Measurement of the transit time for cells through the epidermis and stratum corneum of the mouse and guinea-pig

Abstract A new approach to determine the transit time through the epidermis is presented, involving a gentle washing of the skin surface to collect the loosely attached surface corneocytes. This, it is believed, will be less likely to stimulate the system than tape-stripping or scraping. Radioactively labelled thymidine and iododeoxyuridine have been used to label cells in the basal layer and various labelled amino acids (glycine, cystine and methionine) have been used to label the metabolically viable cell layers (up to and including the granular layer). The resulting changes in surface radioactivity levels have been interpreted to provide a basal to surface transit time of 8–9.5 days for hairless and haired mouse epidermis and about 13.5 days for guinea-pigs. The basal to granular layer transit time, which probably includes some basal layer residence time, is about 4.5 days in the mouse and 8 days in the guinea-pig. The granular to surface time in mice is about 5 days. The results also suggest that when nuclear and cytoplasmic organelles are degraded in the granular layer, material is released that can diffuse rapidly through the stratum corneum to the surface. Some of this can be shown by chromatography to be thymidine. Hence, the stratum corneum is pervious to molecules such as nucleosides. This rapid diffusion outwards through the skin can also be detected shortly after injecting [¹²⁵I]-iododeoxyuridine.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.     

Darier's disease: from dyskeratosis to endoplasmic reticulum calcium ATPase deficiency

The skin is the body's largest organ and has an essential barrier protective function against physical, chemical, and pathogen aggressions and prevents fluid loss. The outer layer of the skin, known as the epidermis, plays a key role in this protection, through a tightly regulated differentiation programme from basal keratinocytes to the stratum corneum at the skin surface. During this process, keratinocytes from the base of the epidermis undergo major morphological and functional changes during their migration through the spinous and granular layers, to become terminally differentiated corneocytes which will be shed from the skin's surface. The role of extracellular Ca2+ in cell-to-cell adhesion and in epidermal differentiation was known to be important, but the identification of the sarco/endoplasmic reticulum Ca2+ transport ATPase (ATP2A2) as the defective gene in a rare genetic skin disease known as Darier's disease, came as a surprise and shed light on the key role of Ca2+ signaling in the homeostasis of the epidermis.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Epidermal cell proliferation. I. Changes with time in the proportion of isolated, paired and clustered labelled cells in sheets of murine epidermis

A new technical approach to analysing labelled cells in sheets of epidermis is presented. The changes in the proportion of isolated single labelled cells, paired or clusters of 3, 4, or more than 4, labelled cells in sheets of epidermis from the back of the mouse have been analysed at various times up to 500 h after 3HTdR administration at either 03.00 h or 15.00 h. The technique is not dependent on the relative number of labelled cells (i.e. the labelling index) but on the spatial distribution of labelled cells. The data cannot be adequately explained on the basis of a simple homogeneous stem cell population in the basal layer but can be better understood on the basis of an hierarchical stem cell-dividing transit proliferative model. The data are consistent with an average cell cycle time of about 100 h but there are suggestions of considerable cell kinetic heterogeneity. The data also suggest that the amount of lateral cell movement within the basal layer is small. The results may suggest that some stem cells either loose label in a manner similar to that suggested by Cairns (1975) i.e. through a process of selective segregation of their DNA strands, or that they have an extremely short S phase duration as postulated earlier (Potten et al. 1982). The present data have been extensively mathematically modelled in an accompanying paper. The model which best fits all the data is an hierarchical scheme with three cell divisions in the transit population but some branches of the lineage may be prematurely terminated by the early production of post-mitotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation in the epidermis of chelonians and growth of the horny scutes

The proliferation of the epidermis in soft skin, claws, and scutes of the carapace and plastron in the tortoise (Testudo hermanni) and the turtle (Chrysemys picta) were studied using autoradiographic and immunocytochemical methods. During the growing season, a basal keratinocyte in the epidermis of soft skin and claws takes 5-9 days to migrate into the corneous layer. In the tortoise, during fall/winter (resting season) a few alpha-keratin cells are produced in soft epidermis and hinge regions among scutes and occasional beta-keratin cells in the outer scute surface. When growth is resumed in spring (growing season), cell proliferation is intense, mainly around hinge regions and tips of marginal scutes. No scute shedding occurs and numerous beta-keratin cells are produced around the hinge regions, while alpha-keratin cells disappear. Beta-cells form a new thick corneous layer around the hinge regions, which constitute the growing rings of scutes. Beta-keratin cells produced in more central parts of scutes maintain a homogeneous thickness of the corneous layer along the whole scute surface. In the turtle, a more complicated process of scute growth occurs than in the tortoise. At the end of the growing season (late fall) the last keratinocytes formed beneath the old stratum corneum of the outer scale surface and hinge regions produce more alpha- than beta-keratin. These thin alpha-keratin cells form a scission layer below the old stratum corneum, which extends from the hinge regions toward the center of scutes and the tip of marginal scutes. In the resting season (fall/winter) most cells remain within the germinative layer of the carapace and plastron and a few alpha-cells move in 7-9 days into the corneous layer above hinge regions. In the following spring/summer (growing season) a new generation of beta-keratin cells is produced beneath the scission layer from the hinge region and more central part of the scutes. The epidermis of the inner surface of scutes and hinge regions contains most of the cells incorporating thymidine and histidine, while the remaining outer scute surface is less active. It takes 5-9 days for a newly produced beta-cell to migrate into the corneous layer. These cells form a new corneous layer that extends the whole scute surface underneath the maturing scission layer. The latter contains lipids and eventually flakes off, determining shedding of the above outer corneous layer in late spring or summer.     

In vitro measurement of the mechanical properties of skin by nano/microindentation methods

The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

A histoquantitative study of the effect of maternal hypervitaminosis A on the differentiation of the foetal mouse skin.

The skin of albino mouse foetuses aged 13, 15, 17, 19 and 21 days was studied histologically and quantitatively. The skin of foetuses aged 21 days after maternal hypervitaminosis A, was compared with that of 21 days controls. On the 13th day, the epidermis consisted of one layer of cuboidal cells. The stratum intermedium appeared on the 15th day, the stratum granulosum on the 19th day and the stratum corneum on the 21st day of intrauterine life. The quantitative study showed that although the epidermis increased more rapidly in thickness in the interval between the 13th and 17th day than in the subsequent 4 days, yet in the latter period differentiation of the stratum granulosum and corneum took place. On the other hand, the rate of proliferation of the epithelial cells concerned in the follicle formation was more rapid in the last two days of intrauterine life than in any previous prenatal stage. After maternal hypervitaminosis A, the whole thickness of the epidermis was reduced by 50% and the dermis showed an oedematous appearance. The hair follicle primordia showed a decreased volume.     

Changes in the proliferation rate of mouse epidermis after irradiat-on continuous labelling studies.

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiography. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).     

Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes

Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.     

Characterization of label-retaining cells in the epidermis of a human skin equivalent

Abstract. The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast-populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long-term labelling of the cells with tritiated thymidine ([³H]dT), followed by irradiation with ultraviolet light. [³H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m² UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m². The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label-retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow-cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.     

CHANGES IN THE PROLIFERATION RATE OF MOUSE EPIDERMIS AFTER IRRADIATION: CONTINUOUS LABELLING STUDIES

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiographs. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and 85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).     

Biological and physical factors influencing the successful engraftment of a cultured human skin substitute

Skin tissue may be engineered in a variety of ways. Our cultured skin substitute (Graftskin, living skin equivalent or G-LSE), Apligraftrade mark, is an organotypic culture of skin, containing both a "dermis" and "epidermis." The epidermis is an important functional component of skin, responsible for biologic wound closure. The epidermis possesses a stratum corneum which develops with time in culture. The stratum corneum provides barrier function properties and gives the LSE improved strength and handling characteristics. Clinical experience indicated that the stratum corneum might play an important role in improving the clinical utility of the LSE. Handling and physical characteristics improved with time in culture. We examined the LSE at different stages of epidermal maturation for barrier function and ability to persist as a graft. LSE grafted onto athymic mice before significant development of barrier function did not withstand bandage removal at 7 days postgraft. LSE grafted after barrier function had been established in vitro were able to withstand bandage removal at day 7. Corneum lipid composition and structure are critical components for barrier function. Media modifications were used in an attempt to improve the fatty acid composition of the stratum corneum. The barrier developed more rapidly and was improved in a serum-free, lipid-supplemented condition. Lipid lamellar structure was improved with 10% of the stratum corneum exhibiting broad-narrow-broad lipid lamellar arrangements similar to human skin. Fatty acid metabolism was not appreciably altered. Barrier function in vitro was 4- to 10-fold more permeable than human skin. Epidermal differentiation does not compromise engraftment or the wound healing ability of the epidermis. The stratum corneum provides features beneficial for engraftment and clinical use. (c) 1996 John Wiley & Sons, Inc.     

Different populations of pig epidermal cells: isolation and lipid composition.

Preparations representing populations of (a) basal and spinous cells, (b) granular cells, and (c) stratum corneum cells were obtained by successive treatments of epidermal slices from pig skin with dilute buffered trypsin solutions. Total lipids accounted for about 8% of the cell dry weight in each of the three populations. Phospholipids, which predominated in the basal and spinous cells, accounted for only 21% of the total lipids in the granular cells and less than 0.1% in the stratum corneum. The latter cells contained more cholesterol (23% of total lipid) than either the granular cells (18%) or the basal and spinous cells (8%). The proportion of ceramide was also much higher in the stratum corneum (17%) and granular cells (9%) than in the basal and spinous cells (1%). The relative amounts of glycosphingolipid (glucosylceramide) and cholesteryl sulfate in the total lipids of stratum corneum cells were less than half those in the granular cells and basal and spinous cells. A novel phospholipid was a major component (26% of total) of the phospholipids from granular cells. The compound, which was partially characterized, contained phosphorus, fatty acids, and glycerol (molar ratio 1:3:2) and appeared to be a neutral derivative of phosphatidic acid.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6–9 h lag period. A number of putative precursor HRPs in the 70–300 K molecular weight range were initially identified using short pulse labelling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [³H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100°C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.