bluuebunny/crossref_metadata_2025 huggingface dataset:
Thiols in the Melanocyte
Authors: RORSMAN H. , ALBERTSSON E. , EDHOLM L. E. , HANSSON C. , ÓGREN L. , ROSENGREN E.
Published: July 2006

Melanocytes contain several substances formed by the nucleophilic addition of cysteine to dopaquinone. 5‐S‐Cysteinyldopa is the quantitatively dominant catecholic amino acid belonging to this group of compounds. Glutathione is the thiol most abundantly present in all cells studied, and the reactivity of the SH‐group of this tripeptide with dopaquinone is about one‐third that of cysteine. However, the amount of glutathionyldopa is at least two orders of magnitude less than that of cysteinyldopa in the melanocyte. A rapid metabolism of glutathionyldopa has therefore been suggested as an explanation for the above‐mentioned findings. The enzyme responsible for hydrolysis of the γ‐glutamyl bond of glutathione, γ‐glutamyltranspeptidase, is present in the melanocyte, but in small quantities. Furthermore, S‐cysteinylglycinyldopa, which is the product of hydrolysis by γ‐glutamyltranspeptidase, is found in only very small amounts. These facts taken together contradict the hypothesis that S‐cysteinyldopas in the melanocyte are formed from S‐glutathionyldopas. The present investigation on IGR1 melanoma cells was performed by in situ derivatization of thiols with monobromobimane. Quantitation of the stable bimane adducts of cysteine and glutathione was achieved by reverse‐phase high‐performance liquid chromatography with fluorimetric detection. The concentration of reduced cysteine in the melanocytes was found to be a few percent of that of reduced glutathione. The quantities of 5‐S‐cysteinyldopa, 5‐S‐glutathionyldopa, cysteine, and glutathione observed in the cultured melanoma cells could best be explained by a pronounced compartmentalization of cysteine within the melanocyte, with a high cysteine concentration at the site of the dopaquinone formation.
Chromatophoromas in a Pine Snake
Authors: JACOBSON E.R. , FERRIS W. , BAGNARA J.T. , IVERSON W.O.
Published: July 2006

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment‐containing cells. Pigment cells within abnormal‐appearing white scales had needle‐shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber‐colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1½ years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.
Phenolic Melanin Precursors Provide a Rational Approach to the Design of Antitumor Agents for Melanoma
Authors: JIMBOW KOWICHI , MIURA TAKAKO , ITO SHOSUKE , ISHIKAWA KIICHI
Published: July 2006

A unique biological property of the melanocyte, melanin synthesis may permit a rational approach to design agents for better management of malignant melanoma. This in vivo and in vitro study examined the selective melanocytotoxicity and antimelanoma effects of phenolic compounds, cysteinylphenol (CP), cysteaminylphenol (CAP), and related compounds, and found (1) that both 4‐S‐CP and 4‐S‐CAP are melanin precursors, (2) that 4‐S‐CAP possesses a marked depigmenting potency with selective destruction of melanocytes in black follicles, and (3) a significant inhibition in the protein synthesis and tumor growth of B16 melanoma. Importantly, a whole body autoradiography indicated that these phenolic melanin precursors are selectively incorporated into melanoma tissues after i.p. administration.
Eye Color Pigment Granules in Drosophila mauritiana: Mosaics Produced by Excision of a Transposable Element
Authors: STARK WILLIAM S. , SAPP RANDALL , HAYMER DAVID S.
Published: July 2006

Compound eyes of the white‐peach (wpch) mutant strain of Drosophila mauritiana have some pigment and receptor cells with wild‐type eye color pigmentation. These eyes are mosaic, because excision of a transposable element reverts wpch to wild type during the development of somatic cells. Wild‐type patches have three types of pigment granule residing in three respective cell types: primary pigment cells, secondary pigment cells, and retinula (visual receptor) cells. Most aspects of these granules, as well as all other aspects of compound eye ultrastructure, are exactly as in the better studied sibling species D. melanogaster. In the wpch parts of the eye, small and giant unpigmented “pigment granules” reside in secondary pigment cells. These white granules are just like the corresponding granules of w mutant D. melanogaster. Small vs. large patches of pigmented cells likely represent excision events occurring late vs. early respectively during development. Mosaics of eye color markers have been important in developmental analyses; the ease of constructing mosaics of D. mauritiana gives this preparation advantages for mosaic analyses
Melanogenesis in the Pigment Cells of Rana esculenta L. Liver: Evidence for Tyrosinase‐Like Activity in the Melanosome Protein Fraction
Authors: CICERO ROSA , MALLARDI ANTONIA , MAIDA IMMACOLATA , GALLONE ANNA , PINTUCCI GIUSEPPE
Published: July 2006

This work demonstrates the presence of a tyrosinase‐like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.
Effect of the Barring Gene on Eye Pigmentation in the Fowl
Authors: SCHRECK RANDALL E. , BOWERS ROGER R.
Published: July 2006

Pigment cells of the iris, pecten, retinal pigment epithelium, and choroid of the wild‐type jungle fowl (JF) and the barred Plymouth rock (BPR) breeds of adult chickens were studied at both light and electron microscopic levels. BPR choroidal tissues had 2.8 times fewer melanophores than the JF choroid, and BPR melanophores also contained 2.4 times fewer melanosomes, which tended to clump together in variously sized clusters. The melanosomes were often irregular in shape, smaller in diameter, and less mature (stage III) than those granules in the JF. The retinal pigment epithelium of both JF and BPR breeds contained a single epithelial layer of columnar cells. Rod‐shaped melanosomes were present in the more apical regions of this cell type in both breeds. Both JF and BPR irides contained a multilayered posterior pigmented epithelium of columnar‐shaped cells that were densely filled with large spherical granules. Intercellular spaces with interdigitating cytoplasmic projections were present between pigment cells of both breeds. The pecten melanophores of both breeds were dendritic with melanosomes that were larger and fewer in numbers than those pigment cells of the iris and choroid. Intercellular spaces were present between cells in both breeds, with numerous villous‐like pigment cell extensions. Choroid melanophores contained very little, if any, acid phosphatase activity. Approximately one‐half of the retinal pigment epithelial cells observed contained small amounts of diffuse acid phosphatase activity in both breeds. The iris and pecten melanophores of both breeds contained profuse acid phosphatase activity scattered throughout their cytoplasms. Sparse tyrosinase activity was seen in iris and pecten pigment cells, whereas no tyrosine activity was observed in choroid melanophores or in retinal pigment epithelial cells in the two breeds, indicating that little new melanogenesis occurs in adult pigmented eye tissues. The results show that the barring gene reduces the number and melanin content of the choroidal melanophores in homozygous male BPR chickens as compared to the wild‐type JF chickens. Whether this gene prevents the initial migration of embryonic neural crest cells (future melanophores) to the choroid or whether some of the choroidal melanophores prematurely degenerate in the embryo of young birds is yet to be determined. If the latter is the case, this choroid system may serve as a model for a genetic hypomelanotic disease such as vitiligo.
Syngeneic Monoclonal Antimelanoma Antibodies and Their Application for Analysis of Tumor Antigens, Gene Cloning, and In Vitro/In Vivo Diagnosis
Authors: TANIGUCHI MASARU , WAKABAYASHI SEIJI , TAGAWA MASATOSHI
Published: July 2006

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)‐like sugar moiety. The GM3 structure expresses the cross‐species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 102‐103 cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross‐species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal antimelanoma M562 and the fluorescence‐activated cell sorter. We also found that genomic DNA possesses transformation‐related activity in NIH3T3 cells.
Boron Neutron Capture Therapy for Malignant Melanoma: An Experimental Approach
Authors: LARSSON BENGT S. , LARSSON BÖRJE , ROBERTO AMILCAR
Published: July 2006

Previous studies have shown that some thioamides, e.g., thiouracil, are incorporated as false precursors into melanin during its synthesis. If boronated analogs of the thioamides share this property, the melanin of melanotic melanomas offers a possibility for specific tumoural uptake and retention of boron as a basis for neutron capture therapy. We report on the synthesis of boronated 1H‐1,2,4‐triazole‐3‐thiol (B‐TZT), boronated 5‐carboxy‐2‐thiouracil (B‐CTU), and boronated 5‐diethylaminomethyl‐2‐thiouracil (B‐DEAMTU) and the localization of these substances in melanotic melanomas transplanted to mice. The distribution in the mice was studied by boron neutron capture radiography. B‐TZT and B‐CTU showed the highest tumour:normal tissue concentration ratios, with tumour:liver ratios of about 4 and tumour:muscle ratios of about 14; B‐DEAMTU showed corresponding ratios of 1.4 and 5, respectively. The absolute concentration of boron in the tumours, however, was more than three times higher in the mice injected with B‐TZT, compared with B‐CTU. The results suggest that B‐TZT may be the most promising compound of the three tested with regard to possible therapy of melanotic melanomas.
Electron Spin Resonance Study of Photoinduced Radicals in Various Eumelanins in Solid Matrix
Authors: BIBANG RENÉ , ARNAUD RENÉ , LEMAIRE JACQUES , DEFLANDRE ANDRÉ , LANG GÉRARD
Published: July 2006

The photoinduced radicals formed in eumelanin from hair at various excitation wavelengths, in KBr matrix, are compared with the photoinduced radicals observed with bovine eye iris and squid ink eumelanins. Qualitative similarities and quantitative differences are observed, specially on exposure at short wavelengths.
Biochemical Analysis of Metastasis‐Related Ax Actin in B16 Mouse Melanoma Cells After Chemical Reversional Modulation and of Tumor Progression‐Related A′ Actin in the Ontogeny of Human Malignant Melanoma
Authors: SASASE AKIHIRO , MISHIMA YUKATA , ICHIHASHI MASAMITSU , TANIGUCHI SHUN'ICHIRO
Published: July 2006

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A′, actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metasasis of established highly metastatic B16‐F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A′ actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16‐F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long‐term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A′ actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A′ actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.
Quantitative In Vitro Assay for Crustacean Chromatophorotropins and Other Pigment Cell Agonists
Authors: BRITTO ANA LUCIA M. , CASTRUCCI ANA MARIA DE L. , VISCONTI MARIA APARECIDA , JOSEFSSON LARS
Published: July 2006

An in vitro crustacean (freshwater shrimp, Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 ± 10−12M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the Ionophore‐induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore‐treated erythrophores. The potencies of α‐PDH and β‐PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure‐activity studies of the various chromatophorotropins and their analogs.
Action of Light on Frog Pigment Cells in Culture
Authors: DANIOLOS ATHENA , LERNER AARON B. , LERNER MICHAEL R.
Published: July 2006

Solar radiation induces numerous biologic effects in skin but the mechanism underlying these responses is poorly understood. To study the etiology of these phenomena, we investigated the effect of light on cultured Xenopus laevis melanophores. Visible light stimulated a marked increase in intracellular cAMP levels within the first minute of irradiation. This light‐induced elevation in cAMP was blocked by melatonin and was not seen in fibroblasts irradiated in a similar manner. These data show that the photoresponse of pigment cells from amphibian skin can be mediated by a cAMP‐dependent mechanism and suggest that a unique member of the rhodopsin family is involved in this process.
Transport of L‐Tyrosine by B16/F10 Malignant Melanocytes: Characterization of the Process
Authors: JARA J.R. , MARTINEZ‐LIARTE J.H. , SOLANO F.
Published: July 2006

The main characteristics of L‐tyrosine (L‐Tyr) uptake by B16/F10 malignant melanocytes are reported. This amino acid can be taken up by two systems, both of them being saturable. The first one would be system L. This system can be studied in cells preloaded with amino acids that are a good substrate for system L, such as L‐methionine or L‐tryptophan. The kinetic parameters for L‐Tyr uptake by this transport system are Vm= 6.5 pmol L‐Tyr/103 cell·smin and Km around 130 μM. The second system, probably the system ASC, shows lower capacity but higher affinity than the former. This system can be detected only in cells previously depleted of amino acids, showing approximate kinetic values of Vm 0.05 pmol L‐Tyr/103 cell·smin and Km around 5 μM. It is shown that the increase in cell density yields a decrease in the rate of L‐Tyr uptake by system L, but this increase does not affect the high affinity system. α‐MSH does not affect significantly the L‐Tyr uptake by both systems. 2‐Amino bicyclo‐(2,2,l)‐heptane‐2‐carboxylic acid produces a remarkable inhibition of the rate of L‐Tyr uptake, but α‐methylaminoisobutyric acid does not affect the rate of transport of this amino acid. The absence of sodium produces a slight but reliable decrease in the rate of L‐Tyr uptake, supporting the involvement of two different transport systems. The ionophores monensin and nigericin enhance the transport by system L, but this effect is suppressed by the presence of ouabain. This finding indicates that the (Na + ‐K+)‐ATPase is essential for the stimulating action of ionophores. Finally, it is shown that γ‐glutamyl cycle is not involved in L‐Tyr uptake, since the inhibition of γ‐glutamyl transpeptidase by periodate treatment does not affect the rate of transport.
Contributions of the Gordon‐Kosswig Melanoma System to the Present Concept of Neoplasia
Authors: ANDERS FRITZ
Published: July 2006

Modern cancerology is based on the oncogene concept. This is rather new. The idea of the oncogene, however, is old, and can be traced back to two sources, namely to “cancer families,” reported in 1866 by P. Broka, and to “virus induced” neoplasia, detected by P. Rous in 1911. A gene which is—to my knowledge—the first reported oncogene by definition was detected in the little ornamental Mexican fish Xiphophorus by Myron Gordon, Curt Kosswig, and Georg Häussler in 1928 when they observed the terrible hereditary melanomas that we are now coming to understand and to compare with other kinds of neoplasms in Xiphophorus and in mammals, including humans. Although the Xiphophorus model was always modest in its claims, it has—sometimes too early in its history—contributed many facts to the present concept of neoplasia.
Comparative Analysis of Hair Melanins by Chemical and Electron Spin Resonance Methods
Authors: VSEVOLODOV EDUARD B. , ITO SHOSUKE , WAKAMATSU KAZUMASA , KUCHINA IRINA I. , LATYPOV IGOR F.
Published: July 2006

Eighteen hair samples from Karakul newborn lambs with various colors were estimated for eumelanin and pheomelanin contents (Ce and Cp, respectively) by electron spin resonance (ESR) spectrometry and by high‐performance liquid chromatography (HPLC). Correlation coefficients between the values estimated by the ESR and HPLC methods were 0.96, 0.93, and 0.99 for Ce, Cp, and Ce/Cp, respectively. The high correlation coefficients show that both methods fit well for estimation of relative values of these parameters. The absolute values of Ce and Ce/Cp coincide rather well when Ce is high, but considerable discrepancies appear when Ce is low. The reasons for these discrepancies are discussed. The HPLC method appears to be more sensitive for detection of low concentrations of pheomelanin, while the ESR method fits well for mass selection purposes.
An Estimate of Melanosome Concentration in Pigment Tissues
Authors: BOROVANSKÝ JAN , VEDRALOVÁ ELIŠKA , HACH PETR
Published: July 2006

Concentration of melanosomes in various tissues has been unknown because of the impracticability of their direct quantification. Using an indirect approach comprising the estimation of melanin both in freeze‐dried tissue samples and in isolated melanosomes, we obtained data on the amount of melanosomes in various pigment tissues. The concentrations of melanosomes found in the tissues were relatively high, not only reflecting the dark color of pigment tissues but also explaining their capacity to perform various functions ascribed to the presence of melanin.
The Protein‐Phosphatase Inhibitor Okadaic Acid Mimics MSH‐Induced and Melatonin‐Reversible Melanosome Dispersion in Xenopus laevis Melanophores
Authors: COZZI BRUNO , ROLLAG MARK D.
Published: July 2006

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a‐melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein‐phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 μM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin‐induced pigment aggregation, but instead induce aggregation on their own.
Functional Properties of Cloned Melanogenic Proteins
Authors: HEARING VINCENT J. , TSUKAMOTO KATSUHIKO , URABE KAZUNORI , KAMEYAMA KOICHIRO , MONTAGUE PAUL M. , JACKSON IAN J.
Published: July 2006

Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct catalytic activities—tyrosine hydroxylase, 3,4‐dihydroxyphenylalanine (DOPA) oxidase and DHI (5,6‐dihydroxyindole) oxidase. The brown locus encodes TRP‐l (tyrosinase‐related protein‐I), which has the same, but greatly reduced, catalytic potential. The slaty locus encodes TRP‐2, another tyrosinase related‐protein, which has DOPAchrome tautomerase activity. In this study we have examined the enzymatic interactions of these proteins, and their regulation by a novel melanogenic inhibitor. We observed that tyrosinase activity is more stable in the presence of TRP‐l and/or TRP‐2, but that the catalytic function of TRP‐2 is not affected by the presence of TRP‐1 or tyrosinase. Other factors also may influence melanogenesis and a unique melanogenic inhibitor suppresses tyrosinase and DOPAchrome tautomerase activities, but does not affect the spontaneous rate of DOPAchrome decarboxylation to DHI. The results demonstrate the catalytic functions of these proteins and how they stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized by the melanocyte.
Molecular Characterization of the pun Allele of the Mouse Pink‐Eyed Dilution Locus
Authors: BRILLIANT MURRAY H. , GONDO YOICHI
Published: July 2006

The mouse pink eyed dilution locus, p, located on chromosome 7, mediates coat and eye color. The human correlate of this gene may underlie some forms of tyrosinase‐positive oculocutaneous albinism. Mutations at the p locus result in a reduction in pigmentation of the eyes and coat. Although most mutant p alleles (including all spontaneous mutations) affect only pigmentation, several mutant alleles (all radiation induced) are also associated with a variety of other phenotypes. We have focused our attention on the pun mutant allele, a spontaneous mutation, exhibiting one of the highest reversion frequencies reported for a mammalian mutation. Using a new technique, genome scanning, we have cloned fragments of genomic DNA from the p locus that are associated with a DNA duplication in pun DNA. These fragments can now be used to locate the p gene‐encoding sequences and aid in the molecular characterization of complex mutant p alleles.
Melanocyte‐Stimulating Properties of Arachidonic Acid Metabolites: Possible Role in Postinflammatory Pigmentation
Authors: TOMITA YASUSHI , MAEDA KAZUHISA , TAGAMI HACHIRO
Published: July 2006

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b‐locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D2, leukotriene (LT) B4, LTC4, LTD4, LTE4, thromboxane (TX) B2 and 12‐hydroxy eicosatetraenoic acid (12‐HETE). The effect of LTC4 was particularly strong compared to that of PGE2, about which we have previously reported. On the other hand, PGE1, PGF2α and 6‐ketoPGF1α did not show any significant stimulatory effect. These data suggest that arachidonate‐derived chemical mediators, especially LTC4, may be responsible for the induction of post‐inflammatory hyperpigmentation of the skin.
Control of Melanocyte Proliferation and Differentiation in the Mouse Epidermis
Authors: HIROBE TOMOHISA
Published: July 2006

Melanocyte‐stimulating hormone plays an important role in the regulation of melanocyte differentiation in the mouse epidermis by inducing tyrosinase activity, melanosome formation, translocation of melanosomes, and increased dendritogenesis. The proliferative activity of differentiating epidermal melanocytes of newborn mice during the healing of skin wounds is regulated by semi‐dominant genes, suggesting that the genes are involved in regulating the proliferative activity of epidermal melanocytes during differentiation. From the results of serum‐free culture of epidermal cell suspensions from neonatal mouse skin, basic fibroblast growth factor is shown to stimulate the sustained proliferation of melanoblasts in the presence of dibutyryl adenosine 3′,5′‐cyclic monophosphate and keratinocyte‐derived factors. Moreover, each step of melanocyte differentiation is controlled by numerous coat color genes. These genes control melanocyte differentiation by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating transcription and/or translation of the tyrosinase gene in the differentiating melanocytes. These results suggest that melanocyte proliferation and differentiation in the mouse epidermis are controlled by both genetic factors and local tissue environment.
In Vivo and In Vitro Expression of Octamer Binding Proteins in Human Melanoma Metastases, Brain Tissue, and Fibroblasts
Authors: THOMSON J. ANGUS F. , PARSONS PETER G. , STURM RICHARD A.
Published: July 2006

The pattern of octamer sequence‐specific DNA binding proteins expressed in human melanoma was examined in nuclear extracts of seven surgically‐isolated tumors, short‐term cultures of these tumors, and 25 human melanoma cell lines to determine the in vivo and in vitro distribution of the melanocytic‐associated Oct‐M1 and Oct‐M2 octamer binding activities. In the biopsy tissue and cultured melanoma cells of a metastasis from the cerebellum, two other binding activities (N‐Oct‐2 and N‐Oct‐6) in addition to the Oct‐M1, Oct‐M2 and the generally expressed Oct‐1 protein were detected; this profile was consistent with that seen in normal human and mouse brain tissue. Melanoma tissue removed from lymph nodes and cell lines established from them also showed Oct‐1, Oct‐M1, Oct‐M2, and N‐Oct‐2. N‐Oct‐2 was distinguished from the comigrating Oct‐2A activity by failure to react with Oct‐2A‐specific antibody. All but one of the 25 melanoma cell lines exhibited Oct‐1, Oct‐M1, and Oct‐M2 and/or N‐Oct‐2 activity, whereas cultured normal melanocytes expressed only Oct‐1 and Oct‐M1. In contrast to murine fibroblasts, which express only Oct‐1, human fibroblast strains also expressed Oct‐2A binding activity, which was confirmed by reactivity with Oct‐2A antibody and the presence of Oct‐2A mRNA and indicated that Oct‐2A has a more general role than that of a lymphoid‐specific transcription factor. Overall, the results indicate that expression of neural‐specific Oct factors in human melanoma is (1) aberrant compared with normal melanocytes, (2) can be modulated by the surrounding tissue in a brain metastasis, and (3) may be part of the altered program of differentiation accompanying transformation.
In Situ Detection of Immunocompetent Cells in Murine B16 Melanoma Locally Treated With Interleukin‐2 or Microwaval Hyperthermia
Authors: NAKAYAMA JUICHIRO , URABE ATSUMICHI , TERAO HIROSHI , TANIGUCHI SHUN'ICHIRO , HORI YOSHIAKI
Published: July 2006

Fidler's B16‐F10 melanoma was locally treated either with recombinant interleukin‐2 (rIL‐2) or microwaval hyperthermia, and immunological responses of the host to the melanoma after each treatment were investigated by immunofluorescent staining of the tissue. It was found that the local injection of rIL‐2 either into the base of the tumor or into the upper part of the tumor directly caused infiltration of mainly NK cells and macrophages in the interstitials and/or in the tumor nests. T cells were also observed but the extent of infiltration was less in both treatments. Local microwaval hyperthermia of melanoma at 42°C for 30 min or at 43°C for 15 min also caused infiltration of NK cells and macrophages. Positive staining of the melanoma tissue with anti‐ICAM‐1 antibody after hyperthermia was seen in the interstitials adjacent to melanoma nests containing necrotic melanoma cells caused by hyperthermia. Based on the results, the rationality of combination of hyperthermia with local injection of rIL‐2 will be discussed.
Preparation of Purified Tyrosinase Devoid of Dopachrome Tautomerase From Mammalian Malignant Melanocytes
Authors: VALVERDE P. , JIMÉNEZ‐CERVANTES C. , SALINAS C. , GARCÍA‐BORRÓN J.C. , SOLANO F. , LOZANO J.A.
Published: July 2006

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60°C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT‐free tyrosinase from mammalian malignant melanocytes grown in culture.
Mechanistic Aspects of the Control of Tyrosinase Activity
Authors: RILEY P.A.
Published: July 2006

There is now much evidence suggesting that there are multiple control points in the process of melanin production. The most fundamental process of melanogenesis is centered on the oxidative activity of the enzyme tyrosinase. Tyrosinase is a highly unusual enzyme in that it apparently catalyses two processes, i.e., the oxidation of tyrosine and the dehydrogenation of dihydroxyphenylalanine (Dopa), at the same active site. The reactions involved account for the unusual kinetics of tyrosine oxidation and suggest biochemical mechanisms whereby the activity of the enzyme and the process of melanogenesis may be modified. It is proposed that the oxidative engine of melanogenesis resides in an oxidation/reduction cycle involving Dopa and dopaquinone and that this can be modified by processes that result in the removal of dopaquinone or Dopa from the reaction system.
Light (Blt), a Mutation That Causes Melanocyte Death, Affects Stria Vascularis Function in the Mouse Inner Ear
Authors: CABLE JOANNE , JACKSON IAN J. , STEEL KAREN P.
Published: July 2006

The Light mutation (Blt) is a dominant allele of the b‐locus on mouse chromosome 4 which causes progressive dilution of coat colour. Melanocytes within the hair follicles of mutant mice develop normally but later degenerate, due to the accumulation of a toxic product, so that the hair becomes lighter with age. Previous studies on W‐locus spotting mutants, from which melanocytes are absent, have shown that melanocytes in the stria vascularis of the inner ear are essential for the development and/or maintenance of the endocochlear potential (EP) which is normally around 100 mV. In this study, physiological recordings from the ears of Light mutants were correlated with strial ultrastructure. EPs recorded from all b/b controls and young homozygous and heterozygous mutants (20–22 days old) were normal (77 to 113 mV), but were reduced (19 to 59 mV) in about 30% of ears from older mutants (Blt/Blt and Blt/b). Strial function therefore appears to develop normally but later degenerates in some mutants. This suggests that strial melanocytes are affected by the Light allele and that the continued presence of melanocytes is necessary for strial function. There was no obvious association between the recorded EP value and the ultrastructural appearance of the stria. No structural abnormalities of the stria were noted in control or mutant mice aged 20 days to 4 months including those which had a reduced EP. Strial atrophy was common in old controls and mutants (1–2 years), and appeared to be an age‐related process rather than an effect of the Light mutation. Similarly, pigment build‐up was common in all strial cells of old mice. However, the accumulations of lipofuscin‐like pigment were much larger and more abundant in aged brown non‐agouti mice than those observed in old agouti mice, which suggests that this age‐related process also has a genetic component.
Cytoskeletal Architecture of Dermal Chromatophores of the Freshwater Teleost Oryzias latipes
Authors: OBIKA MASATAKA , FUKUZAWA TOSHIHIKO
Published: July 2006

Cytoskeletal construction of dermal chromatophores of Orgzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval‐shaped leucophores exhibited the least‐developed microtubule system, where the limited number of microtubules formed a loose basket‐like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin‐immunoreactive. Examination of doubly‐labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.
Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock
Authors: SEIKAI TADAHISA , HIROSE EUICHI , MATSUMOTO JIRO
Published: July 2006

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca2+–, Mg2+–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.
Goldfish Tyrosinase Related Protein I (TRP‐1): Deduced Amino Acid Sequence From cDNA and Comments on Structural Features
Authors: PENG GANG , TAYLOR JOHN D. , TCHEN T.T.
Published: July 2006

Previous workers have shown that mammals have tyrosinase and tyrosinase related proteins (TRPs) that share common structural domains, all of which are not present in microbial tyrosinases. We report here the deduced amino acid sequence of a TRP from fish that is highly homologous to mammalian TRP‐1. Examination of the structures of these vertebrate tyrosinases and TRPs shows that, aside from the conserved cysteine‐rich and histidine‐rich domains previously noted, there are a large number of conserved prolines and glycines, leading to an abundance of turns and few conserved helical regions. These tyrosinases and TRP‐1s also have in their cytosolic tails a consensus sequence that is not present in any other protein. It is proposed that this sequence may participate in directing these proteins to the melanosomes.
Genetics and Molecular Biology of Mouse Pigmentation
Authors: JACKSON IAN J. , BUDD PETER , HORN JACQUELINE M. , JOHNSON RUTH , RAYMOND SOPHIE , STEEL KAREN
Published: July 2006

The formation of mouse coat color is a relatively complex developmental process that is affected by a large number of mutations, both naturally occurring and induced. The cloning of the genes in which these mutations occur and the elucidation of the mechanisms by which these mutations disrupt the normal pigmentation pattern is leading to an understanding of the way interactions between gene products lead to a final phenotype.
Nuclear Onco‐Protein Targets of Signal Transduction Pathways
Authors: WOODGETT JAMES R. , PULVERER BERND J. , PLYTE SIMON , HUGHES KENNETH , NIKOLAKAKI ELENI
Published: July 2006

Irradiation of mammalian cells with ultraviolet light (200‐400 nm) results in the activation of a number of genes, the so‐called “UV response” (Herrlich et al., Rev. Physiol. Biochem. Pharmacol., 119:187‐223, 1992). Many of the UV responsive genes are also transcriptionally activated by growth factors and mitogens. Two transcription factors have been demonstrated to acutely respond to these stimuli, namely AP‐1 and NF‐kB. Whereas NF‐kB proteins are primarily controlled via proteolysis of regulatory domains which prevent nuclear translocation, the AP‐1 proteins are regulated at several levels including transcription and posttranslational modification. Here, we discuss progress in the identification of the components of pathways acutely regulating these transcription factors.
Melanoma Prevention: Is It Possible to Change a Population's Behavior in the Sun?
Authors: MARKS ROBIN
Published: July 2006

With the highest incidence and mortality rates of melanoma in the world, Australia has been running carefully designed primary prevention programs for more than 10 years. These have included public education programs aimed at all ages but concentrating particularly on young people. The programs have also included structural changes such as provision of shade in open space, rescheduling work and sport activities, removal of sales tax from approved sunscreens, and provision of cheap sunscreens. There have been substantial changes in knowledge, attitudes, and beliefs about melanoma, sunlight, and suntans. There have been significant increases in sun protective behavior reflected in the reduced proportions of people sunburnt at the weekend in summer in recent years. The incidence rate of melanoma continues to rise, as expected. Although there has been considerable success so far, we still have a long way to go.
Proliferation and Propagation of Human Melanocytes in Vitro Are Affected by Donor Age and Anatomical Site
Authors: ABDEL‐MALEK ZALFA A. , SWOPE VIKI B. , NORDLUND JAMES J. , MEDRANO ESTELA E.
Published: July 2006

We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin. Two different growth media have been used for this purpose. Medium I supports the long‐term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC. We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages. However, adult MC behaved differently in the two growth media. In very early passages (passages 1‐5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non‐foreskin MC in medium II. The non‐foreskin adult MC had the least proliferative rate in medium II. Unlike neonatal MC, both adult foreskin and non‐foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9‐10) passages in culture. While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX). These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived. We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.
DHICA Oxidase Activity of TRP1 and Interactions With Other Melanogenic Enzymes
Authors: KOBAYASHI TAKESHI , URABE KAZUNORI , WINDER ALISON , TSUKAMOTO KATSUHIKO , BREWINGTON TIMOTHY , IMOKAWA GENJI , POTTERF BRIAN , HEARING VINCENT J.
Published: July 2006

Tyrosinase‐related protein 1 (TRP1) maps to the brown locus in mice. Although the specific function of TRP1 has been in dispute, mutations in its structural gene result in the formation of brown rather than black melanin. We have investigated the melanogenic function of TRP1 by using immune‐affinity purification of the protein and also by using transfection of its gene into fibroblasts to study its characteristics. We show that TRP1 has the ability to oxidize DHICA, a melanogenic intermediate derived from DOPAchrome. In addition, TRP1 has the ability to interact with tyrosinase and significantly stabilize the latter's catalytic function.
Demonstration of Voltage‐Dependent and TTX‐Sensitive Na+‐Channels in Human Melanocytes
Authors: EKMEHAG BJÖRN , PERSSON BERTIL , RORSMAN PATRIK , RORSMAN HANS
Published: July 2006

The electrophysiological properties of cultured human melanocytes were investigated using the whole‐cell configuration of the patch‐clamp technique. Depolarizations to membrane potentials more positive than ‐30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage‐gated Na+‐channels, and disappeared when Na+ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K+‐current. The first type, observed in every cell, was highly sensitive (Ki 1 mM) to the K+‐channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4‐aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage‐dependent Na+‐channels, a delayed rectifying K+‐current and a K+‐current similar to the A‐current in neurones.