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.

Altered biochemical properties of actin in normal skin fibroblasts from individuals predisposed to dominantly inherited cancers.

The data presented here show that normal skin fibroblasts from individuals with dominantly inherited retinoblastoma, polyposis coli, and nevoid basal cell carcinoma (predisposed cells), grown in the presence of [35S]methionine, contain more than 2.5-fold [35S]methionine-labeled actin as compared to normal fibroblasts from individuals without a prior history of predisposition to cancer (normal cells). The rate of incorporation of [35S]methionine into actin in predisposed cells is rapid and is not correlated with an increase in the total protein and actin contents of the cells, in the intracellular pool size of [35S]methionine, or in the synthesis of beta-actin-specific mRNA, as compared to normal cells. However, the half-life of actin in predisposed cells is less than 5 h, as compared to at least 48 h for normal cells. The significantly reduced half-life of actin and an increased incorporation of [35S]methionine specifically into actin in all predisposed cells studied may represent an inherited biochemical defect which leads to cytoskeletal disorganization previously observed in these cells. It can be speculated that the altered properties of actin in predisposed cells may be caused by the same genetic lesion(s) which is responsible for the induction of dominantly inherited cancers.

Bovine type I collagen: A study of cross-linking in various mature tissues.

The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000.

Loss of retinol-binding properties for plasma retinol-binding protein in normal human epidermis.

Terminal differentiation of the keratinocytes (cornification) has been linked to a restricted supply of retinol. Retinol is distributed to target cells by the retinol-binding protein (RBP), which circulates in the plasma in complex with transthyretin (TTR). In this study we have addressed the question of retinol delivery to the epidermis via RBP. Retinol radiobinding assays, affinity chromatography with TTR coupled to Sepharose beads, polyacrylamide gel electrophoresis, and immunoblotting techniques were used to show that epidermal extracts contain retinol binding sites with no affinity for TTR. Immunoreactive RBP was detected in the epidermal extracts. The RBP in the epidermis was in the apoform (without retinol) in contrast to serum where the majority of RBP is in the holoform (with retinol). Epidermal RBP was converted in vitro into the holoform only after addition of 20 times more retinol, which was needed to reconstitute holoforms of RBP in dermal extracts, human buccal mucosal extracts, and delipidized normal serum or purified delipidized RBP. Moreover, several immunoreactive RBP bands (possibly degradation products) were identified in the epidermal but not in dermal extracts. Retinol-binding protein from nonkeratinizing human oral mucosa showed different immunoblotting patterns when compared to epidermal RBP. These results suggest that degradation of RBP within the epidermis may result in a decreased retinol supply to the keratinocytes, and may lead to the cornification of the epidermis.

Bovine keratohyalin: anatomical, histochemical, ultrastructural, immunologic, and biochemical studies.

Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.

Characterization and quantification of epidermal and dermal glucocorticoid receptors in the rat.

The interaction between [3H]triamcinolone acetonide and cytosol proteins was studied in separated epidermis and dermis of neonatal rats. Both tissues possessed a binding site with a high affinity (dissociation constant = 0.2 nM) and a low capacity (150-220 fmol/mg protein) for triamcinolone acetonide. The binding site in each tissue cytosol was inactivated by heating at 37 degrees C or by incubating with either trypsin, Triton X-100 or NaCl. The second-order rate constant of association was 1.99 X 10(6)M-1min-1 for the epidermal, and 1.65 X 10(6)M-1min-1 for the dermal receptor binding site. Quantitatively there was a difference between the two tissues with 82% of the receptor being found in the dermis and 18% in the epidermis.

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues.

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.

Analysis of epidermal proteins for DNA-binding activity.

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.

Differential effects of collagen in oral tissues and skin by protein-energy malnourished newborn rats and lactating dams.

Effects of protein-energy malnutrition were studied in newborn rats and their dams. Upon delivery, dams received 6%, 12% or 20% protein diets. At Day 15 pups received 14C(U)-proline. The posterior tongue, hard palatal mucosa, soft palatal mucosa and skin were analyzed for collagen and counts incorporated (collagen synthesis, that is, the rate of 14C-proline converted to 14C-hydroxy-proline into gingival collagen). These regions of the dams were also removed to study collagen content. Although soft palatal mucosal collagen of newborns in the 6% and 12% protein groups was decreased, that of skin in the 6% protein group was increased. No such differences were observed in tongue and hard palatal mucosa. Counts incorporated was decreased in the tongue, soft palatal mucosa and skin, but not in hard palatal mucosa. Collagen contents of tongue and hard palatal mucosa and skin of the dams showed no differences, whereas that of soft palatal mucosa in the 6% protein groups was increased. Effects of protein-energy malnutrition on oral tissues and skin were therefore different between newborns and lactating dams. Furthermore, one part of oral mucosa is affected differently from other parts of mucosa and in both, degree of malnutrition has different influences.

Chemical nature of collagen in the placoid-scale dentine of the blue shark, Prionace glauca L.

The dentine fraction was obtained from powdered placoid scales by differential density-flotation, and demineralized with 0.5 M EDTA. Monomeric alpha 1 and alpha 2 chains of collagen were extracted from the residual organic matrix, and the two alpha chains purified by chromatography. The two alpha chains were also isolated from shark-skin collagen. The corresponding alpha chains from shark dentine and skin collagens resembled each other closely in chromatographic and electrophoretic behaviour and in their CNBr-peptide maps but differed from calf-skin alpha chains. The differences in the chemical composition between shark dentine and skin were due only to post-translational modification (hydroxylation and phosphorylation), indicating that the collagens are of the same type. The hydroxylation of prolyl and lysyl residues occurred more in the dentine alpha chains than in the skin chains. Among the four alpha chains, the phosphate content was the highest in the alpha 2 chain of the dentine collagen. These differences in hydroxylation and phosphorylation have been observed among alpha chains of mammalian mineralized and unmineralized tissues. The preferential dimerization to form alpha 1-alpha 2, characteristic of shark-skin collagen, was not observed in the dentine collagen. Other dimers were hardly detectable in the latter.

Immunofluorescent localization of fibronectin in human oral mucosa.

Fibronectin (FN) with an immunohistochemical fluorescence technique using anti-human FN and anti-rat FN sera was localized in the basement membrane of lip, buccal mucosa, palate, tongue, around ectopic sebaceous glands and around acini and ducts of labial salivary glands though in different amounts. In the mucosal lamina propria, FN was present in a net-like pattern. The highest concentrations of FN were in the palatal and tongue connective tissues. Much FN was present in the walls of small blood vessels and in perineural sheaths of peripheral nerves. No fluorescence was seen in the acini of labial salivary glands. In their striated ducts however, some cells were intensely fluorescent; the significance of this is unknown.

Microscopy and X-ray microanalysis of subcutaneously implanted conventional and high-copper dental amalgam powders in the guinea pig.

With each material, there was only a mild early inflammatory response; particles were taken up by macrophages and giant cells. By 1.5 to 3 months, chronic granulomas had developed. With conventional amalgam, there were early changes in the intracellular material, associated with the rapid degradation of Sn-Hg particles, corresponding to the gamma 2 phase. Thereafter, intracellular particles from both types of amalgam underwent progressive degradation, producing halos of secondary material containing Ag and S. Apart from an initial loss of Cu and Sn from some high-Cu amalgam particles, there were no qualitative differences in the later changes between the two materials, although conventional amalgam particles appeared to degrade faster. With both, vast numbers of fine secondary particles containing Ag and S became widely distributed throughout the lesions and gave rise to macroscopic tattooing of the skin. Secondary material and small degrading primary particles from both types of amalgam were detected in the submandibular lymph nodes where they caused localized staining.

Biochemistry of glycosaminoglycans in the skin and oral mucosa of the rat.

Glycosaminoglycans (GAG) were extracted by digestion with papain followed by ultrafiltration and separated by cellulose-acetate electrophoresis and by chromatography of their cetyl-pyridinium complexes on cellulose microcolumns. The uronic-acid content of the tissues ranged from 0.8 to 2.4 mg/g of dry defatted tissue. Hyaluronic acid and dermatan sulphate were found in all tissues with chondroitin-4-sulphate also in skin, palatal mucosa and gingiva. There was 3-fold more hyaluronic acid in palatal mucosa than in any other tissue; it was concentrated in the antemolar rugae. A substance of presumptive salivary origin staining with alcian blue was found in cheek and floor of mouth mucosa. It migrated differently from reference GAG by electrophoresis and was not degraded by testicular hyaluronidase.

A new look at osteogenesis imperfecta. A clinical, radiological and biochemical study of forty-two patients.

In a clinical, radiological and biochemical study of forty-two patients from Oxford with osteogenesis imperfecta, it was found that patients could be divided simply into mild, moderate and severe groups according to deformity of long bones. In the severe group (seventeen patients) a family history of affected members was uncommon and fractures began earlier and were more frequent than in the mild group (twenty-two patients); sixteen patients in the severe group had scoliosis and eleven had white sclerae; no patients in the mild group had white sclerae or scoliosis. Radiological examination of the femur showed only minor modelling defects in patients in the mild group, whereas in the severe group five distinct appearances of bone (thin, thick, cystic and buttressed bones, and those with hyperplastic callus) were seen. The polymeric (structural) collagen from skin was unstable to depolymerisation in patients in the severe group, but normal in amount, whereas the reverse was found in the mild group. This division according to long bone deformity may provide, a basis for future research more useful than previous classifications.

The 43 kDa inhibitor of human skin and other epithelia.

We have used immunohistochemical and quantitative immunochemical techniques to study the tissue distribution of the 43 kDa papain inhibiting protein, originally isolated from psoriatic scale. High amounts of the inhibitor were found in the epidermis only in several skin diseases where the epidermal cell proliferation was increased (psoriasis, various eczemas). The inhibitor was also found in the basal cells of the bronchial epithelium and in the squamous epithelia of mouth and oesophagus, and the Hassal's corpuscles of the thymus. Sera of patients suffering from several skin diseases did not contain the inhibitor, but antibodies against it were found in the sera of two patients suffering from leg ulceration. The inhibitor was immunologically unrelated to filaggrin, involucrin, or keratins.

cAMP levels in reactive tissues around dimethylpolysiloxane solid implants.

By the aid of radioimmunologic assay, the authors measured the cAMP content of the reactive capsules around solid dimethylpolysiloxane implants in mice at different times; they also measured the same substance in some human connective tissues (granulation tissue and normal dermis) and compared together all the values they obtained. Different concentrations of cAMP in different tissues seem to reflect correspondent histologic findings, since they vary according to them. These values also seem to indicate a close correspondence between the development of the process of wound healing and of the foreign-body reaction following the implantation of alloplastic materials. On the basis of these data, the authors suggest an experimental therapeutic trial to enhance peripheral cAMP synthesis in order to control the process of reactive capsule constitution.

Evaporation and skin penetration characteristics of mosquito repellent formulations.

Formulations of the mosquito repellent N,N-diethyl-3-methylbenzamide (deet) in combination with a variety of additives were developed to control repellent evaporation and percutaneous penetration. Deet was also formulated with the repellent dimethyl phthalate to study the interaction of the two compounds on the skin. The evaporation and penetration processes were evaluated on whole and split-thickness pig skin using radiolabeled repellents with an in vitro apparatus. Under essentially still air and air flow conditions, one of the deet formulations resulted in significantly reduced total evaporation and percutaneous penetration of deet as compared to unformulated repellent. When deet and dimethyl phthalate were combined, neither repellent affected the total amount of evaporation and penetration of the other compound. However, initial percutaneous penetration and evaporation rates were slightly less and decayed less rapidly than when both chemicals were tested separately at the same dose. These results indicated a degree of competition of the two compounds for the same avenues of loss.

The U.S. Transuranium Registry report of the 241Am content of a whole body. Part IV: Preparation and analysis of the tissues and bones.

Los Alamos National Laboratory has analyzed autopsy tissue for the USTR, as a part of its study of the uptake, distribution and retention of Pu and other transuranic elements in occupationally exposed workers since 1978. In April 1979, Los Alamos received the internal organs and bone samples from the first whole-body donation to the USTR. The donor was known to have an internal deposition of 241Am. All soft tissue, the bones from the right half of the skeleton, and the odd-numbered vertebrae were received at Los Alamos in February 1980. The bones were subdivided along anatomical areas of interest. All soft tissues and bone specimens were analyzed for their 241Am content. A total deposition of 147.4 nCi 241Am was measured. Approximately 18% of the 241Am remaining in the body (disregarding that in the left hand), was found in the soft tissues, and 82% was in the bones and teeth. The soft tissues and organs containing the largest amounts of 241Am were the combined soft tissue (striated muscle, connective tissue and skin) 8.8%; liver, 6.4% and respiratory tract, 1.5%. The remaining organs accounted for 0.9% of the systemic burden.

[Use of Vladipore cellulose acetate ultrafiltration membranes for isolation of small peptides]. / Primenenie atsetiltselliuloznykh ul'trafil'tratsionnykh membran Vladipor v vydelenii korotkikh peptidov.

Retintion of some amino acids and peptides by standard ultrafiltration membranes "Vladipore" was studied. Membranes UAM-30M and UAM-50M may be used for concentration and desalting of peptides containing not less than 3-4 amino acid residues. An increase in NaCl concentration in the samples led to a decrease in the peptide retention coefficient. The ultrafiltration membranes were used for isolation of small peptides from blood, urine and extracts of normal skin, after thermal trauma and irradiation of the tissue.