Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo.

Exposure of normal human skin in vivo to ultraviolet irradiation at wavelengths shorter than 320 nanometers stimtulated an unscheduled DNA synthesis in all of the cell layers of the epidermis and in the upper dermnial fibrocytes. The skin of patients with xeroderma pigmentosum did not show this response. correlation of these findings with previous tissue culture studies suggests that the defect in repair of the damaged DNA in xeroderma cells occurs in vivo as well as in vitro.

Role of sweat in accumulation of orally administered griseofulvin in skin.

Griseofulvin, an orally effective antimicrobial agent, appears in the stratum corneum within 4-8 h after oral administration. Griseofulvin distribution was found to be highest in the outermost layers of the stratum corneum (level I, 20.8+/-1.5 ng/mg) and lowest inside (level II, 10.0+/-1.5; level III, 7.5+/-2.2 ng/mg). In order to study the precise mechanism of griseofulvin transfer to stratum corneum, the role of sweat in the accumulation of griseofulvin was considered. Heat-induced total body sweating decreased the mean stratum corneum concentration of griseofulvin by 55%, and 200-300 ng of griseofulvin accumulated per ml of sweat. A silicone hydrophobic resin was used to differentiate between "wash-off" and carrier properties of sweat for griseofulvin. Prevention of transepidermal water and sweat loss by (a) topical application of formaldehyde-releasing cream to one palm, (b) occlusion by a 2 x 2-cm patch on one arm, and (c) wearing a rubber glove for 24 h, showed a lower griseofulvin concentration when compared to control areas in the same subjects. The results of the gloved hand experiment show that a complete equilibrium is established at all three levels of stratum corneum, thereby removing the reversed gradient. These results support the hypothesis that a "wick effect" is responsible for the observed reversed drug gradient within the stratum corneum. The results of the experiments suggest that sweat and transepidermal fluid loss play an important role in griseofulvin transfer in stratum corneum.

In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Purification and characterization of bradykinin-hydrolyzing enzyme from 2-day-old rat epidermis.

Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.

Immunochemical studies of proteins in epidermal cornified cells of human and newborn rat.

1. Proteins were extracted from cornified cells of newborn rats and human palm with 8 M urea containing 0.1 M beta-mercaptoethanol. Two fractions, rat FIIIa and human F5.5 were obtained by acid precipitation for further study. 2. Antibodies raised in rabbits by injection of rat FIIIa gave two precipitin lines by agarose diffusion against rat FIIIa, but only one line against human F5.5. One of the antigenic determinants of rat FIIIa was found to be a protein of approximately 66 000 daltons. The other seems to be formed with two polypeptides in the range of 60 000 and 66 000 daltons. The antigenic determinant of human F5.5 was a protein of approximately 64 000 daltons which immunologically cross-reacted only with the antiserum to a protein of 66 000 daltons in rat FIIIa. 3. The antisera also cross-reacted with proteins extracted from epidermis of guinea pig, hamster, hairless mouse, dog ear, dog snout and dog foot pad, but did not react with the epidermis of either rabbit immunized with rat FIIIa or non-treated normal rabbit. 4. Indirect immunofluorescence demonstrated a reaction of rabbit anti-rat FIIIa serum over cornified cells as well as over granular, spinous and basal cells of the epidermis of newborn rat and human.

Purification and some properties of bound cysteine proteinase inhibitor from cornified cells of newborn rat epidermis.

Cysteine proteinase inhibitor exists in two forms in terminally differentiated keratinocytes. One is readily soluble in 20 mM sodium phosphate buffer but the other is bound to the plasma membrane and is poorly soluble. The cysteine proteinase inhibitor (CPI) from the membrane was extracted from cornified epidermal layers of 2-day-old rats and its properties were compared with those of soluble CPI. This CPI (bound CPI) was solubilized in alkaline 8 M urea containing 2-mercaptoethanol from the residual tissue exhaustively treated with buffered 4 M urea. CPI was separated from keratin by ammonium sulfate precipitation and purified by means of papain affinity chromatography, ion exchange column chromatography and gel filtration. Bound CPI had an Mr value of about 16,000, a pI value of 3.8 and was unstable at above 80 degrees C, while soluble CPI was of Mr 13,000 and stable at above 80 degrees C. Both CPIs were stable at 4 degrees C in the range of 3.0-9.0. Bound CPI contained half cystine and the ratio of acidic-to-basic amino acids was 3.18. Bound CPI inhibited rat liver cathepsins B, H, and L but did not inhibit the activity of noncysteine proteinases. Papain activity was inhibited by bound CPI at three sites, noncompetitively, and the Ki value was calculated to be 0.11 nM.

Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells.

(1) Combination of techniques for extraction and purification of histidine rich protein established by several investigators were employed for comparison of histidine-rich protein in granular cells and cornified cells of newborn rats. (2) Histidine-rich protein extracted from the same cell fraction by two different techniques either in 1 M potassium phosphate buffer (Ugel) or in 4 M urea (Dale) showed identical elution profiles on CM 52 cellulose ion exchange chromatography and the same SDS polyacrylamide gel electrophoretic patterns. (3) Histidine-rich protein from granular cells contained polypeptides of larger molecular sizes than those in histidine-rich protein from cornfield cells, although amino acid composition of the two histidine-rich protein was non-distinguishable (histidine residue was more than 7%). (4) Antibodies raised in rabbits by injection of histidine rich protein from granular cells and that from cornfield cells immunologically cross-reacted. Furthermore, the antisera were found to be reactive over both keratohyalin granules and cornified cells, but not epidermal cells of the lower strata.

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis.

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis.

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells.

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.

Light-scattering studies of cation-stimulated filament assembly of newborn rat epidermal keratin.

Effects of CaCl2 on in vitro polymerization of keratin extracted from cornified cells of newborn rat were investigated by means of light-scattering and supramolecular structures. Elongation and parallel assembly of filaments occurred with addition of CaCl2 to dialyzed keratin solutions and was detected by an increase in light-scattering intensity. Nonfibrous aggregates which occurred in higher buffer concentrations and in lower pH were also recorded as intensity increased. MgCl2, ZnCl2, and GdCl3 demonstrated similar effects, but NaCl and KCl showed no effect.

Identification of the mouse calcium-binding proteins, MRP 8 and MRP 14, in Schistosoma mansoni-induced granulomas: biochemical and functional characterization.

Granuloma initiation factor (GIF), which elicits a granulomatous reaction in naive murine skin, contains low-molecular-weight proteins partially purified from organized granulomas developed in livers of mice with schistosomiasis. In this study, we found that 10- and 14-kDa proteins in the GIF are highly homologous to mouse migration inhibitory factor-related protein (MRP) 8 and MRP 14. Compared with the N-terminal amino acid sequence deduced from each corresponding cDNA, the 10-kDa protein from the granuloma lacks the first methionine, whereas the 14 kDa misses methionine, alanine, and asparagine. Immunohistochemically, cells expressing MRP 8 and MRP 14 considerably increased in different murine tissues after Schistosoma mansoni infection and concentrated in liver around the dilated blood vessels and at the edge of granulomas. The staining of differentiated macrophages and epithelioid cells located in the center of the granulomas was negative. Immunoreactivity of peritoneal exudate cells also was found to gradually disappear with time in cell culture. Furthermore, in vivo effects of the recombinant proteins in murine skin were described histologically. Both MRPs caused severe infiltration of neutrophils and monocytes during 7-14 days. The reaction resulting from individual MRP implantation became minimal after 50 days but inoculation of the Ca2+-dependent heterodimers showed an extensive eosinophil accumulation.

Heterogenous proteins in keratohyaline granules studied by quantitative autoradiography.

Electron microscopic autoradiography with [3H]histidine, [3H]cystine, I13H]arginine, and [3H]proline was used to study protein synthesis in keratohyaline granules of newborn rats. All 3H-amino acids were incorporated into proteins in the granular cells, and the radioactive proteins appeared in the keratohyaline granules. However, the amount of radioactivity associated with the granules and the pattern of ultrastructural localization of the radioactive proteins differed considerably for each 3H-amino acid. "Histidine-labeled" protein was located mainly in the matrix portion of keratohyaline granules whereas "cystine-labeled" protein accumulated in the dense homogenous deposits. "Arginine-labeled" protein was distributed more diffusely in the organelles of granular cells, but that associated with keratohyaline granules seemed to localize mostly with "histidine-labeled" protein and partly with "cystine-labeled" protein. Large amounts of "proline-labeled" protein were also present in other areas of the cytoplasm than keratohyaline granules. This protein localized in the dense homogeneous deposits, but it seemed to turn over more rapidly than "cystine-labeled" protein, an indication that the dense homogenous deposits consist of at least two different polypeptide chains, one of which contains higher cystine and the other higher proline.

Epilogue as prologue. The skin: continual source of biologic problems and discoveries.

In much the same way as the survival of many species depends upon their abundant production of such genetic materials as pollens and spermatozoa, so does scientific progress depend upon a continual, lavish production of basic scientific works. In stimulating assembling, and disseminating various scientific studies related to the skin, the Montagna Symposia on the Biology of Skin have ensured progress not only in the understanding and management of skin was, is, and probably will always will be one of the richest sources of basic biologic information. No scientific discovery is wasted, no accurate clinical observation useless. No one can foresee when or how some seemingly irrelevant discovery in pure science or some seemingly insignificant clinical observation will yield fruits of incalculable practicable value. We cite here a few examples of some of the best results from apparently unrelated research and isolated clinical findings. In dermatologic research, the circular rhythm from patient with cutaneous disease to laboratory and from laboratory back to patient creates a centrifugal force that often spins off valuable discoveries.

New cell formation in human sebaceous glands.

Intradermal injection of thymidine-H3 fol- lowed by biopsy removal 40 minutes to 36 days later revealed a characteristic pattern of human sebaceous gland activity. Cells lying on or near the basement membrane divide, and daughter cells move centrally, produce lipid, and undergo nuclear degeneration. The steady state replenishment from the periphery con- tinues for at least 2 to 4 weeks. The average renewal time for the human sebaceous gland in the 4 specimens examined is 7.4 days.

The induction of human multinucleated monocytes in culture.

The formation of multinucleated giant cells in vitro has been studied using human monocyte cultures. Multinucleated monocytes appeared early in the course of the culture, indicating that they are not purely a phenomenon found only in aging cultures. In 4 of the 33 normal adults, more than 20% of multinucleated forms appeared in the cultures. Supernatants from early monocyte cultures of those subjects having a high incidence of multinucleated cells induced the formation of multinucleated cells in other cultures. This effect did not occur when supernatant from subjects with low counts of multinucleated cells were incubated with other monocyte cultures.

Inhibition of the in vivo effects of concanavalin-A on mammalian epidermis by alpha-methyl-D-glucopyranoside.

Concanavalin-A (Con-A) injected intradermally into newborn rats produces inhibition of granular-cell formation, accumulation of spinous cells, glycogen deposition, and a decrease followed by an increase in the number of basal cells in DNA synthesis. These changes were maximal with a dose of 0.1 mg Con-A, although 0.005, 0.01, and 0.05 mg caused some epidermal changes. The Con-A effects were partially blocked when 0.1 ml of 0.3 or 0.1 M alpha-methyl-D-glucopyranoside (alphaMG) solution was injected 2 hr after 0.1 mg Con-A and completely inhibited by injection of 0.1 ml of 3.0 M alphaMG solution. The inhibitory effects were not seen after injection of 0.1 ml of 3.0 M N-acetyl-galactosamine saline solution, or 0.1 ml normal saline. Injection of alphaMG alone did not cause any changes in epidermal cells. These results indicate that specific sugar inhibits Con-A effects on mammalian epidermis in vivo.

Dibutyryl cyclic AMP-induced differentiation of epidermal cells in tissue culture.

Histochemical and biochemical techniques have been used to compare the effects of dibutyryl cyclic AMP on epidermal cells and dermal cells in primary tissue culture. Rhodamin B staining showed only scattered positive cells in nontreated epidermal cells and a few contaminating keratinizing cell foci in both nontreated and treated dermal cell cultures. In contrast, treated epidermal cells stained strongly and had many keratinizing cell foci. A significant increase in histidine, cystine, and arginine incorporation was noted in epidermal cells treated with dibutyryl cyclic AMP as compared to untreated epidermal cells and to dermal cell cultures both treated and untreated. Dibutyryl cyclic AMP had no significant effect on leucine and phenylalanine incorporation. These results seem to suggest that the intracellular level of cyclic AMP not only controls the synthesis of DNA by epidermal cells in culture but also induces the process of differentiation toward keratinization.

Eosinophil response in guinea pig skin to a low molecular weight eosinophil chemotactic factor extracted from livers of mice with schistosomiasis.

A low-molecular weight eosinophil chemotactic factor (ECF-G), isolated and partially purified from livers of mice with schistosomiasis, was injected intradermally into guinea pigs. Biopsies obtained were studied for inflammatory cell accumulation in the injected sites and compared with those in the control sites injected with phosphate buffered saline. Tissue eosinophilia was seen as early as 1 hr after injection of ECF-G, but not in the control site. The increase of eosinophilia appeared biphasic with peaks at 6 and 24 hr. Mast cells increased in both ECF-G and saline injected sites and the increase was still found at 120 hr after injection. Neutrophils also increased in both ECF-G and saline injected sites but disappeared within 48 hr. These findings indicate that ECF-G is a tissue and species nonspecific eosinophil chemotactic factor, and injection of ECF-G initiates interaction of eosinophils and mast cells in the skin.