bluuebunny/crossref_metadata_2025 huggingface dataset:
The Expression of Tyrosinase, Tyrosinase‐Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein, and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities
Authors: KAMEYAMA KOICHIRO , SAKAI CHIE , KUGE SAKAE , NISHIYAMA SHIGEO , TOMITA YASHUSI , ITO SHOSUKE , WAKAMATSU KAZUMASA , HEARING VINCENT J.
Published: July 2006

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase‐related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM‐IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM‐TY cells, possessed significant amounts of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6‐dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm‐1/4 cells, whereas DHI was stable in amelanotic human SK‐MEL‐24 cells. A melanogenic inhibitor that has been purified from SK‐MEL‐24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.
Seasonal Variation in Serum Concentration of 5‐S‐Cysteinyldopa and 6‐Hydroxy‐5‐Methoxyindole‐2‐Carboxylic Acid in Healthy Japanese
Authors: WAKAMATSU KAZUMASA , ITO SHOSUKE
Published: July 2006

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.
Characterization of the Melanogenic System in Vibrio cholerae, ATCC 14035
Authors: RUZAFA CAROLINA , SANCHEZ‐AMAT ANTONIO , SOLANO FRANCISCO
Published: July 2006

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin‐like pigment when cultured in the presence of L‐tyrosine. Other phenolic chemicals related to L‐tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami‐nases that transforms L‐tyrosine into p‐hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p‐quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper‐Mason pathway, but by a L‐tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu‐ and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.
Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB
Authors: BESSOU SANDRINE , SURLÈVE‐BAZEILLE JEAN‐ETIENNE , SORBIER EVELYNE , TAÏEB ALAIN
Published: July 2006

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air‐liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm2× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.
Ca2+ and UVB Radiation Have No Effect on E‐Cadherin‐Mediated Melanocyte‐Keratinocyte Adhesion
Authors: NAKAZAWA KEIKO , NAKAZAWA HISAYOSHI , BONNARD MICHELLE , DAMOUR ODILE , COLLOMBEL CHRISTIAN
Published: July 2006

Direct cell‐cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium‐dependent epithelium‐specific cell adhesion molecule, E‐cadherin, in melanocyte‐keratinocyte adhesion was suggested previously. To further clarify regulation of E‐cadherin‐mediated melanocyte‐keratinocyte interactions, we investigated the effects of physiological (Ca2+) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E‐cadherin in melanocyte‐keratinocyte adhesion. Expression of E‐cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti‐human and anti‐mouse‐E‐cadherin antibodies (anti‐uvomorulin and ECCD‐2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E‐cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca2+ during the binding assay. Addition of anti‐mouse‐E‐cadherin antibody (ECCD‐1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti‐P‐cadherin antibody (PCD‐1) had no effect. The levels of E‐cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm2) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte‐keratinocyte adhesion. These results demonstrate that E‐cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E‐cadherin in the overall regulation of the skin pigmentary system.
Melanocytes in the Stria Vascularis and Vestibular Labyrinth of the Mongolian Gerbil (Meriones unguiculatus)
Authors: ESCOBAR CARLOS , ZUASTI ADELINA , FERRER CONCEPCION , GARCIA‐ORTEGA FELIX
Published: July 2006

Inner ear melanocytes are mainly present in the cochlea, vestibular organ, and endolymphatic sac, but their exact biological function has not been determined. In this investigation, we study the pigment cells in the membranous labyrinth of the gerbil. The inner ear melanocytes of M. unguiculatus show an irregular dendritic shape with cytoplasmic processes. These cells are disposed following the distribution of striai marginal and vestibular dark cells that have an important metabolic activity. Gerbil inner ear melanocytes are characterized by the presence of melanosomes, which are homogeneously dense organelles, of variable size and shape, that are surrounded by a membrane. In these cells, the Golgi apparatus plays a important role in melanin synthesis. When melanocytes were incubated in L‐DOPA solution, the vesicles and cisterns of the Golgi apparatus exhibited a positive tyrosinase reaction. An interesting observation is the relation between melanocytes and inner ear capillaries. Sometimes, near to sensory vestibular areas, the melanocytes were in contact with Schwann cells and with myelinated fibres of vestibular nerve. The ultrastructural findings of this investigation are consistent with the hypothesis that melanocytes may have functional significance in the inner ear.
Agouti Alleles Influence Thiol Concentrations in Hair Follicles and Extrafollicular Tissues of Mice (Ay/a, AwJ/AwJ, a/a)
Authors: GRANHOLM DAVID E. , REESE R. NEIL , GRANHOLM NELS H.
Published: July 2006

Agouti protein (AP) expression in the wild‐type agouti mouse (AwJ/AwJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (Ay/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of Ay/a, AwJ/AwJ, and a/a (black) mice, and 2) adipose and spleen tissues of Ay/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. Ay/a hair follicles and serum showed highest cysteine and lowest GSH levels. AwJ/AwJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed Ay/a > a/a (P > 0.01). An Ay‐induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.
Incomplete Expression of the Tyrosinase Gene Family (Tyrosinase, TRP‐1, and TRP‐2) in Human Malignant Melanoma Cells In Vitro
Authors: EBERLE JÜRGEN , GARBE CLAUS , WANG NANPING , ORFANOS CONSTANTIN E.
Published: July 2006

Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP‐1 gene and 6F5 with TRP‐2 containing a long untranslated 3’ end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The Northern blots were probed with cDNA fragments specific for TRP‐1, TRP‐2, and tyrosinase, for nested tyrosinase‐PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP‐2 genes were found always to be expressed together, and TRP‐1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT‐PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP‐2 on one hand and TRP‐1 on the other. Overall, it seemed that RT‐PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP‐1, TRP‐2, and other, additional markers may be required.
A Transmission Electron Microscopic (TEM) Method for Determining Structural Colors Reflected by Lizard Iridophores
Authors: MORRISON RANDALL L.
Published: July 2006

Iridescent tissue colors are thought to be produced by iridophores through the optical phenomenon of thin‐layer interference. Land and others have shown that structural features, predominantly reflecting platelet width and the cytoplasmic spacing between layers of platelets, determine the wavelength of light maximally reflected by this mechanism in iridophores. Some researchers have used interference microscopy to estimate these structural parameters, but the most direct measurement technique should be transmission electron microscopy (TEM). Transmission electron microscopy (TEM) has associated processing artifacts (particularly cytoplasmic shrinkage) that preclude direct measurement of ultrastructure, but if a number of assumptions are made, reflected wave‐lengths can be predicted. A thin‐layer interference model and its associated assumptions were tested using TEM measurements of iridophores from several brightly colored tissues of each of three lizards (Sceloporus jarroui, S. undulatus erythrocheilus, and S. magister). In all the instances examined when the contribution of the pigments present were accounted for, tissue color corresponded with predicted iridophore reflectances from the model. Finally, if the model and its assumptions are assumed to be correct, the amount of iridophore cytoplasmic shrinkage as a result of TEM processing can be calculated.
The Identification and Partial Cloning by PCR of the Gene for Tyrosinase‐Related Protein‐1 in the Mexican Axolotl
Authors: MASON KENNETH A. , MASON SALLY K.
Published: July 2006

The tyrosinase gene family is currently composed of three members, tyrosinase and two tyrosinase‐related proteins, TRP‐1 and TRP‐2. These three gene products have all been found to act in the synthesis of melanin pigments with the enzyme tyrosinase catalyzing the initial rate‐limiting steps. Thus far these genes have primarily been analyzed in higher vertebrates. We have used degenerate PCR primers to isolate a large fragment of an axolotl tyrosinase‐related protein. Sequence analysis of the entire 1,057‐bp fragment isolated indicates a high degree of similarity to the mouse TRP‐1, the product of the brown locus. Phylogenetic analysis supports the conclusion that the fragment isolated corresponds to the axolotl TRP‐1 homolog. This is the first TRP‐1 gene to be identified in an amphibian species.
Effects of Extracellular Matrix Molecules on Subepidermal Neural Crest Cell Migration in Wild Type and White Mutant (dd) Axolotl Embryos
Authors: OLSSON LENNART , SVENSSON KRISTIAN , PERRIS ROBERTO
Published: July 2006

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168–181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/‐) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin‐nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/‐ embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control micro‐carriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultra‐structure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three‐dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.
Yellow Marking and Pteridine Contents in the Integument of Albino Armadillidium vulgare
Authors: NEGISHI SUMIKO , SUEOKA TERUMI , HASEGAWA YURIKO , KATOH SETSUKO
Published: July 2006

The albino mutant strain in the woodlice, Armadillidium vulgare, was investigated with respect to the yellow patterns on the dorsal integument. Pigment cells were observed with electron microscope in order to determine the cell types of yellow markings. Quantitative analyses of pteridines in the albino were carried out by HPLC. The result indicated that the albino integument contain sepiapterin, biopterin, pterin, isoxanthopterin as in the wild type and the red mutant strain. The total amount of the four pteridines in the albino was about half as much as that in the red phenotype for both males and females, respectively. Males and females showed almost the same totals and ratios of the four pteridines in the albino and red phenotypes. Therefore, pteridine contents in both phenotypes of A. vulgare may not be related to the activity of androgenic gland hormone. Yellow chromatophores of the albino and red phenotypes were morphologically identical, emitting a yellow fluorescence. These cells contained numerous electron‐lucent pigment organelles which were similar to pteridine granules of the wild type.
Interference by Cellular Melanin With Assay of DNA‐Protein Crosslinks by the Potassium Dodecyl Sulfate Precipitation Method
Authors: GOODMAN ROBERT M. , HILL HELENE Z.
Published: July 2006

The potassium dodecyl sulfate precipitation method was used to quantify DNA‐protein crosslinks (DPCs) in lysates of melanoma cells exposed to ultraviolet radiation. Inducing melanin production in these cells before exposure to ultraviolet radiation decreased the apparent yield of DPCs. The decrease could also be produced by addition of melanin to lysates after exposure to crosslinking conditions. Experimental models could attribute this decrease to neither quenching of scintillations from the tritium label used nor to an effect of single strand breaks of DNA. This assay appears to be inappropriate for quantification of DPCs in melanized cells.
Vitiligo, Psoralen, and Melanogenesis: Some Observations and Understanding
Authors: CHAKRABORTY D.P. , ROY S. , CHAKRABORTY A.K.
Published: July 2006

Since the etiology of vitiligo is still unknown, we searched for some abnormal biochemical parameters, if any, in subjects with vitiligo. Higher urinary excretion of indole metabolites in vitiliginous patients have been noted, in association with higher dioxygenase, superoxide dismutase, and tyrosine aminotransferase activity in their serum. Similar results have also been found in an animal model, Bufo melanostictus, during induced tyrosinase inhibition. Treatment with psoralen can reverse the parameters, except tyrosine aminotransferase, to a normal level. Although psoralens are not the magic bullet for the therapy of vitiligo, they are still being used as a chemotherapeutic agent against vitiligo on a major scale to date. Tryptophan was found to participate in the pathway of melanogenesis, as a precursor as well as a positive regulator of tyrosinase. Its behavior in this regard is much more similar to the conventional substrates tyrosine and dopa (dihydroxyphenylalanine). In consideration of combined participation of tyrosine and tryptophan in the synthesis of melanin and its breakdown, the possible influence of different enzymatic reactions, like mono‐oxygenase, dioxygenase, and deamination, has been suggested.
Pheomelanin Production by the Lipoxygenase‐Catalyzed Oxidation of 5‐S‐Cysteinyldopa and 5‐S‐Cysteinyldopamine
Authors: MOSCA LUCIANA , FOPPOLI CESIRA , COCCIA RAFFAELLA , ROSEI MARIA ANNA
Published: July 2006

5‐S‐cysteinyl‐dopa (cysdopa) and 5‐S‐cysteinyl‐dopamine (cysdopamine) are oxidized in vitro by soybean lipoxygenase (LOX) in the presence of hydrogen peroxide giving rise to the corresponding pheomelanins. The reaction is activated by caffeic acid and other catechols, suggesting a cofactor role for these compounds. The activating effect is proportional to the concentration of the cofactor, with a saturation profile. The activation extent of the various cofactors is directly related to LOX affinity for the same compounds. The possible implications of the peroxidative action of LOX in Parkinson's disease and in aging are discussed.
The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines
Authors: BIZIK JOZEF , BESSOU SANDRINE , FELNEROVA DIANA , VAHERI ANTTI , TAÏEB ALAIN
Published: July 2006

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low‐serum medium deprived of phorbol esters, cholera toxin, and other non‐physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue‐type plasminogen activator and utilised it to generate cell‐bound plasmin. No urokinase‐type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide‐spectrum proteinase inhibitor alpha‐2‐macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines.

The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.
Different Populations of Melanocytes Are Present in Hair Follicles and Epidermis
Authors: TOBIN DESMOND J. , BYSTRYN JEAN‐CLAUDE
Published: July 2006

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured. In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub‐populations of cells all reacted with melanocyte‐specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus, antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.
Signal Transduction by the Oncogenic Receptor Tyrosine Kinase Xmrk in Melanoma Formation of Xiphophorus
Authors: WELLBROCK CLAUDIA , GÓMEZ ANA , SCHARTL MANFRED
Published: July 2006

Melanoma formation in Xiphophorus is initiated by overexpression of an oncogenic version of the EGFR‐related receptor tyrosine kinase Xmrk (Xiphophorus melanoma receptor kinase). High steady‐state levels of Xmrk oncogene mRNA are found in malignant melanoma; however, this overabundance of transcripts appears to be not sufficient for manifestation of the full oncogenic potential of Xmrk. In addition, several amino acid exchanges cause the oncogenic receptor to be highly active, resulting in a strong tyrosine phosphorylation even without growth factor stimulation. Besides the receptor itself a Xmrk‐specific signal transduction seems to be a critical part of the transformation machinery. Expression experiments in transgenic fish indicate that the Xmrk‐mediated intracellular signalling is contributing to the cell‐type specificity in development of hereditary melanoma in Xiphophorus.
Melanocytes In Vitro: How Do They Undergo Mitosis?
Authors: KIPPENBERGER STEFAN , BERND AUGUST , BEREITER‐HAHN JÜRGEN , RAMIREZ‐BOSCA ANA , KAUFMANN ROLAND
Published: July 2006

Human melanocytes of the adult skin are slow‐cycling cells with a highly dendritic morphology. Nevertheless in vitro proliferation can be achieved using adequate stimulators. Time lapse studies revealed the morphologic changes during melanocyte mitosis: dendrites are drawn back into the cell body, the cell becomes spherical and detaches from the support. Cell division takes place while the cell is suspended. Consecutively the two cells reattach to the support and form new dendrites.

About 1% cells per culture are detached from the support and ca. 70% of these cells are viable and putative within mitosis. By every medium change mitotic cells become withdrawn supporting selection of G0‐cells, Therefore we recommend centrifugation of exhausted medium in order to add mitotic cells back to the culture.
Effect of Maltol on the Oxidation of DL‐DOPA, Dopamine, N‐Acetyldopamine (NADA), and Norepinephrine by Mushroom Tyrosinase
Authors: KAHN VARDA , BEN‐SHALOM NOAH
Published: July 2006

Maltol (3‐hydroxy‐2‐methyl‐4H‐pyran‐4‐one) appears to inhibit the rate of oxidation of DL‐DOPA, dopamine, NADA and epinephrine by tyrosinase when assayed spectropho‐tometrically but not when assayed polarographically. Maltol has an effect on the spectrum of product(s) formed when each catecholamine was oxidized by tyrosinase showing that maltol hastens the disappearance of the quinones, possibly by conjugating with them. Indeed, at relatively high concentrations, maltol prevented the conversion of DL‐DOPA, dopamine, and norepinephrine to their corresponding melanins via tyrosinase.
Clinical, Pathological, and Etiologic Aspects of Acquired Dermal Melanocytosis
Authors: MIZOGUCHI MASAKO , MURAKAMI FUMIKO , ITO MASARU , ASANO MARI , BABA TAKAKO , KAWA YOKO , KUBOTA YASUO
Published: July 2006

To study the pathogenesis of acquired dermal melanocytosis (ADM), we reviewed the clinical, immunohistochemical, and ultrastructual features of 34 cases (female, 33, and male, 1) of ADM. The patients’ ages at onset ranged from 8 to 51 years and averaged 26.8+12.7 years. There was a positive family history. Gray‐brown macules were mostly recognized on the face. Not only active dermal melanocytes but also non‐pigmented c‐KIT‐and TRP‐2‐positive immature melanocytes were detected in the dermis. Taken together those clinical and histological findings, activation of pre‐existing immature melanocytes by sunlight, estrogen, and/or progesterone, and some other factors, may be the most likely mode of the development of ADM. Moreover, using cultured murine neural crest cells as a model of c‐KIT‐positive immature melanocytes, we confirmed that endothelin‐1, which is produced and secreted by keratinocytes after UV‐irradiation, affects melanocytes and accelerated melanogenesis.
The Role of Endothelin‐1 in Epidermal Hyperpigmentation and Signaling Mechanisms of Mitogenesis and Melanogenesis
Authors: IMOKAWA G. , KOBAYASHI T. , MIYAGISHI M. , HIGASHI K. , YADA Y.
Published: July 2006

The paracrine linkage of endothelins (ET) between keratinocytes and melanocytes suggested that ETs are intrinsic mediators for human melanocytes in UVB‐induced pigmentation. In this study, the role of ET‐1 in the epidermal hyperpigmentation was investigated in vivo and in vitro. The addition of 10 nM ET‐1 induced a H‐7 (10 μM) suppressible‐increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase‐related protein‐1 mRNA expression as shown by Northern blotting. Analysis of signaling mechanisms leading to tyrosinase activation demonstrated the involvements of quick translocation of PKC, the H‐7 (10 μM) suppressible‐phosphorylation of the threonine residue of several proteins, and highly elevated level of cyclic AMP (4‐fold over control). Reverse transcription polymerase chain reaction (RT‐PCR) of RNA isolated from the epidermis of human skin exposed to UVB revealed that UVB irradiation with a dose of 2 MED caused a significant increase in the expressions of ET 1, IL‐1α, and tyrosinase mRNA signals 5 days after irradiation. The involvement of ET‐1 in UVB‐pigmentation was also corroborated by the experiments that the extracts of M. Chamomilla, which can act as an antagonist for ET receptor binding‐mediated signaling but has no inhibitory effect on tyrosinase activity in culture, had a significant inhibitory effect on UVB‐induced pigmentation in vivo when daily applied immediately after UVB exposure to human skin. These findings suggest that ET‐1 is an important mediator in the epidermis for UVB‐induced pigmentation in vivo.
Biosynthesis, Structure, and Function of Neuromelanin and its Relation to Parkinson's Disease: A Critical Update
Authors: D'ISCHIA MARCO , PROTA GIUSEPPE
Published: July 2006

No longer dismissed as just a mere curiosity in the family of melanin pigments, neuromelanin is attracting increasing interest as a central constituent of certain populations of dopaminergic neurons in the human substantia nigra, which may hold the key for the understanding of neuron functioning and degeneration in aging and in Parkinson's disease. It is the purpose of this article to provide a concise review of the most significant data on the origin, structure, and functional significance of neuromelanin that accrued over the past few years. It also aims at critically surveying the currently debated views regarding the role of such intriguing pigment in the etiology and biochemical pathology of Parkinson's disease.
A Big Gene Linked to Small Eyes Encodes Multiple Mitf Isoforms: Many Promoters Make Light Work
Authors: YASUMOTO KEN‐ICHI , AMAE SHINTARO , UDONO TETSUO , FUSE NOBUO , TAKEDA KAZUHISA , SHIBAHARA SHIGEKI
Published: July 2006

Among more than 80 different loci related to mouse coat color, microphthalmia‐associated transcription factor (Mitf) encoded at the mouse microphthalmia locus is one of the most exciting molecules that regulates the development and survival of many cell types, including melanocyte, retinal pigment epithelium (RPE), and mast cells. Mitf and its human homolog MITF consist of at least three isoforms, referred to as Mitf‐A/ MITF‐A, the heart‐type Mitf‐H/MITF‐H, and the melanocyte lineage‐specific Mitf‐M/ MITF‐M, respectively. These isoforms differ in the amino‐terminal domains but share a transactivation domain and a basic helix‐loop‐helix and leucine‐zipper structure that is required for DNA binding and dimerization. MITF‐M is exclusively expressed in melanocytes and melanoma cells, but not in other cell types, including RPE cells. In contrast, MITF‐A mRNA is widely expressed in many cell types. These three isoform mRNAs are possibly generated by differential usage of the gene promoters and by alternative splicing. We predict that the entire MITF gene spans about 200 kb of DNA. Like MITF‐M, MITF‐A is able to activate the two melanogenesis gene promoters, tyrosinase and tyrosi‐nase‐related protein 1. These results suggest that melanogenesis may be regulated by different MITF isoforms in melanocyte and RPE. Possible implications of the multiplicity in Mitf/MITF isoforms are discussed.
Arbutin Increases the Pigmentation of Cultured Human Melanocytes Through Mechanisms Other Than the Induction of Tyrosinase Activity
Authors: NAKAJIMA MITSUNARI , SHINODA ICHIZO , FUKUWATARI YASUO , HAYASAWA HIROTOSHI
Published: July 2006

We assessed the effects of arbutin on the pigmentation of cultured normal human melanocytes. As indicated by a cell‐blotting assay, arbutin at concentrations in the range of 0.5–8 mM increased the pigmentation of the cultured melanocytes, while kojic acid at concentrations in the range of 0.5–4 mM decreased the pigmentation. The pigmentation‐augmenting effect of arbutin was further confirmed by the results of a cellpelleting assay, the traditional method of assessment. Treatment of the cells with arbutin increased the melanin content of the cells and the protein content as well. On the other hand, the tyrosinase activity in the cells was reduced by arbutin treatment. The levels of transcription of tyrosinase and tyrosinase related protein‐1 genes were not affected by arbutin treatment as indicated by a semi‐quantitative reverse transcription‐polymerase chain reaction assay. These results demonstrate that arbutin promotes an increase in pigmentation of cultured human melanocytes that is not mediated by augmented tyrosinase activity.
In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes
Authors: SHOJI TORU , PARK HEE‐YOUNG , JALBERT NICOLE , BHAWAN JAG , BYERS H. RANDOLPH
Published: July 2006

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC‐α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ.

Immunohistochemical staining for PKC‐α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3‐fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC‐α and Mel‐5 (tyrosinase related protein‐1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC‐α showed strong PKC‐α mRNA expression in cultured melanocytes, whereas PKC‐α mRNA in cultured non‐stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC‐α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10‐fold more PKC‐α mRNA and approximately 6‐fold more PKC‐α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5‐fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC‐α provides further evidence for cell type specificity in the balance of PKC‐α expression and may implicate differential PKC isoform signaling pathways in neuro‐ectodermally derived cells.
N‐Acetyl‐L‐Tyrosine (NAT) as a Substrate for Mushroom Tyrosinase
Authors: KAHN VARDA , BEN‐SHALOM NOAH
Published: July 2006

N‐acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N‐acetyl dopa formed is oxidized by the enzyme to N‐acetyl dopaquinone (λmax= 390 ± 10 nm). H2O2 and NH2OH each shortened the lag period of NAT hydroxylation by the enzyme. H2O2 had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H2O2 converts N‐acetyl dopaquinone to a pink‐violet product(s) (λmax= 490 nm), whereas such a product(s) was not formed in the absence of H2O2.

A pink‐violet product(s) (λmax 490 ± 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH2OH or para amino benzoic acid (PABA), probably as a result of an interaction between N‐acetyl dopaquinone and NH2OH or PABA forming mono‐ or di‐oximes.

Kojic acid (5‐hydroxy‐2‐hydroxymethyl)‐4H‐pyran‐4‐one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H2O2. When NAT was oxidized by the enzyme in the absence of kojic acid, N‐acetyl dopaquinone was formed at once and a shoulder at 490–530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320±10 nm, was detected, suggesting that N‐acetyl dopaquinone oxidizes kojic acid to the yellow product(s).

Maltol(3‐hydroxy‐2‐methyl‐4H‐pyran‐4‐one), a γ‐pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N‐acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N‐acetyl dopaquinone alone.

The final brown‐red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H2O2.
The Mechanism of Melanocyte Dendrite Formation: The Impact of Differentiating Keratinocytes
Authors: KIPPENBERGER STEFAN , BERND AUGUST , BEREITER‐HAHN JÜGEN , RAMIREZ‐BOSCA ANA , KAUFMANN ROLAND
Published: July 2006

In human epidermis one dendritic melanocyte interacts with about 36 keratinocytes and supplies them with melanin. In contrast to the vivo situation melanocytes in culture are far less dendritic.

In the present study different culture systems were tested in order to observe the mechanism of melanocyte dendrite formation. In particular, we focused on the role of keratinocytes in this process. Time lapse studies revealed that only differentiated keratinocytes enhance melanocyte dendricity. Differentiated keratinocytes form connected cell sheets, which attach to part of the melanocyte plasma membrane. By contraction and retraction of keratinocyte units, new dendrites were drawn out from the melanocytes. Melanocytes remain passive during this process, which is indicated by the observation that sometimes extended dendrites could not withstand the tension and shear.
Tyrosinase Autoactivation and the Problem of the Lag Period
Authors: NAISH‐BYFIELD SANDRA , RILEY PATRICK A.
Published: July 2006

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4‐hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS‐PAGE separation showed labelling of the enzyme when incubated with 14C ring‐labelled 4‐hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met‐tyrosinase and explains the lag kinetics of tyrosinase oxidation of non‐cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met‐enzyme‐recruiting substrate.
Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro
Authors: EBERLE J. , WAGNER M. , MACNEIL S.
Published: July 2006

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase‐related genes [tyrosinase, tyrosinase‐related protein‐1 (TRP‐1) and tyrosinase‐related protein‐2 (TRP‐2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase‐related genes in these cells in culture. Four of the cell lines were also stimulated with α‐MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes.

There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α‐MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α‐MSH was without effect; and TRP‐1 and TRP‐2 expression were largely unaffected by IBMX or α‐MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase‐related genes.
Does α‐MSH Have a Role in Regulating Skin Pigmentation in Humans?
Authors: THODY ANTHONY J. , GRAHAM ALISON
Published: July 2006

Over the years there has been much debate as to whether α‐MSH has a role as a pigmentary hormone in humans. There are two main reasons for this. First, despite the observations in the 1960s that α‐MSH increased skin darkening in humans, there are reports that the peptide has no effect on melanogenesis in cultured human melanocytes. Second, the human pituitary, unlike that of most mammals, secretes very little α‐MSH and circulatory levels of the peptide in humans are extremely low. However, there is now evidence from several groups that α‐MSH is capable of stimulating melanogenesis in cultured human melanocytes. Rather than producing an overall increase in melanin production, it appears that the peptide acts specifically to increase the synthesis of eumelanin. Such an action could well explain the previously observed skin darkening effects of α‐MSH. It is also now known that α‐MSH is not produced exclusively in the pituitary but has been found at numerous sites, including the skin where it is produced by several cell types. Related Proopiomelanocortin (POMC) peptides such as ACTH are also produced in human skin. The ACTH peptides act at the same receptor (MC‐1) as α‐MSH and certain of these would appear to be more potent than α‐MSH in stimulating melanogenesis. The ACTH peptides are also present in greater amounts than α‐MSH in human epidermis and it is likely that they play an important role in regulating pigmentary responses. These POMC peptides are released from keratinocytes in response to ultraviolet radiation (UVR) and it has been proposed that they serve as paracrine factors in mediating UV induced pigmentation. Their production by keratinocytes could therefore be critical in determining pigmentary responses and any changes in the availability of these POMC peptides might explain the variations in tanning ability seen in different individuals. However, the possibility that tanning ability is also dependent upon differences at the level of the MC‐1 receptor cannot be ruled out and it has been suggested that an inability to tan may depend upon the presence of non‐functional changes at the MC‐1 receptor. α‐MSH does, of course, affect human melanocytes in several ways and its stimulation of melanogenesis could be the consequence of some other fundamental action in the melanocyte. The peptide also has many other target sites in the skin and while it may have a role in regulating skin pigmentation in humans, it should not be viewed solely as a pigmentary peptide. α‐MSH clearly has many different actions and its primary role in the skin may be to maintain homeostasis.
Enhanced Melanogenesis Induced by Tyrosinase Gene‐Transfer Increases Boron‐Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma
Authors: TSUBOI TATSUYA , KONDOH HIROFUMI , HIRATSUKA JUNICHI , MISHIMA YUTAKA
Published: July 2006

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, 10B‐p‐boronophenylalanine (10B‐BPA), has been established, but amelanotic melanoma is insufficiently responsive to 10B‐BPA BNCT in comparison with actively melanin‐producing melanoma. Although the accumulation mechanism of 10B‐BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in 10B‐BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase‐related protein (TRP)‐l and TRP‐2 are well expressed. Thus, by using a similarly tyrosinase‐lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase‐cDNA into these cells. TA1059 cells acquired higher DOPA‐oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher p‐boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by 10B‐BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene‐transfer effectively improves the treatment of amelanotic melanoma by BNCT.