).

The DACM staining of the epidermis showed the same results as previously described [5,6,10) . -SH groups are present moderately in the cytoplasm of the living cells and strongly along the membranous part of the lowest part of the horny cells [5,6,10] (Fig la, b). The living layers are not rich in S-S linkages, but the membranous part of _t~e horny cells ~bows a brilliant fluorescence by DACM stammg for S-S lmkages [5,6,10] (Fig 1c, d). The nuclei of these living cells seem to lac~ both -SH groups and S-S linkages except for th_eir nucleoli, which seem to contain -SH groups but not S-S hnkages [10] (Fig la-d).

Over 100 sebaceous glands were examined by the DACM staining method. In the sebaceous duct ~pi~helium a di~tribu­t ion of -SH groups and S-S linkages stmtlar to that m the epidermis was observed. -SH groups ~ere p~esent in the cells in all layers of the epithelium, but not m cormfied cells released in the duct lumen (Fig 2a, b). 8-S linkages were present only in the cornified cells including ones released in the lumen (Fig 3a b). These cornified cells were very thin and it was almost im,possible to obtain their complete cross-sections; therefore, it could not be confirmed whether their fluorescence showed a membranous pattern or not. The nuclei, including nucleoli, seemed to display no fluorescence by either -SH (Fig 2a, b) or S-S staining (Fig 3a, b) .

The peripheral§ and differentiating cells~ of sebaceous glands

§Germinative cells of sebaceous glands. ~ Sebaceous cells producing lipid droplets.

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FIG 2. -SH distribution in sebaceous glands. Deparaffinized section of rabbit ear skin. a, DACM staining for -SH. b, Hematoxylin-eosin staining. -SH is present in the cytoplasm and nuclei (long arrows) of the peripheral and differentiating cells. A strong -SH fluorescence is present in the condensed cells (arrowheads) including their pyknotic nuclei. The cytoplasm of the sebaceous duct epithelial cells (sd) includ­ing the cornified cells (thick arrows) contains -SH, but their nuclei (small arrows) and the cornified cells (stars) released into the duct lumen (L) do not. X530.

showed a moderate fluorescence in their nuclei and cytoplasm (except for lipid droplets of the latter cells) by DACM staining for -SH groups (Fig 2a). These fluorescent structures could be conftrmed in Fig 2b. A brilliant fluorescence of -SH groups by DACM staining was found in cytoplasm and nuclei of the condensed cells (Fig 2a), which were located in the center of sebaceous glands and the cytoplasm of which was homogene­ously stained with eosin (Fig 2b).

On the other hand, 8 -S linkages were almost absent in the peripheral and differentiating layers, except for the slightly condensed cells with pyknotic nuclei of the latter layer just before a strong condensation (Fig 3a, b) . The condensed cells however, possessed a large amount of S-S linkages in their cytoplasm and nuclei as shown by a strong fluorescence in Fig 3a. These cells were located in the central parts of sebaceous glands and homogeneously stained with eosin (Fig 3b).

Lipid droplets in all sebaceous cells contained neither -SH groups (Fig 2a) nor S-S linkages (Fig 3a).

Ultrastructural Observations

Over 30 complete sebaceous glands were ultrastructurally observed. The serial morphologic changes of the nuclei of sebaceous cells during the differentiation were observed: The peripheral cells, which lacked lipid droplets, had flattened nuclei (Fig 4), while the differentiating cells with a large number

April1984

FIG 3. S-S distribution in sebaceous glands. Deparaffinized se~tion of rabbit ear skin. a, DACM stammg for 8-8. b, Hematoxylin-eosin staining. 8-8 is pres­ent in the condensed cells (arrowheads) and the pyknotic nuclei (long arrows) of differentiating cells. The cornified cells (thick arrows) of the duct epithelium, including released ones (stars) into the lumen (L), contain 8-8, but the nuclei (small arrows) and cytoplasm of noncor­nified cells of the duct epithelium (sd) do not. x670.

FIG 4. Electron micrograph of a sebaceous gland of rabbit ear skin. Uranyl acetate- lead citrate staining. The peripheral (germinative) cells (P) possess ellipsoidal nuclei (NuP). The differentiating cells (D) are producing many lipid droplets and the nucleus indicated by NuD shows an irregular shape. T = terminally differentiated (condensed) cell; L = sebaceous duct lumen.

PROTEINS OF SEBACEOUS GLANDS 383

of lipid droplets had large ellipsoidal or small, irregularly shaped nuclei. The more differentiated cells seemed to have the latter type of nuclei (Fig 4). All of these nuclei are rich in -SH groups from the findings of DACM (see above) .

The cytoplasm of the peripheral and differentiating cells, where -SH groups were richly seen by DACM staining (see above), contained free ribosomes and a small amount of tono­filaments. The tonofilaments were located mainly in the pe­ripheral part of their cytoplasm and seemed to increase gradu­ally in amount with the differentiation of the sebaceous cell (Figs 5, 6). No cementsomes [12), which are produced in epi­dermal keratinocytes before keratinization, were seen in these sebaceous cells.

The terminally differentiated cells** of sebaceous glands, which light microscopically appeared as condensed cells by hematoxylin-eosin staining (Figs 2b, 3b ), seemed to be formed abruptly by cellular condensation from the differentiating cells and had homogenization of cytoplasm and nucleus (Figs 4-6) . This abrupt change was very similar to that of epidermal keratinocytes when keratinization took place between granular and horny cells. Occasionally, between the differentiating and terminally differentiated cells of sebaceous glands, intermedi­ately electron-dense cells with slightly pyknotic nuclei were observed. In the terminally differentiated cells of sebaceous glands, which corresponded to horny cells of the epidermis, there were neither keratin filaments nor marginal bands (Fig 6) as in those of epidermal horny cells or nail cells [13]. Very electron-dense and irregularly shaped small pyknotic nuclei still remained in t he terminally differentiated cells of sebaceous glands (Fig 5), whereas there were no nuclei in the epidermal horny cells. The fairly electron-dense cytoplasm and nuclei of the terminal cells of sebaceous glands are very rich in both -SH groups and S-S linkages from the histochemical data described above. The dense, homogenized cellular components partially and entirely became disintegrated into a nonstructural less dense mass, which was discharged into the lumen of the sebaceous duct (Figs 4, 5) and then into the hair canal.

-SH and S-S distributions in the epidermis, sebaceous duct epithelium, and sebaceous glands are summarized in Table I.

** Although the final form of sebaceous cells is an oily liquid, "terminally differentiated cells" are called the final cells which still morphologically keep their cell shapes.

384 lTO ET AL

FIG 5. Abrupt change of sebaceous cells. Enlargement of the area indicated by T in the central part of the sebaceous gland in Fig 4. Uranyl acetate-lead citrate staining. The cells (D) with sebaceous differentiation just before cellular condensation contain cytoplasmic organelles such as tonofilaments (arrowheads), mitochondria (arrows), and many lipid droplets (ld); whereas the terminally differentiated cells (Tl, T2, T3) are composed of fragmented cytoplasm and pyknotic nuclei (NuT), both of which are homogeneously very electron dense, and their lipid droplets (ldT) fuse to each other and show irregular shapes. The cells Tl and T2 still keep their cellular shapes, while the cell T3 becomes partly disintegrated into a less dense mass (asterish), facing the sebaceous duct lumen. X6300.

DISCUSSION

It has been believed that lysosomal enzymes may play an important role in the holocrine secretion of sebaceous glands which requires cellular lysis. Lysosomal enzymes were detected histo- and cytochemically [2,3] and biochemically [4] in the sebaceous cells; the physiologic cell autolysis by lysosomal enzymes may be one of the mechanisms that causes the holo­crine secretion. We believe that a mechanism similar to kera­tinization is involved in sebaceous glands.

Curtis and Cowden reported the presence of -SI;i groups only in the developing glandular cells, using preputial (modified sebaceous) glands of mice [8]. However, as shown in the current study, the cells in all layers, including the condensed (termi­nally differentiated) cells, of sebaceous glands are rich in -SH groups. On the other hand, S-S linkages are practically absent in the peripheral and differentiating cells of sebaceous glands but richly present in the condensed cells as shown in Fig 3a. These findings indicate that -SH groups of proteins in the sebaceous cells are converted to S-S linkages during the ter­minal cellular differentiation. This phenomenon is very similar to the -SH to S-S conversion during epidermal keratinization [5,6], although the final product of sebaceous cells (lipid) is much different from that of epidermal keratinocytes (keratin). Interestingly, the conversion takes place in the nuclei and cytoplasm (except for lipid droplets) of sebaceous cells, whereas in the epidermal keratinocytes such conversion takes place only along the cell periphery [5,6] . The -SH groups, which are present in largest amount in the lowest cornified cells of the epidermis, gradually decrease in amount toward the upper horny cells as shown in Fig la [5,6]; whereas in sebaceous glands condensed cells with -SH and S-S eventually break down to a less dense amorphous mass (Figs 4, 5).

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FIG 6. Abrupt cytoplasmic change between differentiating and ter­minally differentiated cells. Sebaceous gland in rabbit ear skin. Uranyl acetate-lead citrate staining. The cytoplasm of the terminally differ­entiated cell (T) is very electron dense and contains neither keratin filaments nor marginal bands. Arrowheads = cell membrane of the differentiating cell (D); f = tonofilaments; star= ribosomes. X75,000.

TABLE I. -SHand S-S distributions in the epidermis, sebaceous duct epithelium, and sebaceous glands

Epidermis Living cells

Cytoplasm Nucleus Nucleolus

Lower horny cells Cellular envelope Cytoplasm

Upper horny cells Cellular envelope Cytoplasm

Sebaceous duct epithelium Living cells

Cytoplasm Nucleus

Cornified cells Sebaceous glands

Peripheral and differentiating cells Cytoplasm Nucleus

Terminally differentiated (condensed) cells Cytoplasm Nucleus

+ = present; - = absent.

-SH S-S

+

+

++ ++

+-- ++

+

+-- ++

+ +

++ ++ ++ ++

Ultrastructurally, Brandes et al described in rat that the partial cytoplasmic lysis (condensation) of a sebaceous cell might occur during cellular death [2]. In the present ultrastruc­tural study, however, no feature of such partial condensation of a differentiating sebaceous cell was seen in 30 specimens examined. The differentiating cells seem to become homoge­neously electron dense. Intermediately electron-dense cells are occasionally present between the differentiating and terminally differentiated cells. This serial structural change of sebaceous cells also resembles that of epidermal keratinocytes during keratinization.

April1984

The epidermal keratinocytes in the living layers also produce lysosomal enzymes, which are thought to play a role in the keratinization process [14,15]. As shown in recent studies [16,17], lysosomal acid proteinases in the granular layer may activate epidermal transglutaminase which catalyzes the for­mation of intermolecular ~- ( ')'-glutamyl)lysine bonds. Other than the ~-(1'-glutamyl)lysine bonds, the formation of S-S linkages from -SH groups in proteins are also important for epidermal keratinization [5,6]. This reaction seems to be cata­lyzed by sulfhydryl oxidase, which has recently been found in rat epidermis [18]. The cornified envelopes of the epidermis contain both ~ -(1' -glutamyl)lysine [19-21] and S-S [20,21] cross-linkages. It is unknown whether or not the -SH to S-S conversion of cellular proteins of sebaceous cells observed in t he present study is also catalyzed by these enzymes since no biochemical data are available. The present study demonstrated histochemically that the -SH to S-S conversion occurred and suggested that it might be an important cytochemical change in the sebaceous cells before holocrine secretion.

On holocrine secretion, the terminally differentiated (con­densed) cells finally lose the integrity of their cellular boundary, discharging their cellular content (lipid) into a sebaceous duct lumen as shown in Figs 4 and 5. In the current study, the mechanism of such breakdown of the terminally differentiated cells, which contain S-S linkages probably as a cross-linking structure of cell proteins, is still unclear. Since in t he epidermis the envelope of the upper horny cells may also be broken and t heir cell content (keratin filaments) may be released to outside of the cells, a common mechanism of the breakdown of the cross-linked proteins may be present in both epidermal horny cells and sebaceous condensed cells. To clarify this problem further studies are required.

Another new finding noted in the current study is the pres­ence of -SH groups in the nuclei of sebaceous cells. Several studies on various tissues using DACM staining [5-8,10] showed that nuclei lacked both -SH groups and S-S linkages, although -SH groups were shown to be present in a moderate amount in nucleoli [7,10] and in a small amount in the mitotic chromosomes and condensed chromatin [8]. Apparently seba­ceous cells are different from others in this respect , although it was difficult to demonstrate fluorescence of nucleoli because a brilliant -SH fluorescence of nuclei masks their nucleoli. Bio­chemically, it is known that a fraction of extracted histones [22,23] and some other nuclear nonhistone proteins [24-26] contain cysteine as an amino acid component. There may be variations in amount of -SH groups in nuclear proteins between different tissues.

Finally, the use of deparaffinized tissue sections for DACM staining with our modification has several advantages over the frozen tissue sections [10]; (i) fine and sharp fluorescence images can be obtained with deparaffinized sections compared with frozen sections, (ii) the deparaffinized sections are much better stained with hematoxylin-eosin with a sharper definition after DACM staining, and (iii) old specimens stored on file can be utilized.

REFERENCES 1. Plewig G, Chris top hers E: Renewal rate of human sebaceous glands.

Acta Derm Venereol (Stockh) 54:177- 182, 1974 2. Brandes D, Bertini F, Smith EW: Role of lysosomes in cellular

lytic processes. II. Cell death during holocrine secretion in se-

PROTEINS OF SEBACEOUS GLANDS 385

baceous glands. Exp Mol Pathol 4:245-265, 1965 3. Bell M: A comparative study of the ult rastructure of t he sebaceous

glands of man and other primates. J Invest Dermatol 62:132-143, 1974

4. Im MJC, Hoopes JE: Enzymes of carbohydrate metabolism in normal human sebaceous glands. J Invest Dermatol62:153- 160, 1974

5. Ogawa H, Taneda A, Sekine T, Kanaoka Y: Distribution and characteristic mobilit ies of sulfhydryl residues and cross- linkages during keratinization process. New histochemical staining method. Jpn J Dermatol 88:525-528, 1978

6. Ogawa H, Taneda A, Kanaoka Y, Sekine T: The histochemical distribution of protein bound sulfhydryl groups in human epi­dermis by the new staining method. J Histochem Cytochem 27:942-946, 1979

7. Ogawa H, Taneda A: Characteristic distribution of sulfhydryl groups in human epidermal cancer by t he new histochemical staining method. Arch Dermatol Res 264:77-81, 1979

8. Curt is SK, Cowden RR: Demonstration of sulfhydryl and disulfide groups by a fluorescent maleimide procedure. Histochemistry 68:23- 28, 1980

9. Taneda A, Ogawa H, Hashimoto K: The histochemical demonstra­tion of protein-bound sulfhydryl groups and disulfide bonds in human hair by a new staining method (DACM staining). J Invest Dermatol 75:365-369, 1980

10. Tanizawa M, Ito M, Maruyama T, Sato Y: Distribution of -SHand S-S in epidermis and eccrine glands: comparison of the findings based on the routine and N-(7-dimethylamino-4-methyl-3-cou­marinyl)maleimide (DACM) stainings. Biomed Res (Tokyo) 4:41-50, 1983

11. Reynolds ES: The use of lead cit rate at high pH as an electron­opaque stain in electron microscopy. J Cell Biol17:208-212, 1963

12. Hashimoto K: Cementsome, a new interpretation of the membrane­coating granule. Arch Dermatol Forsch 240:349-364, 1971

13. Hashimoto K: The marginal band. A demonstration of the thick­ened cellular envelope of t he human nail cell wit h the aid of lanthanum staining. Arch Dermatol 103:387-393, 1971

14. Wolff K, Schreiner E : Epidermal lysosomes. Electron microscopic cytochemica l studies. Arch Dermatol 101:276-286, 1970

15. Lazarus GS, Hatcher VB, Levine N: Lysosomes and the skin. J Invest Dermatol 65:259-271, 1975

16. Negi M, Matsu i T, Ogawa H: Possible activation mechanisms of transglutaminase in epidermis. Arch Dermatol Res .271:101- 105, 1981

17. Negi M, Matsui T , Ogawa H: Mechanism of regulation of human epidermal transglutaminase. J Invest Derma to! 77:389-392, 1981

18. Ta kamori K, Thorpe JM, Goldsmith LA: Skin sulfhydryl oxidase. Purification and some properties. Biochim Biophys Acta 615: 309-323, 1980

19. Rice RH, Green H: The cornified envelope of terminally differen­tiated huma n epidermal keratinocytes consists of cross-linked protein. Cell 11:417-422, 1977

20. Hirotani T , Manabe M, Ogawa H, Murayama K, Sugawara T: Isolation and characte rization of horny cell membrane. Arch Dermatol Res 274: 169-177, 1982

21. Ogawa H, Manabe M, Hirotani T, Takamori K, Hattori M: Com­parative studies of the marginal band and plasma membrane of the epidermis. (Chemical structures of the marginal band), Nor­mal and Abnormal Epidermal Differentiation. Edited by M Seiji, IA Bernstein. Tokyo, Univ of Tokyo Press, 1983, pp 265-276

22. Shimada T, Okihama Y, Murata C, Shukuya R: Occurrence of Hl "­like protein and protein A24 in t he chromatin of bullfrog eryth­rocytes lacking histone 5. J Bioi Chern 256:10577-10582, 1981

23. Lattman E, Burlingame R, Hatch C, Moudrianakis EN: Crystalli ­zation of t he tetramer of histones H3 and H4. Science 216:1016-1018, 1982

24. James GT, Yeoman LC, Matsui S, Goldberg AH, Bush H: Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synt hesis. Biochemistry 16:2384- 2389, 1977

25. Brown E, Goodwin GH, Mayers ELV, Hasting JRB, Johns EW: Heterogeneity of proteins resembling high-mobility-group pro­tein HMG-T in t rout testes nuclei. Biochem J 191:661-664, 1980

26. Conner BJ , Comings DE: Isolation of a non-histone chromosomal high mobility group protein from mouse liver nuclei by hydro­phobic chromatography. J Bioi Chern 256:3283-3291, 1981