bluuebunny/crossref_metadata_2025 huggingface dataset:
Melanin Biosynthesis From Dopamine. II. A Mass Spectrometric and Collisional Spectroscopic Investigation
Authors: COSTA CARLO , BERTAZZO ANTONELLA , ALLEGRI GRAZIELLA , TOFFANO GINO , CURCURUTO ORNELLA , TRALDI PIETRO
Published: July 2006

Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry together with collisional activation (CA) experiments were applied to the study of the oxidation pathway of dopamine by tyrosinase. In order to prevent attachment of the protein to the highly reactive intermediates, ultrafiltration was employed to remove the enzyme at different reaction times. FAB, privileging molecular species formation, was successfully used for identification of transient intermediates and their relative concentrations with respect to time, directly in the reaction mixture. The presence of isobaric molecular species made chromatographic separation necessary. Further EI mass spectrometry and collision spectroscopy led to structural identification of pure components. Of these, dopamine‐o‐quinone, leucoaminochrome, and aminochrome semiquinone were characterized for the first time as real intermediates in dopamine melanogenesis, in agreement with previous hypotheses.

This approach elucidated the pathway of dopamine melanogenesis.
Molecular Genetics and the Ontogeny of Pigment Patterns in Mammals
Authors: QUEVEDO WALTER C. , HOLSTEIN THOMAS J.
Published: July 2006

The conclusion that animal development is guided by a hierarchical system of gene expression and interaction has gained considerable support from recent molecular genetic studies on fruit flies (Drosophila melanogaster) and mice (Mus musculus). They demonstrate that the patterns of organization revealed by terminal differentiation of cells is anticipated by a myriad of transient prepatterns that channel the developing embryo toward its genetically‐programmed target. The numerous white spotting mutants in mice exhibit some of the most dramatic and variable patterns of cutaneous melanin pigmentation. Until recently, the mechanisms of action of white spotting genes and their relationship to the developmental genetic hierarchy remained unknown. It now appears that certain white spotting genes may encode growth factors essential for melanoblast development. Others may be related to homeobox genes that play a number of developmental roles, the primary one being the determination of regional organization along the anterior‐posterior axis of the early embryo. The patterns of homeobox gene expression are consistent with several of the developmental models for white spotting in mice and other mammals. It is evident that white spotting genes are not solely concerned with the terminal differentiation of melanoblasts into melanocytes. They are heterogeneous with regard to action and level of expression within the developmental hierarchy.
Pigment Cell Refugia in Homeotherms—The Unique Evolutionary Position of the Iris
Authors: OLIPHANT LYNN W. , HUDON JOCELYN , BAGNARA JOSEPH T.
Published: July 2006

Homeotherms are generally considered to lack classical active dermal pigment cells (chromatophores) in their integument, attributable to the development of an outer covering coat of hair or feathers. However, bright colored dermal pigment cells, comparable to chromatophores of lower vertebrates, are found in the irides of many birds. We propose that, because of its exposed location, the iris is an area in which color from pigment cells has sustained a selective advantage and appears to have evolved independently of the general integument. In birds, the iris appears to have retained the potential for the complete expression of all dermal chromatophore types. Differences in cell morphology and the presence of unusual pigments in birds are suggested to be the result of evolutionary changes that followed the divergence of birds from reptiles. By comparison, mammals appear to have lost the potential for producing iridophores, xanthophores, or erythrophores comparable to those of lower vertebrates, even though some species possess brightly colored irides. It is proposed that at least one species of mammal (the domestic cat) has recruited a novel iridial reflecting pigment organelle originally developed in the choroidal tapetum lucidum. The potential presence of classical chromatophores in mammals remains open, as few species with bright irides have been examined.
Differentiation of New Metastatic Variants of B16 Melanoma Under Different Culture Conditions
Authors: AUBERT CHRISTIAN , ALI‐MEHIDI SMAIL , ROUGE FRANCOISE , VOULOT CHRISTIAN
Published: July 2006

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow Ay/a, albino c/c, and C+/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5‐S‐CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C+/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.
Muscarinic Cholinoceptors That Mediate Pigment Aggregation Are Present in the Melanophores of Cyprinids (Zacco spp.)
Authors: HAYASHI HIROSHI , FUJII RYOZO
Published: July 2006

Like melanophores of many teleosts, those of the dark chub, Zacco temmincki, and the common minnow, Z. platypus (Cyprinidae, Cypriniformes) responded to norepinephrine (NE) by the aggregation of pigment. It was further found that some melanophores were responsive to acetylcholine (ACh) in the same way. The response to NE was blocked by an alpha‐adrenergic blocker, phentolamine, whereas the response to ACh was not. By contrast, two muscarinic cholinoceptor antagonists, namely, atropine and scopolamine, effectively blocked the action of ACh. The pigment aggregation due to the liberated sympathetic neurotransmitter was blocked by phentolamine but not by cholinergic blockers. These results suggest that, although the melanophores of these species are controlled in an orthodox manner by the sympathetic nervous system, some of them possess extra muscarinic cholinoceptors that also mediate the aggregation of pigment. The present report is the first to describe the presence of cholinoceptors on the chromatophores in species of fish other than those that belong to the order Siluriformes. The evolutionary implications of these findings are discussed.
Vitiligo: Where Do We Stand?
Authors: ORTONNE JEAN‐PAUL , BOSE SUMIT KUMAR
Published: July 2006

Vitiligo is a puzzling disorder characterized by a disappearance of epidermal and/or follicular melanocytes by unknown mechanisms. This very common disorder involving 1–4% of the world population is thus of great importance for the practicing dermatologist. The cellular and molecular mechanisms leading to the destruction of melanocytes in this disorder have not yet been elucidated, making it of major interest for the cell biologist involved in melanocyte research.

Recent advances in this field, due largely to the availability of techniques for culturing normal human melanocytes, opened new perspectives in the understanding of vitiligo. Although vitiligo has long been considered a disorder confined to the skin, there is now good evidence that it also involves the extracutaneous compartment of the “melanocyte organ.” It is also clear that vitiligo is not only a melanocyte disorder, but that it also involves cells, such as keratinocytes and Langerhans cells, found in the epidermis and follicular epithelium.

The three prevailing theories of the pathogenesis of vitiligo are the immune hypothesis, the neural hypothesis, and the self‐destruct hypothesis. New hypotheses suggest that vitiligo may be due to (1) a deficiency in an unidentified melanocyte growth factor, (2) an intrinsic defect of the structure and function of the rough endoplasmic reticulum in vitiligo melanocytes, (3) abnormalities in a putative melatonin receptor on melanocytes and (4) a breakdown in free radical defense in the epidermis. None of these hypotheses has been demonstrated, and according to the available data, it is likely that the loss of epidermal and follicular melanocytes in vitiligo may be the result of several different pathogenetic mechanisms.
Atypical Granules in the Eyes of the White Mutant of Drosophila melanogaster Are Lysosome‐Related Organelles
Authors: SCHRAERMEYER ULRICH , DOHMS MARIANNE
Published: July 2006

In the pigment cells of the white mutant of Drosophila melanogaster, as described earlier, two types of abnormal granules are found by conventional electron microscopy. However, both types of abnormal granules, in addition to those in pigment cell invaginations, are also present in the cytoplasm of the photoreceptor cells.

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) are localized within the eyes of wild type and white mutant Drosophila melanogaster by electron microscopy. Peroxidase activity is present in lamellar bodies close to the rhabdomeral microvilli of both fly types. However the organelles containing peroxidase activity are 6‐fold more frequent in the wild type than in the mutant. Acid phosphatase is present in lamellar bodies between and at the bases of the rhabdomeral microvilli of the wild type, as well as in ommochrome granules of the photoreceptor cells. In the white mutant, however, acid phosphatase was located in electron lucent vacuoles in the cytoplasm of the receptor cells. These acid phosphatase‐positive vacuoles also contained both types of abnormal granules. The latter result indicates that abnormal granules in the receptor cells originate from lysosomal degradation and that targeting of lysosomal enzymes is altered in the white mutant.

Due to the tyrosinase activity in the hemolymph of flies, the extracellular spaces are electron dense after DOPA incubation. Since some abnormal granules within the photoreceptor cells are not surrounded by an extracellular space, they can be assumed to originate within the photoreceptor cells.
Application of Photoacoustic Phase Angle Spectroscopy (φAS) to Eumelanins and Pheomelanins
Authors: CAITI ELENA , CRIPPA PIER RAIMONDO , VIAPPIANI CRISTIANO
Published: July 2006

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment.

Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.
Chemical and Electron Microscopic Studies of Cattle (Bos taurus) With Four Types of Phenotypic Pigmentation
Authors: RENIERI CARLO , CECCARELLI PIERO , GARGIULO ANNA MARIA , LAUVERGNE JEAN‐JACQUES , MONACELLI GIACOMO
Published: July 2006

The biological behavior of the pigmentary phenotypes of four breeds of cattle has been analysed: the black pigmentation of Holstein Friesian; the red pigmentation of Limousin; the dilution in Charolais; and the postnatal disappearance of red pigmentation in Chianina. The analytic techniques included the characterization of melanins by high‐performance liquid chromatography, the examination of follicular melanocytes by light microscopy, and the examination of melanosomes by electron microscopy. The black phenotype was very strongly eumelanogenic. The red phenotype in Limousin is polymorphic: individual follicular melanocytes contain both mature eumelanosomes and pheomelanosomes. Charolais and Chianina cattle exhibited a dramatic reduction in melanogenic activity, which was characterized by the almost exclusive presence of prephaoemelanosomes in Charolais and of immature premelanosomes in Chianina. In the dilute Charolais phenotype, the density of distribution of follicular melanocytes also seemed to be reduced. The genes that are responsible for these four phenotypes seem to act on the maturation, differentiation, and density of distribution of the melanosomes.
Evolution of the Tyrosinase Related Gene (TYRL) in Primates
Authors: OETTING W.S. , STINE O.C. , TOWNSEND D. , KING R.A.
Published: July 2006

Tyrosinase is the major enzyme responsible for the formation of melanin pigment and is found throughout the animal kingdom. In humans, the tyrosinase gene (TYR) maps to the long arm of chromosome 11 at band q14→q21, while a tyrosinase related gene (TYRL) maps to the short arm of chromosome 11 at pll.2°Cen. We and others have found that the TYRL locus contains sequences that are similar to exons IV and V of the authentic tyrosinase gene but lacks sequences of exons I, II, and III. In an attempt to understand the evolution of the human tyrosinase gene, we have analyzed TYR and TYRL in primates and have found that exons IV and V of the chimpanzee and gorilla TYR are very similar to the human, with the gorilla sequence being more similar than the chimpanzee. We have also found that the gorilla but not the chimpanzee contains a TYRL locus similar to the human TYRL locus.
Pteridines as Reflecting Pigments and Components of Reflecting Organelles in Vertebrates
Authors: OLIPHANT LYNN W. , HUDON JOCELYN
Published: July 2006

This paper reviews evidence for the presence of pteridines in iridophores, leucophores, and xanthophores in a wide variety of vertebrate chromatophores, and argues that the chemical and functional distinction between pterinosomes and reflecting platelets is not as clear‐cut as previously believed. Observations indicate that: (1) Pteridines may, either alone or in conjunction with purines, form pigment granules that reflect light, (2) these pigment granules are highly variable ranging from fibrous pterinosomes to typical reflecting platelets and may be colored, reflect white light, or be iridescent, and (3) many “leucophores” probably contain typical pterinosomes and presumed associated colorless pteridines and are therefore more closely related to erythrophores and xanthophores than to iridophores with which they are usually classified. We propose that the classification of pigment cells should be modified to reflect these facts.
Role of Melanin as a Scavenger of Active Oxygen Species
Authors: BUSTAMANTE JUANITA , BREDESTON LUIS , MALANGA GABRIELA , MORDOH JOSE
Published: July 2006

The protective role of melanin, either synthetic or derived from a metastatic lung melanoma nodule, was studied in terms of its ability to interact with active oxygen species (O2, H2O2, RO, ROO, etc.). Both melanins showed the ability to react with O2. The superoxide dismutase‐like activity corresponds to 21 and 10 U/mg for synthetic and tumor melanin, respectively. The latter value accounts for about 8% of the superoxide dismutase activity of cultured melanoma cells. Neither type of melanin showed catalase‐like or glutathione peroxidase‐like activity. Both types of melanin reacted with RO and ROO radicals as determined by inhibition of the lipid peroxidation reaction of rat liver homogenates. The spontaneous lipid peroxidation of rat liver homogenate was inhibited up to 90% and 80% by synthetic and tumor melanin with half‐maximal effects at 2.5 and 5.5 μg melanin/ml, respectively. The 2,2‐azobis‐(2 amidino propane) (AAPH)‐initiated lipid peroxidation of rat liver homogenate was inhibited up to 3% and 20% by synthetic and tumor melanin, with half maximal effect at 120 and 500 μg melanin/ml, respectively. Both types of melanin were able to protect the in vitro inactivation of glucose oxidase, which occurs in the presence of AAPH‐generated radicals.
An Improved Modification of Permanganate Oxidation of Eumelanin That Gives a Constant Yield of Pyrrole‐2,3,5‐Tricarboxylic Acid
Authors: ITO SHOSUKE , WAKAMATSU KAZUMASA
Published: July 2006

Microanalysis of eumelanin is based on the formation of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) on permanganate oxidation followed by its HPLC determination. A problem in this method was that the oxidation gave concave, exponential curves when the amounts of PTCA formed were plotted against the amounts of sample oxidized. The problem has been mostly overcome by adding a homogenate of 5 mg of a mouse liver to the oxidation medium. Sepia melanin, C57BL black mouse hair, B16 mouse melanoma, and MM418 human melanoma cells were oxidized in the absence or presence of the liver homogenate. The yields of PTCA increased about 1.5‐fold by adding the liver homogenate and the calibration curves became linear or almost linear. With the improved method the PTCA values from various types of samples can be reliably compared.
Pharmacological Profiles of the Subtypes of Muscarinic Cholinoceptors That Mediate Aggregation of Pigment in the Melanophores of Two Species of Catfish
Authors: HAYASHI HIROSHI , FUJII RYOZO
Published: July 2006

Using selective antagonists, including pirenzepine, adiphenine, AF‐DX116, gallamine, and 4‐DAMP we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M3 receptor‐selective antagonist, 4‐DAMP, potently inhibited the acetylcholine‐induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M3 muscarinic subtype.
Premature Avian Melanocyte Death Due to Low Antioxidant Levels of Protection: Fowl Model for Vitiligo
Authors: BOWERS ROGER R. , LUJAN JASON , BIBOSO ARCADIO , KRIDEL STEVEN , VARKEY CIBBY
Published: July 2006

Feather melanocytes in the Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens die prematurely in vivo when compared to the wild type Jungle Fowl (JF) chicken. Since these mutant melanocytes live in vitro, an environmental factor in the feather must precipitate their death. Results show that the addition of selected antioxidants, glutathione (GSH) and superoxide dismutase (SOD), can rescue these mutant melanocytes in vitro that have been placed under stress conditions that cause their premature cell death. Measurements of in vivo levels of GSH, catalase, and SOD show no significant difference in catalase activity between the JF, BPR, and WL feathers but do show a significant reduction in GSH activity in both the BPR and WL feathers to approximately 66% of the GSH concentration found in JF feathers. SOD activity in the BPR tissue is reduced significantly to approximately 50% of the JF activity and the WL SOD activity is reduced significantly to approximately 50% of the BPR SOD activity. Preliminary results of measurements of glutathione peroxidase activity indicate there is no difference in the levels of this enzyme in JF, BPR and WL feathers. A working hypothesis, based on current results, is proposed for premature cell death in BPR and WL feather melanocytes. The BPR melanocytes are genetically sensitive due to a defect in their SOD and GSH levels caused by the barring gene (B) and their death, due to reactive species of oxygen radicals, is precipitated in the poorly vascularized feather by the accumulation of oxygen radicals due to the low turnover of tissue fluids. The WL chicken carries the dominant white gene (I) in addition to the B gene. This gene directs the further reduction of the level of SOD and, when combined with the cell death mechanism already present in the BPR chicken, causes the WL feather melanocytes to die much earlier than the BPR feather melanocytes which in turn die much earlier than the wild type JF melanocytes. This same mechanistic hypothesis could apply as a cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their oxygen radical protection system.
Increasing Melanoma Incidence: Putatively Explainable by Retrotransposons Experimental Contributions of the Xiphophorine Gordon‐Kosswig Melanoma System
Authors: ANDERS ANNEROSE , PETRY HARALD , FLEMING CHRISTINE , PETRY KERSTIN , BRIX PETRA , LÜKE WOLFGANG , GRÖGER HARALD , SCHNEIDER ECKART , KIEFER JÜRGEN , ANDERS FRITZ
Published: July 2006

The worldwide accelerating increase of neoplasia in humans is difficult to explain. We use the Xiphophorus tumor model to approach this problem by melanoma provocation with X‐rays. Melanoma develops following inappropriate expression of x‐erb B‐conducted developmental genes and their controllers. These oncodeterminants are inherited according to Mendelian rules. We detected a new type of oncodeterminants that, following a single treatment of embryos with X‐rays, generates a self‐generating non‐Mendelian melanoma transmission and accelerating increase of its incidence in succeeding generations (e.g., 0→18→33→52%). To localize these oncodeterminants, we crossed nonirradiated fish having half of their chromosomes irradiated with nonirradiated fish having none of, half of, or all of their chromosomes irradiated. Because tumor rate and expression in the following generations correspond to the rates of treated chromosomes, we conclude that the new oncodeterminants are distributed over the chromosomes of the fish, where they may increase in the changing generations. By means of xiphophorine‐specific retroviral DNA, we isolated two retrotransposons that behave hereditarily like the new transgenerational oncodeterminants. Sequence analysis revealed three ORFs flanked by LTRs containing motives of regulatory sequences typical for known retroviral and retrotransposal LTRs. Pol‐ and env‐resembling sequences are lacking. Southern and in situ hybridization showed their multiple and repetitive nature distributed throughout the chromosomes and indications for their capability to increase in number without further treatment. Their transcripts are expressed in concert with those of most of the other known xiphophorine tumor determinants. Their expression is extremely high in cell cultures from tumorous embryos derived from ancestors treated as embryos with X‐rays.
Tissue Distribution of an Immunogenic Melanoma Associated Antigen, D‐1
Authors: HAYASHIBE KAZUHITO , NAGAI HIROSHI , KAKO SATORU , TSUKAMOTO HIDEKAZU , ICHIHASHI MASAMITSU
Published: July 2006

A human melanoma‐associated antigen immunogenic in patients was recently identified by screening an expression cDNA library constructed from cultured human melanoma cell line with sera from patients with melanoma. The nucleic acid sequence of the cloned D‐1 cDNA has no significant homology with previously reported mammalian genes. The cDNA D‐1 encodes a peptide of about 37 kDa, which showed fivefold higher reactivity with sera from patients with melanoma than with sera from normal donors. In order to detect D‐1 gene expression in vivo, in‐situ hybridization and immunostaining with cRNA probe and murine anti‐D‐1 sera were carried out on surgically removed tissues. Digoxigenin‐labeled cRNA D‐1 was exclusively hybridized with mRNA in the cytoplasm of melanoma cells but not with keratinocytes and fibroblasts adjacent to melanoma nests. Polyclonal anti‐D‐1 antibodies were obtained by immunization of Balb/c mice with recombinant D‐1 peptide and clearly reacted with melanoma cells but not with keratinocytes and fibroblasts, similar to the results of in‐situ hybridization. The above information will help to assess the suitability of recombinant D‐1 peptide to implement active specific immunotherapy in patients with melanoma.
Identification of Nuclear Factors That Bind to the Mouse Tyrosinase Gene Regulatory Region
Authors: SATO SHIGERU , MIURA HIROHITO , YAMAMOTO HIROAKI , TAKEUCHI TAKUJI
Published: July 2006

Several nuclear factors that interact with sequences in the 5’ flanking region of the mouse tyrosinase gene were identified using band shift and methylation interference assays. One of these factors bind to an AT‐rich sequence, TATCAAITAG, located at –183 base pairs upstream of the transcription start site. To isolate cDNA clone encoding this DNA binding protein, we have screened a λgt11 cDNA expression library prepared from mouse melanocyte cell line with a labeled oligonucleotide probe containing its binding site. Complementary DNA clones encoding mouse high mobility group protein HMG‐I and its isoform HMG‐Y were obtained. HMG‐I(Y) is a low molecular size, basic nuclear protein that binds specifically to AT‐rich region of double‐stranded DNA in vitro. In Northern blot analysis the level of HMG‐I(Y) mRNA expression did not correlate with that of tyrosinase or TRP‐1. Although the amount of HMG‐I(Y) transcripts has no apparent influence on the mouse tyrosinase gene expression, it is possible that HMG‐I(Y) binds to the 5’ flanking sequence of the tyrosinase gene as an auxiliary factor, and facilitates the binding and activity of other transcription factors.
Tyrosinase Isoenzymes: Two Melanosomal Tyrosinases With Different Kinetic Properties and Susceptibility to Inhibition by Calcium
Authors: JIMÉNEZ‐CERVANTES C. , SOLANO F , LOZANO J.A. , GARCIA‐BORRÓN J.C.
Published: July 2006

Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS‐PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, proving that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme, Immunoprecipitation and Western blots performed with the specific antibody αPEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca2+ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca2+ concentrations for LEMT. Moreover, binding of Ca2+ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.
The DHICA Oxidase Activity of the Melanosomal Tyrosinases LEMT and HEMT
Authors: JIMÉNEZ‐CERVANTES C. , SOLANO F. , LOZANO J.A. , GARCÍA‐BORRÓN J.C.
Published: July 2006

Although melanins can be formed in vitro by the unique action of tyrosinase on L‐tyrosine, it is now well accepted that other enzymes termed tyrosinase‐related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L‐dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez‐Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA‐melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Opposite to HEMT, the true tyrosinase encoded by the albino locus, its role in melanogenesis would be related to the incorporation of DHICA into eumelanin rather than to the first steps of the pathway.
Role of β1 Integrins in Cell Spreading and Migration of Human Nevomelanocytes and Dysplastic Nevi Cells on Collagen Type IV and Laminin
Authors: VINK JACQUELINE , DEKKER SYBREN K. , VAN LEEUWEN ROBERT L. , VERMEER BERT J. , BRUIJN JAN ANTHONIE , BYERS H. RANDOLPH
Published: July 2006

We characterized β1 integrin subunit expression on three different cultures of benign human nevomelanocytes (NMC) and on four different cell cultures of human dysplastic nevus (DN) cells by flow cytometry analysis and examined their role in mediating cell spreading and migration on collagen type IV (CN IV) and laminin (LN) coated substrates by using a quantitative video image analysis system. The seven human NMC and DNC cultures expressed heterogeneous levels of β1, α2, α3 and α6 integrin subunits. Image analysis showed that a significant increase (P<0.001) in cell spreading and migration of the DN cells was induced on increasing coating concentrations of CN IV and LN. However, the NMC did not show an increase in cell spreading or migration on these substrates when compared to the substrates coated with denatured BSA only. The CN IV‐induced cell spreading of the DN cells was significantly inhibited by anti‐β1 mAb (AIIB2), anti‐α2 mAb (P1E6), or anti‐α3 mAb (P1B5), but not by mAb against α6 integrin subunit (GoH3). The DN cell spreading on LN was not significantly inhibited by these mAbs. In contrast, the migration of the DN on CN IV and LN was significantly inhibited by anti‐β1 mAb, anti‐α2 mAb, anti‐α3 mAb and anti‐α6 mAb. These data suggest that the α2 and α3 subunit are important for cell spreading of the DN on CN IV, although they are less important in cell spreading on the extracellular matrix component LN. The α2, α3 and α6 integrin subunits are important for the migration of DN cells on both CN IV and LN.
211At‐Methylene Blue in Targeted Radiotherapy of Disseminated Melanoma
Authors: LINK EVA M. , MICHALOWSKI ADAM S. , RÖSCH FRANK
Published: July 2006

Targeted radiotherapy with 211At‐methylene blue (211At‐MTB) is a systemic treatment selectively directed at melanoma due to a high affinity of MTB to melanin synthesized in the tumor cells. Since MTB forms a strong complex with melanin, it is an effective carrier for a number of radioisotopes to be addressed to the tumor deposits of any size including individually dispersed melanoma cells. Thus, appropriately radiolabeled MTB can be used for either diagnosis or therapy of the neoplasm. As predicted and found in animal experiments, 211At‐MTB is most effective therapeutically. Histopathological investigations showed that the highly pigmented 211At‐MTB‐treated tumors were characterized initially by perivascular oedema and hydropic degeneration of tumor cells followed by gradual development of extensive areas of coagulative necrosis. The necrotic tumor areas contained microvessels occluded by thrombi and tended to undergo microfocal calcification. Although melanoma‐bearing animals successfully treated with 211At‐MTB did not reveal any adverse effects of the therapy, detailed toxicological studies were undertaken. No serious macro‐ or microscopic lesions were observed in normal organs of 211At‐MTB treated mice. Only the relative number of small lymphocytes in the groin lymph nodes in a minority of animals was variably reduced, most often in conjunction with the treatment of highly, but not poorly, pigmented tumors.
Inhibitory Effects of Melanin Monomers, Dihydroxyindole‐2‐Carboxylic Acid (DHICA) and Dihydroxyindole (DHI) on Mammalian Tyrosinase, With a Special Reference to the Role of DHICA/DHI Ratio in Melanogenesis
Authors: WILCZEK ADAM , MISHIMA YUTAKA
Published: July 2006

DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase‐controlled, melanogenic reaction, namely conversion of L‐tyrosine to DOPAchrome (2‐carboxy‐2,3‐dihydroindole‐5,6‐quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase‐rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L‐tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase—tyrosine‐hydroxylation and DOPA‐oxidation—more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase‐catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase‐catalyzed early phase of melanogenesis.
Corticotropin Expression by Human Melanocytes in the Skin
Authors: IYENGAR BHANU
Published: July 2006

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G2‐phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV‐sensitivity of the melanocytes.
The Melanotropic Peptide, [Nle4, d‐Phe7]α‐MSH, Stimulates Human Melanoma Tyrosinase Activity and Inhibits Cell Proliferation
Authors: JIANG JINWEN , SHARMA SHUBH D. , NAKAMURA SHELLEY , LAI JENG‐YU , FINK JODY L. , HRUBY VICTOR J. , HADLEY MAC E.
Published: July 2006

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle4,d‐Phe7]α‐MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called “amelanotic” (tyrosinase‐negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle4,d‐Phe7]α‐MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and “amelanotic” cell lines incubated with [Nle4,d‐Phe7]α‐MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle4,d‐Phe7]α‐MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.
Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis
Authors: NAKAGAWA HIDEMI , IMOKAWA GENJI
Published: July 2006

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10.

On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.
Transcription of Melanogenesis Enzymes in Melanocytes: Dependence upon Culture Conditions and Co‐Cultivation With Keratinocytes
Authors: KIPPENBERGER STEFAN , BERND AUGUST , BEREITER‐HAHN JÜRGEN , RAMIREZ‐BOSCA ANA , KAUFMANN ROLAND , HOLZMANN HANS
Published: July 2006

Eumelanogenesis of human skin melanocytes requires at least three enzymes: tyrosinase, TRP 1, and TRP 2. The regulation of these enzymes on transcriptional level was detected in a semiquantitative attempt. The total RNA of melanocytes was reverse‐transcripted and followed by a PCR with degenerated primers for all three enzymes. The amplification products were related to each other densitometrically. We examined five different culture conditions: 1) melanocytes in a popular phorbolester containing F‐10‐medium, 2) melanocytes in a co‐culture medium with EGF, 3) melanocytes in a co‐culture medium with high calcium, 4) melanocytes co‐cultured with keratinocytes in EGF containing co‐culture medium, and 5) melanocytes co‐cultured with keratinocytes in co‐culture medium with high calcium. Melanocytes cultured in phorbolester containing F‐10‐medium featured transcripts of tyrosinase, TRP 1, and TRP 2 in the ratio 45:45:10. The same results were obtained for melanocytes co‐cultured with keratinocytes under the two different culture conditions. In melanocytes cultured alone in co‐culture media only TRP 1‐transcripts were present. It is likely that under co‐culture conditions a keratinocyte‐derived factor supports the transcription of all three enzymes. For melanocytes in the phorbolester‐containing melanocyte medium a proteinkinase C dependent regulation of transcription seems possible.
Human Epidermal Melanocyte and Keratinocyte Melanocortin Receptors: Visualization by Melanotropic Peptide Conjugated Microspheres (Latex Beads)
Authors: JIANG JINWEN , SHARMA SHUBH D. , HRUBY VICTOR J. , BENTLEY DAVID L. , FINK JODY L. , HADLEY MAC E.
Published: July 2006

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle4,D‐Phe7]α‐MSH, a superpotent analog of α‐melanocyte stimulating hormone (α‐MSH), were conjugated to a macromo‐lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin‐conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM.

Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro‐spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance‐P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small‐cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.
The Amphibian Melanization Inhibiting Factor (MIF) Blocks the α‐MSH Effect on Mouse Malignant Melanocytes
Authors: LÓPEZ‐CONTRERAS ANA M. , MARTÍNEZ‐LIARTE JOSÉ H. , SOLANO FRANCISCO , SAMARAWEERA PREMINDA , NEWTON J.M. , BAGNARA JOSEPH T.
Published: July 2006

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S‐91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of α‐MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH‐mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.
Evidence to Suggest That Expression of MITF Induces Melanocyte Differentiation and Haploinsufficiency of MITF Causes Waardenburg Syndrome Type 2A
Authors: TACHIBANA MASAYOSHI
Published: July 2006

MITF (microphthalmia‐associated transcription factor) encodes a transcription factor with a basic‐helix‐loop‐helix‐leucine zipper (bHLH‐Zip) motif. Ectopic expression of MITF is found to convert NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci, which superficially resembled those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase‐related protein 1. Such properties were not observed in cells transfected with the closely related gene, TFE3. These findings indicated that MITF is involved in melanocyte differentiation.

Two mutations (C760.T and C895.T) in MITF are found to be associated with individuals with Waardenburg syndrome type 2 (WS2). These mutations create stop codons in exon 7 and 8, respectively, and probably result in truncated proteins lacking HLH‐Zip or Zip structure. To understand how these MITF mutations cause WS2 in heterozygotes, mutant MITF proteins were generated and used for DNA‐binding and luciferase reporter assays. The mutated MITF proteins lose their DNA‐binding activity and fail to transactivate the promoter of the tyrosinase gene. However, these mutated proteins do not appear to interfere with the activity of wild‐type MITF protein in these assays, indicating that they do not show a dominant‐negative effect. These findings suggest that the phenotypes of the two WS2 families are caused by loss‐of‐function mutations in one of the two MITF alleles, resulting in haploinsufficiency of the MITF protein, the transcription factor necessary for normal melanocyte differentiation.
Application of the New Stereological Probes to the Study of the Melanosome in Cloudman S91 Melanoma Cells
Authors: FANG DONG , DOCKERY PETER , WEATHERHEAD BRIAN
Published: July 2006

The relationship between melanosome size and number and melanin content has been investigated in Cloudman S91 melanoma cells growing in vitro using both “model‐based” and “design‐based” stereological procedures. Cells were cultured for 4 days, harvested at daily intervals, and resin‐embedded for light and electron microscopy; one aliquot of each sample of cells was assayed to determine its melanin content.

By comparing their volume‐weighted mean nuclear volume and their number‐weighted mean nuclear volume, we have found that the nuclei of Cloudman melanoma cells form a fairly homogeneous population. The volume fraction and absolute volume of premelanosomes (Vvpm, cell and Vpm) and mature melanosomes (Vvmm, cell and Vmm) were all found to decrease progressively throughout the period of culture as did the number of premelanosomes (Npm) and mature melanosomes (Nmm). Whilst the volume‐weighted mean volume of individual stage I and stage II premelanosomes, (vipm), remained fairly constant at about 10 nm3, the volume of individual stage III and IV mature melanosomes showed significant variation ranging between about 13 nm3 and 32 nm3. The melanin content of the cells decreased progressively over the 4 days of culture. There were, however, considerable variations in both the average melanin content per unit volume of mature melanosomes, in the range 170–600 fg/μm3, and in the melanin content per individual mature melanosome, in the range 3–12 fg.

Our findings show that stereological techniques can provide unbiased and sensitive tools for the study of the morphological basis of melanogenesis; their value will become even more evident when they are combined with techniques for the localization of melanogenic enzymes and their substrates.