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The synthesis of specific proteins in adult mouse epidermis during phases of proliferation and differentiation induced by the tumor promoter TPA, and in basal and differentiating layers of neonatal mouse epidermis.

Mouse epidermis can be induced to undergo sequential waves of proliferation and keratinization by treatment with the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate). This model system offers a unique possibility to study the synthesis of differentiation-specific proteins in the epidermis. During these studies it could be shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that several previously undetected proteins appear in the epidermis during the phase of differentiation. These have been fractionated according to solubility in 1 M phosphate buffer, pH 7.0, or 0.1 M sodium citrate buffer, pH 2.6. Of the soluble proteins, a band of approximate molecular weight 13,500 (band 1) appears early during the differentiative phase (24 hr after treatment with TPA). Another previously undetected protein of approximate molecular weight 27,000 (band 2) appears in the soluble fraction after 48 hr, at a time when many of the typical manifestations of increased keratinization can be observed. Both of these soluble proteins, although not detectable in normal adult mouse epidermis, are shown to be present in newborn mouse epidermis, which demonstrates histologically a well-developed layer of keratinizing and cornified cells. In addition, by separation of neonatal and TPA-treated adult mouse epidermis into "basal" and "cornified" layers, it could be demonstrated that these two proteins are localized exclusively in the upper layer of the epidermis.

Animals

Epidermotropically metastatic malignant melanoma. Differentiating malignant melanoma metastatic to the epidermis from malignant melanoma primary in the epidermis.

In four instances, metastases to epidermis from primary cutaneous malignant melanomas at different sites showed histological features similar to those of cutaneous malignant melanoma primary in the epidermis. In these metastases, atypical melanocytes were present within the epidermis and in the upper part of the dermis much as in primary cutaneous malignant melanoma. Therefore, the presence of atypical melanocytes within the epidermis is not in itself an absolute criterion of malignant melanoma primary in skin. Nor does that finding absolutely deny malignant melanoma metastastic to the skin. Features that may enable histologic differentiation of epidermotropically metastatic malignant melanoma from primary cutaneous malignant melanoma are emphasized.

Adult

Prostaglandins and cyclic AMP in epidermis. Evidence for the independent action of prostaglandins and adrenaline on the adenyl cyclase system of pig and human epidermis, normal and psoriatic.

Prostaglandins E1 and E2 stimulate cyclic AMP accumulation in pig epidermis and in human epidermis from patients with psoriasis. Prostaglandins A1,A2 and F2alpha are relatively ineffective. The fact that this stimulation is not inhibited by a beta-blocker (propranolol) and that the stimulation by prostaglandin E2 and adrenaline is additive indicates that each drug acts independently on the epidermal adenyl cyclase system. In other words, prostaglandins E1 and E2 act on a site other than the beta-receptor of adenyl cyclase in epidermis. The stimulation by prostaglandins E1 and E2 is not additive; hence they probably act on the same site. Concentrations of prostaglandin E above 3X10(-7) M are effective in causing stimulation. This concentration may be within the physilogical range and the contribution of endogenous prostaglandin levels in the control of intracellular cyclic AMP levels cannot be disregarded.

Adenylyl Cyclases

Ultrastructural studies of nuclei and nucleoli in psoriatic epidermis, compared with those in normal epidermis.

Quantitative and qualitative differences of the nuclear and nucleolar structures in normal and psoriatic (involved and uninvolved) epidermis were revealed with the electron microscope. The size of nuclei and nucleoli was increased, the frequency of nuclear bodies was increased, and the relative amount of heterochromatin was decreased in involved skin compared to normal or uninvolved skin. There was no significant difference between normal skin and uninvolved skin of psoriasis. In addition to these changes, multiple fibrillar centers within nucleoli and small masses of chromatin in the epidermal nucleoplasm were observed very frequently in involved skin, while rarely or not at all in normal and uninvolved skin. These results suggest that involved psoriatic keratinocytes have increased metabolic activity and abnormal protein synthesis, having undergone gene derepression.

Adolescent

Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis.

Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in lesional epidermis. The concentrations of prostaglandins E2 and F2alpha, free arachidonic acid, and 12L-hydroxy-5,8,10,14-eicosatetrawnoic acid in specimens of uninvolved and involved epidermis of psoriasis were measured with deuterium-labeled carriers and multiple ion analysis. Snap frozen specimens contained: 1.4 +/- 0.4 mug/g (wet weight) of arachidonic acid in uninvolved in contrast to 36.3 +/- 16.7 mug/g in involved epidermis (P = 0.015); less than 0.05 +/- 0.01 mug/g of hydroxyeicosatetraenoic acid in uninvolved in contrast to 4.1 +/- 1.9 mug/g in involved epidermis (P = 0.015); 23.6 +/- 5.0 ng/g of prostaglandin E2 in uninvolved in contrast to 33.1 +/- 5.7 ng/g in involved epidermis (P less than 0.01); and 21.0 +/- 4.4 ng/g of prostaglandin F2alpha in uninvolved in contrast to 39.0 +/- 5.9 ng/g in involved epidermis (P less than 0.01). The arachidonic acid and hydroxyeicosatetraenoic acid levels in involved epidermis were strongly correlated (r = 0.97). The increased levels of arachidonic acid and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid in involved epidermis may have diagnostic and pathophysiological importance.

Arachidonic Acids

Possible defects in triacylglycerol and phosphatidylcholine metabolism in psoriatic epidermis.

Biopsies of normal skin, psoriatic lesions and the adjacent psoriatic skin were incubated with I-14C-acetate, 32P-orthophosphate and U-14C-glycerol. Total incorporation of I-14C-acetate into psoriatic lesions (17 samples) was 50% higher than in the adjacent uninvolved epidermis (9 samples) and 120% higher than into normal epidermis (10 samples). In the psoriatic lesion a much higher proportion of the total incorporation was into the neutral lipids and was due mainly to a very high incorporation of I-14C-acetate into the triacylglycerols. The I-14C-acetate incorporated into the phospholipids and especially phosphatidylcholine was proportionally much less in the psoriatic lesion and uninvolved psoriatic epidermis than in normal epidermis even though the incorporation of 32P-orthophosphate and U-14C-glycerol, both representing de novo synthesis, into the phospholipids in the psoriatic lesions and uninvolved epidermis were higher (four-fold in lesion) than in normal epidermis. Our findings (1) are evidence for a much increased triacylglycerol synthesis in psoriatic epidermis which would account for the long-known observation of lipid droplet accumulation in psoriatic cells; (2) suggest that in psoriatic epidermis there is a defect in phospholipid metabolism mainly involving phosphatidycholine and the deacylation (phospholipase A)-reacylation (phospholipid acyltransferase) cycle for fatty acid transfer.

Acetates

Epidermal-dermal tissue interactions between mutant and normal embryonic back skin: site of mutant gene activity determining abnormal feathering is in the epidermis.

The site of the scaleless gene's activity in the development of abnormal feathers was determined by reciprocally recombining epidermis and dermis between normal and scaleless chick embryos and culturing the recombinants for seven days on the chorioallantoic membrane. When recombined with a common dermal source, feather development is enhanced by scaleless high line as compared to scaleless low line epidermis. Against a common responding tissue, 7-day normal back epidermis, significant differences were not found in feather inducing ability between normal, scaleless high line and scaleless low line dermis. It was concluded that, in relation to abnormal feathering, these tissue interactions reveal that the site of the scaleless gene's activity is the epidermis. A model of tissue interaction in the development of normal and abnormal feathers is presented. According to the model, the focus of the scaleless mutation and the genes accumulated by selection for high or low feather numbers is the epidermis, the effect being that the reactivity of the epidermis to dermal stimuli is altered. Subsequently, the epidermis controls the morphogenetic organization of the dermis. The scaleless dermis is presumed to contain normal positional information for the determination of feather structure and pattern.

Animals

Demonstration of giant and anular nexus in the psoriatic epidermis.

In the psoriatic skin, nexus were most prominent in the upper layers of the viable epidermis, using the Alcian blue-Lanthanum technique. There, they reached double and manifold the length (giant nexus) of those in normal epidermis. According to the villous transformation of the epidermal cell surface in psoriasis, the nexus showed a great variation in their shape. Undulating nexus were seen beside invagination--and anular nexus. On ther other hand, nexus were lacking in the deepest layers of the psoriatic epidermis in contrast to normal epidermis. From these findings it seems that the intercellular communication by specialized low resistant junctions is reduced in the deepest layers of the psoriatic epidermis. In the upper epidermal layers, however, the extensive formation and the variable configuration of the nexus express a high synthetic activity and a well developed intercellular information in these areas of the psoriatic epidermis.

Epidermis

The phospholipid pattern in the involved and the uninvolved psoriatic epidermis.

The phospholipid pattern of the involved and the uninvolved epidermis from 48 psoriatic patients and of the normal epidermis from 23 healthy controls was determined by thin-layer chromatography. A higher amount of total phospholipids was found only in the involved psoriatic epidermis, whereas significant alterations in the phospholipid pattern were observed in the psoriatic lesion and in the lesion-free epidermis. Namely, the decrease of phosphatidylserine and the increase of phosphatidylinositol were identically present in both the involved and the uninvolved psoriatic epidermis. It seems likely that these alterations in the phospholipid pattern in psoriasis may be related to subclinical alterations of the epidermis in this disease.

Adult

Ultrastructure of lineus ruber (Rhyncocoela) epidermis.

Recent evidence indicates that nemertean epidermis is capable of absorbing certain organic solutes from sea water via mediated transport mechanisms, as well as secreting mucoid substances. Morphological studies suggest that these functions may be restricted to distinct epidermal cell populations. Mucous secretion at the free surface of the epidermis is the result of synthesis and release activites of cells in both the epidermis and dermis (cutis). Secretion of dermal origin passes through the epidermis to the worm's exterior in slender cytoplasmic process (process (canaux d'evacuation) in the form of membrane bound vesicles. A single gland cell type, located entirely within the epidermis, releases externally a granular product histochemically identified as largely protein plus some amount of carbohydrate with low periodic acid-Schiff's reactivity. The close juxtaposition of granular endoplasmic reticulum and Golgi apparati to the secretory material is consistent with the composition of this secretory product. Interstitial cells possess microvilli projecting from their apical surface, in addition to cilia. The outer surface of the plasmalemma covering these ciliary projectons is unadorned, but microvilli possess a fuzzy coat. At the peripheral ends of the microvilli, the coat is filamentous, while at their base the coat consist of foliate structures. Cationic colloidal iron binding suggest that the filamentous portion of the fuzzy coat contains the greatest proportion of the acidic surface charge. The presence of periodic acid-Schiff's positive material in this region suggests that the fuzzy coat also contains carbohydrate. Lateral boundaries of the interstitial cell lacks obvious junctional specializations; however, the apical 150 nm intracellular space narrows to 40 nm and continues in a tortuous interdigitating path to the base of the adjacent interstitial cells. Where the apex of these cells is broad, the interdigitations are shallow, but the basal half of the interstitial cells have deep complex infoldings. Cytoplasmic organelles other than the nucleus, mitochondria and some granular endoplasmic reticulum, are restricted to the apical half of the cytoplasm. The presence of closely apposed Golgi complexes and smooth endoplasmic reticulum, multivesicular bodies, lysosome-like dense vesicles and coated vesicles suggests that these cells may play a role in intracellular digestion of phagocytized particulate matter from the external environment. The amplification of the interstitial cell's free surface suggests that these cells are primarily responsible for mediated solute transport across the epidermis.

Animals

Polyinosinic-polycytidylic acid: inhibition of cell proliferation in carcinogen-treated epidermis and in carcinogen-induced skin tumors in mice.

After administration of the synthetic double-stranded RNA polyinosinic with polycytidylic acid (poly l with poly C) to hairless mice, cell proliferation kinetics were studied in normal epidermis, in epidermis initiated with 3-methylcholanthrene (MCA), in hyperplastic epidermis treated topically with MCA for 15 weeks, and in MCA-induced skin tumors. Poly l with poly C did not influence the mitotic rate or the transit of cells from the G1 phase to the S phase in normal mouse epidermis. Pretreatment of poly l with poly C inhibited cell proliferation in mouse epidermis initiated with MCA for at least 24 hours. In mouse epidermis made hyperplastic by repeated applications of MCA. Poly L with poly C inhibited G1 cells from starting DNA synthesis. Skin tumor DNA synthesis was also altered after poly l with poly C administration. After a short period of enhanced 3-methylthymidine incorporation, tumor DNA synthesis decreased to less than half the control value. The results indicated thatthe antitumorigenic effect of poly l with poly C could be related to its influence on cell proliferation.

Administration, Topical

LDH isozymes in epidermis and skin carcinoma in hamsters.

Lactate dehydrogeanse (LDH) isozymes in the epidermis and in transplantable epidermal carcinoma from Syrian hamsters were studied by polyacrilamide electrophoresis. A 5-band pattern with occasional loss of 1--2 anode fractions in certain tumor samples is typical. In the epidermis of 14 to 15-day-old foetuses, in the regenerating epidermis, and in tumor cells cathode of M subunits predominate, in particular the fifth fraction in the last two cases. In the epidermis from new born, 25-day-old (growing stage of the hairs) and 60 to 80-day-old (resting stage of the hairs) animals a relative increase of the H subunits is striking. These results reveal a foetal-like pattern of LDH isozymes in regenerating and tumorous epidermis, as has been observed in other isozymes and in other tissues.

Animals

Epidermis is the site of action of tabby (Ta) in the mouse.

The site of action of the sex-linked tabby (Ta) locus was analyzed by the technique of dermal-epidermal recombination grafting. Skin components from normal and tabby 14-day embryos were separated, recombined and grown 21 days in testes of histocompatible mice. Grafts of the combinations normal epidermis-normal dermis and normal epidermis-tabby dermis produced predominantly zig-zag hairs. Grafts of the combination tabby epidermis-normal dermis and tabby epidermis-tabby dermis produced hairs with a morphology similar to hairs found in tabby mice. We conclude from these results that the tabby locus acts within the epidermis, and has no effect on the dermis.

Animals

Reversible binding of 5- and 8-methoxypsoralen to human serum proteins (albumin) and to epidermis in vitro.

Binding of the two photosensitizers, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), to serum proteins and to epidermis was measured. 8-MOP binds to serum proteins with an apparent dissociation constant (Kd) of 4 x 10(-5) M. Under conditions of oral therapy, serum concentrations of the photosensitizer 2 h after administration are usually in the range of 100-1000 ng per ml serum. In this concentration range, 75-80% of the drug was found to be reversibly bound to serum proteins. 5-MOP shows a higher binding affinity to serum proteins and 98-99% of the drug is protein bound. The binding of both psoralen derivatives appears to take place mainly to serum albumin. 5-MOP and 8-MOP bind to different and non-interacting sites on serum proteins and the binding of the one has no effect on the binding of the other methoxypsoralen. Both photosensitizers bind reversibly to human epidermis. 8-MOP concentration in the epidermis is increased by ten to twenty fold compared with the equilibrium buffer. 5-MOP shows a higher binding affinity, resulting in a higher tissue concentration of the photosensitizer. As in serum, the two drugs appear to be bound in the epidermis to independent and non-interacting sites. No binding competition was found between the two methoxypsoralens and hydrocortisone, fluocinonide and acetyl salicylic acid, either in serum or in epidermis, using up to 1000 fold higher concentrations as compared with those of 5-MOP and 8-MOP.

Binding, Competitive

Postnatal development of the epidermis in a marsupial, Didelphis virginiana.

At birth the epidermis of the opossum is 43 micron thick and consists of a basal layer of columnar cells, an intermediate layer of fusiform cells, a layer of incompletely cornified cells and a single surface layer of cells (the periderm). The latter shows central nuclei and distinct cell boundaries. Adjacent surface cells. are contiguous and show extensive interdigitations of the lateral cell membranes. The periderm is lost during the first week of postnatal development. The epidermis attains its greatest thickness (58 micron) at the 4.0 cm stage (18 days postnatum), and this is due primarily to an increase in thickness of the spinous layer. After this the epidermis thins to 14 micron in the adult. The epidermis of the adult consists of a thin Malphighian layer and a desquamating cornified layer. Hair follicles begin to differentiate at the 2.5 cm stage (7 days postnatum). They continue to differentiate and develop while the epidermis is increasing, and then decreasing, in thickness. The young are fully furred prior to the time they first venture from the protection of the pouch.

Animals

In vitro examination of cell proliferation in dermatofibroma and in the overlying epidermis.

In nine typical cases of dermatofibroma autoradiographic methods were used to examine the proliferative activity in the tumor and in the overlying epidermis. The number of DNA-synthesizing cells in the dermal nodular lesions was extremely low (0 -- 0.1%). In seven of nine skin lesions acanthosis was to be seen in the overlying epidermis. The quantity of DNA-synthesizing cells in the epidermis overlying the dermatofibroma was not related to the thickness of the epidermis and remained independent, whether the predominant element in the tumor is fibrillar or cellular. In this study we were able to demonstrate the existence of a direct relation between the degree of epidermal proliferation and the distance between the tumor and the epidermis. The duration of DNA-synthesis was constant.

Cell Division

Biochemical and immunological analysis of a basic protein from newborn rat epidermis.

A basic protein, solubilized in buffered salt solutions from keratohyalin granules of newborn rat epidermis, has been purified by ion-exchange chromatography. The relative molecular weight of the protein was determined as 12 800 +/- 200 from its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is relatively rich in lysine, glycine, alanine, and valine which together comprise about 60% of the total amino acid residues. Using an antibody to this protein, which we have designated fraction 4, we have found that it is specific to rat epidermis and is not present in any other rat tissues or in epidermal extracts from other species. The cells of the four epidermal layers were separated and the amount of fraction 4 in each cell layer was measured by radioimmunoassay. The protein is localized mainly in the upper layers of epidermis. The protein, which binds to DNA, appears in the epidermis just prior to birth, increases during the first week of post-natal life and declines sharply thereafter. Fraction 4 represents about 7% of the total solubilized protein in 7-day-old rat epidermis.

Aging

Studies on the soluble and membrane-bound amino acid 2-naphthylamidases in pig and human epidermis.

1. Membrane-bound (particulate) and soluble amino acid 2-naphthylamidases (EC 3.5.1.-) were present in subcellular fractions of epidermis from pig and human. 2. The particulate enzymes exhibited Michaelis-Menten kinetics, with Km 5.1x10(-5) (pig) and Km 7.3x10(-5)M (human) for the substrate L-leucine 2-naphthylamide. They were inhibited by puromycin and partially inhibited by EDTA. They did not require heavy metals and were not inhibited by thiol-group-blocking agents. Their pH optima were 7.0 (human) and 6.6 (pig). The particulate enzyme from pig epidermis retained 50% activity after 30 min at 70 degrees C. 3. The soluble amino acid 2-naphthylamidases gave sigmoidal curves for reaction velocity versus substrate concentration, and the kinetic data suggested that there was positive co-operativity between binding sites. This co-operativity was lost after treatment with 0.1mM-p-hydroxymercuribenzoate and the enzymes showed first-order kinetics at low substrate concentrations. The soluble enzymes were inhibited by puromycin and by thiol-group-blocking agents and activated by dithiothreitol. They were inactivated above 60 degrees C and lost activity on storage, but this could be restored with dithiothreitol. 4. The amino acid 2-naphthylamidases of human epidermis were much more active (2.5 times) towards L-alanine 2-naphthylamide than towards the commonly used substrate L-leucine 2-naphthylamide. 5. The kinetics of both the solube and particulate enzymes from epidermis of some elderly patients with either diabetes or ischaemia showed some differences from the kinetics of enzymes from healthy epidermis from younger individuals.

Adolescent