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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

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

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

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

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

Sphingomyelinase in pig and human epidermis.

The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine.

Animals

Cell proliferation kinetics of epidermis and sebaceous glands in relation to chalone action.

Median S-phase lengths of pinna epidermis and sebaceous glands, and of epithelia from the oesophagus and under surface of the tongue of Albino Swiss S mice were estimated by the percentage labelled mitoses method (PLM). The 18.4 and 18,8 hr for the median length of S-phase for pinna epidermis and sebaceous glands respectively made it possible for these two tissues to be used experimentally for testing tissue specificity in chalone assay experiments. The 10.0 and 11.5 hr for oesophagus ang tongue epithelium respectively made experimental design for chalone assay difficult when pinna epidermis was the target tissue. The results of the Labelling Index measured each hour throughout a 24-hr period showed no distinct single peaked diurnal rhythm for pinna epidermis and sebaceous glands. Instead a circadian rhythm with several small peaks occurred which would be expected if an S-phase of approximately 18 hr was imposed on the diurnal rhythm. This indicates that there may be very little change in the rate of DNA synthesis. The results are given for the assay in vivo of purified epidermal G1 and G2 chalones, and the 72--81% ethanol precipitate of pig skin from which they could be isolated. These experiments were performed over a time period which took into account the diurnal rhythm of activity of the mice as well as the S-phase lengths. Extrapolating the results with time of action of the chalone shows that the G1 chalone acts at the point of entry into DNA synthesis and that the S-phase length was approximately 17 hr for both the pinna epidermis and sebaceous glands. This may be a more correct value since the PLM method overestimates the median S-phase length as it is known that in pinna skin the [3H]TdR is available to the tissues for 2 hr and true flash labelling does not take place. The previous reports that epidermal G1 chalone acts some hours prior to entry into S-phase resulted from experiments on back skin where the S-phase is shorter and there is a pronounced diurnal rhythm which could mask the chalone effect. The epidermal G2 chalone had no effect on DNA synthesis even at different times in the circadian rhythm. Thus the circadian rhythms and S-phase lengths of the test tissues need to be considered when experiments are performed with chalones. Ideally, the target tissues selected for cell line specificity tests should have the same cell kinetics for the easier and more accurate assessment and interpretation of results. When the tissues have markedly different cell kinetics, experimental procedures and results need to be evaluated accordingly. The point of action of G1 chalone can only be assessed if the effect is measured over the peak of incorporation of [3H]TdR into DNA. The results of the effects of skin extracts are analysed in relation to changes in the availability of [3H]TdR for the incorporation into DNA and to the possibility of there being two distinct populations of proliferating cells.

Animals

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.

Animals

Differentiation markers in fetal epidermis: transglutaminase and transpeptidase.

Two members of the transpeptidase family of enzymes, transglutaminase and gamma glutamyl transpeptidase, were assayed histochemically and biochemically in developing rat epidermis from day 15 of gestation through postnatal day 5. Electron microscopic examination of serial skin biopsies enabled precise dating of fetal epidermis and periderm and correlation of ultrastructural details of the cells with marker enzyme activities. Transglutaminase activity appeared histochemically in surface epidermis and in hair follicle inner root sheath on day 18 and day 21 of gestation, respectively, concomitant with the onset of terminal keratinization in these tissues. Enzyme activity was biochemically detectable 2 days before the histochemical stain became positive. Transpeptidase was active in fetal epidermis prior to keratinization but was only detectable in basal cells thereafter. Subsequent to birth, enzyme activity rose geometrically in hair follicles undergoing initial differentiation, and was thereafter found in all anagen hairs. Transglutaminase is active only in cells approaching terminal keratinization, while transpeptidase is associated with early phases of epidermal proliferation and differentiation.

Acyltransferases

Absence of intercellular antigens in the deep layers of the epidermis in pemphigus foliaceus.

12 patients with pemphigus foliaceus, a form of pemphigus with lesions that arise in the intercellular substance in the superficial layers of the epidermis, and 7 patients with pemphigus vulgaris, where lesions are in the deep layers, were studied by immunofluorescence. Circulating antibodies to intercellular antigens (IC antibodies) were found in 11 pemphigus foliaceus and 5 pemphigus vulgaris patients. On direct immunofluorescence of skin lesions 75% (9 of 12), pemphigus foliaceus patients had intercellular deposits of IgG localized solely or predominantly in the superficial epidermal layers, whereas this was not the case in any of the patients with pemphigus vulgaris. Over 70% of the pemphigus foliaceus patients with predominantly superficial IgG deposits lacked in their lesions normal intercellular antigens usually expressed in the deep layers of the epidermis. This was shown by the inability of IC antibodies in autologous or allogeneic sera to bind to intercellular antigens in the lower epidermis of patient's skin, even though the same sera could bind to intercellular antigens in all layers of normal allogeneic skin. Lack of normal intercellular antigens deep in the epidermis may result in circulating IC antibodies binding to the superficial layers, a site which corresponds to, and thus in some patients may account for, the anatomical location of lesions in pemphigus foliaceus.

Antibody Specificity

Presence of two typical DNA-binding nonhistone proteins in psoriatic scales contrary to normal human dermis, epidermis and horny layer.

The composition of DNA-binding proteins (DBP) was shown to be tissue-specific and to vary at different stages of gene expression. As the accelerated epidermopoesis in psoriasis indicates changed gene activities, DBP of psoriatic scales were compared with those of normal human epidermis, dermis and horny layer. Each skin fraction is characterized by its own DBP pattern, indicating different cell species. 1. The DBP of normal human epidermis shows only a small accordance with the DBP of human dermis and implies their difference in origin, function and cell types. 2. Psoriatic scale DBP and epidermal DPB contain more corresponding proteins which can be deduced from the scale's origin from epidermis. However, the composition of all proteins differs to a great extent. This either occurred during parakeratotic keratinization or reflects differences of normal to psoriatic epidermis. Imposing for psoriatic scale DBP are two protein bands with molecular weights of 84,000 and 90,000 daltons. Evidently both are not present in the DBP of other skin layers. 3. The horny layer contains a very small amount of DBP which might represent DNases to a major part. The small DBP content in horny layer confirms the previous supposition of psoriatic scales, to be mostly derived from the preserved nuclei of the parakeratotic scale layer.

Chromosomal Proteins, Non-Histone

[Relationship between mononuclear infiltration of the epidermis and its hyperplasia after auto- and allotransplantation of the full-thickness skin of mice].

Histological and histochemical studies of the back skin were carried out with morphometry on the 1st, 2nd, 3rd, 6th, and 9th days after auto- or allotransplantation of its full thickness graft in 60 lineal (C3H) and random-bred mice. In both kinds of transplantation, mononuclear infiltration of the epidermis of the graft and the surrounding skin was established. A correlation between the intensity of this infiltration and the thickness of the epidermis in the skin surrounding the grafts and in the autograft was established. In allotransplantation, there was no such correlation in the graft. No mononuclear cells were found in regeneration layers of the epidermis. It is suggested that epidermis regeneration is stimulated by autogenous lymphocytes.

Animals

Histochemical evidence of mucosubstances in the metaplastic epidermis and hair follicles produced in vitro in the presence of excess vitamin A.

Pieces of upper lip and lower jaw from 13-day old mouse embryos grown in organ culture with excess vitamin A showed metaplastic changes in epidermis and hair follicles after 7 to 21 days. Changes were less marked in upper lip explants from 15-day old embryos. Areas of keratinizing epidermis showed a much higher incidence of Alcian blue-positive bodies when excess vitamin A was present. Histochemical tests with Alcian blue at critical electrolyte concentrations indicated moderately sulfated acidic mucosubstances in these bodies. Other patches of epidermis were transformed into stratified cuboidal epithelium producing PAS-positive, Alcian blue-negative, mucosubstances. The hair follicles, transformed by vitamin A into glands, developed three types of epithelium with apical granules, luminal borders and occasional goblet cells, all showing mucosubstances. Tests suggest that both metaplastic epidermis and glands secrete either neutral mucosubstances or non-sulfated acidic mucosubstances. Another response to vitamin A was a rapid loss of glycogen, particularly in hair follicles and the epidermal basal layer. It was concluded that excess vitamin A alters not only the morphogenesis but also the distribution and synthesis of polysaccharides in developing skin.

Animals

The distribution of enzymes in the epidermis of the domestic cat.

With the help of enzyme histochemical methods, the distribution and activities of several oxidative and hydrolytic enzymes in the epidermis of the densely-haired domestic cat have been studied. Distinct oxidative enzyme activity could be demonstrated in all epidermal layers except the stratum corneum. Among the hydrolytic enzymes investigated, strong reactions for non-specific esterases were visible especially in the str. granulosum and the basal lamellae of the str. corneum. Positive reactions for cholinesterases could not be observed in the cat epidermis. The results are discussed in relation to corresponding investigations on the epidermis of sparsely-haired mammals, e.g. the domestic pig and man. Generally the enzyme pattern of the cat epidermis only shows limited parallels to man, especially where esterase distribution is concerned.

Animals