Seasonal Variation in Serum Concentration of 5-S-Cysteinyldopa and 6-Hydroxy-5-Methoxyindole-2-Carboxylic Acid in Healthy Japanese

Serum concentrations of 5-S-cysteinyldopa (5-S-CD) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) have been used as biochemical markers of melanoma progression. We examined the effect of solar radiation on serum levels of 5-S-CD and 6H5MI2C in 10 healthy Japanese by measuring these markers every month during a period of 2 years. 5-S-CD levels were higher in early summer and lower in early winter. The difference in the average levels was approximately twofold, but among the 240 samples, no individual values exceeded the upper limit of normal value, 10 nmol/L. A significant correlation (P<0.02) was observed between 5-S-CD level and solar radiation. 6H5MI2C levels showed a smaller variation than 5-S-CD. No correlation was observed between 6H5MI2C level and solar radiation. This study showed that serum 5-S-CD and 6H5MI2C in healthy Japanese did not exceed the upper limit of normal values even in sunny season.     

Characterisation of ACTH Peptides in Human Skin and Their Activation of the Melanocortin-1 Receptor

α-Melanocyte-stimulating hormone (α-MSH) is a proopiomelanocortin (POMC)-derived peptide, which is produced in the pituitary and at other sites including the skin. It has numerous effects and in the skin has a pigmentary action through the activation of the melanocortin-1 (MC-1) receptor, which is expressed by melanocytes. Recent evidence suggests that the related POMC peptides such as adrenocorticotrophin (ACTH), which is the precursor of α-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of α-MSH in human skin where staining was evident in keratinocytes and especially strong in melanocytes and possibly Langerhans cells. ACTH was also present and tended to show the strongest reaction in differentiated keratinocytes. Immunostaining was also observed for the prohormone convertases, PC1 and PC2, which are involved in the formation of ACTH and its cleavage to α-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of α-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, α-MSH, and desacetyl α-MSH in epidermis and in cultured keratinocytes. The ability of these peptides to activate the human MC-1 receptor was examined in HEK 293 cells that had been transfected with the receptor. All peptides increased adenylate cyclase in these cells with the following order of potency: ACTH1-17 > α-MSH > ACTH1-39 > desacetyl α-MSH > acetylated ACTH1-10 > ACTH1-10. ACTH1-17 also increased the dendricity and melanin content of cultured human melanocytes indicating that the peptide was able to activate MC-1 receptors when present in their normal location. However, as found with α-MSH, not all cultures were responsive and, as we have previously suggested, we suspect that this was the result of changes at the MC-1 receptor. Nevertheless, it would appear that ACTH peptides can serve as natural ligands of the MC-1 receptor on human melanocytes and their presence in the skin suggests that, together with α-MSH, they may have a role in the regulation of human melanocytes.     

Cellular Heterogeneity in the Late-onset Form of Hereditary Melanomas in the Xiphophorus Fish Hybrids

The late-onset form of melanomas occurring in the Xiphophorus , fish hybrids carrying a macro-melanophore gene Sp was investigated for its cellular heterogeneity. The melanoma tissues were dissociated enzymatically and cultured for a short term. The cultured melanoma cells were characterized according to cell size, cell shape, pigmentation, and response to epinephrine. The melanoma cells were considerably heterogeneous in these phenotypic traits. Various combinations of these heterogeneous cells gave a great heterogeneity to individual melanomas. The stability of the phenotypic traits was followed during the course of tumor growth. Cell size and cell shape were stable, but pigmentation and response to epinephrine varied. The results are discussed in relation to cell differentiation and tumor progression.     

Chemical Degradation of Melanins: Application to Identification of Dopamine-melanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanins and pheomelanins. These pigments can be quantitatively analyzed by acidic KMnO₄ oxidation or reductive hydrolysis with hydriodic acid (HI) to form pyrrole-2,3,5-tricarboxylic acid (PTCA) or aminohydroxyphenylalanine (AHP), respectively. Dark brown melanin-like pigments are also widespread in nature, for example, in the substantia nigra of humans and primates (neuromelanin), in butterfly wings and in the fungus Cryptococcus neoformans. To characterize such diverse types of melanins, we have improved the alkaline H₂O₂ oxidation method of Napolitano et al. (Tetrahedron, 51: 5913–5920, 1995) and re-examined the HI hydrolysis method of Wakamatsu et al. (Neurosci. Lett., 131: 57–60, 1991). The results obtained with H₂O₂ oxidation show that 1) pyrrole-2,3-dicarboxylic acid (PDCA), a specific marker of 5,6-dihydroxyindole units in melanins, is produced in yields ten times higher than by acidic KMnO₄ oxidation, and 2) PTCA is artificially produced from pheomelanins. The results with HI hydrolysis show that dopamine-melanin produces a 1:1 mixture of 3-amino and 4-amino isomers of aminohydroxyphenylethylamine, while the isomer ratio is about 0.2 in melanins prepared from dopamine and cysteine. These results indicate that alkaline H₂O₂ oxidation is useful in characterizing synthetic and natural eumelanins and that reductive hydrolysis with HI can be applied to analyzing oxidation products of dopamine such as neuromelanin.     

Morphological and anatomical analyses of rheophytic Rhododendron ripense Makino (Ericaceae)

The comparative morphology and anatomy of the leaves of the rheophytic Rhododendron ripense and the closely related inland species Rhododendron macrosepalum were examined. The leaf of R. ripense is thinner than that of R. macrosepalum, with leaf length to width ratios (leaf index) of 2.92 and 1.91, respectively. Moreover, the leaf of R. ripense consists of fewer cells than the leaf of R. macrosepalum, suggesting stenophyllization of R. ripense caused by the decreased number of cells. In addition, leaf thickness and the number of stomata per leaf of R. ripense were significantly greater than those of R. macrosepalum, but the density of the short glandular pilose hairs on the leaf of R. ripense was lower. The observed morphological differences between the two species may be explained by certain aspects of the riparian environment, such as high irradiation and frequent flooding after heavy rainfall, to which R. ripense is exposed.     

Characterization of Vitiligo Antigens

Patients with vitiligo have circulating antibodies directed in part to pigment cell antigens with MWs of approximately 90, 75, and 40-45 kDs. These antigens are denominated VIT 90, VIT 75, and VIT 40, respectively. To further characterize these “vitiligo” antigens, we examined their relation to antigens defined by a panel of 25 monoclonal antibodies (moab) to pigment cell antigens. We found by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled, detergent soluble, human melanocyte macromolecules, that 24 (83%) of 29 patients with vitiligo had antibodies to one or more vitiligo antigens vs. 2 (7%) of 28 control individuals. Seventeen of the 25 moabs did not react with any labelled antigen in the same lysate. Of the remaining eight moabs, only four precipitated an antigen that co-migrated with one of the vitiligo antigens. Moab TA99, HMSA-5, and TMH-1 (all directed to the 75 kD tyrosinase-related protein [TRP1]) co-migrated with VIT 75. Moab W6/32 (directed to class I HLA antigen) co-migrated with VIT 40. Immunodepletion studies with vitiligo antibodies selectively depleted the antigen defined by W6/32 but not the antigen defined by TA99 and HMSA-5, indicating that VIT 75 was not the 75 kD tyrosinase-related protein. The vitiligo antigens were easily labelled by the lactoperoxidase technique but poorly labelled with ³⁵S-methionine, suggesting they are expressed on the cell surface. These studies indicate that VIT 90 and VIT 75 differ from antigens defined by currently available moabs to pigment cell antigens. VIT 40 appears to share a cross-reactive epitope, or be tightly bound to, class I HLA antigen.