bluuebunny/crossref_metadata_2025 huggingface dataset:
Hairless Pigmented Guinea Pigs: A New Model for the Study of Mammalian Pigmentation
Authors: BOLOGNIA JEAN L. , MURRAY MARILYN S. , PAWELEK JOHN M.
Published: July 2006

A stock of hairless pigmented guinea pigs was developed to facilitate studies of mammalian pigmentation. This stock combines the convenience of a hairless animal with a pigmentary system that is similar to human skin. In both human and guinea pig skin, active melanocytes are located in the basal layer of the interfollicular epidermis. Hairless albino guinea pigs on an outbred Hartley background (CrI:IAF/HA(hr/hr)BR; designated hr/hr) were mated with red‐haired guinea pigs (designated Hr/Hr). Red‐haired heterozygotes from the F1 generation (Hr/hr) were then mated with each other or with hairless albino guinea pigs. The F2 generation included hairless pigmented guinea pigs that retained their interfollicular epidermal melanocytes and whose skin was red‐brown in color. Following UV irradiation, there was an increase in cutaneous pigmentation as well as an increase in the number of active epidermal melanocytes. An additional strain of black hairless guinea pigs was developed using black Hr/Hr animals and a similar breeding scheme. These two strains should serve as useful models for studies of the mammalian pigment system.
Coexistence of β1‐ and β2‐Adrenoceptors in the Melanophore of the Goby Tridentiger obscurus*
Authors: KATAYAMA HEIZABURO , MORISHITA FUMIHIRO , MATUSHIMA OSAMU , YAMADA KOJI
Published: July 2006

The effects of β‐adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β‐adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment‐aggregating response of melanophores to norepinephrine. The effects of these β‐agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α‐and β‐adrenoceptors, and that the activation of the β‐adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment‐dispersing effects of two β1‐selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β1‐receptors. By contrast, the pigment‐dispersing effects of two β2‐selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β‐agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β1‐ and β2‐adrenoceptors in the melanophores. The relative numbers of β1‐ and β2‐adrenoreceptors as a percentage of the total population of β‐adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β‐agonists on the melanophores in the presence of butoxamine or metoprolol.
Protein‐Kinase C Mediates MCH Signal Transduction in Teleost, Synbranchus marmoratus, Melanocytes
Authors: ABRÃO MARIA SILVIA , CASTRUCCI ANA MARIA DE L. , HADLEY MAC E. , HRUBY VICTOR J.
Published: July 2006

MCH (melanin concentrating hormone) is a heptadecapeptide, Asp‐Thr‐Met‐Arg‐Cys‐Met‐Val‐Gly‐Arg‐Val‐Tyr‐Arg‐Pro‐Cys‐Tip‐Glu‐Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown.

Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose‐response curve to MCH in the presence of various concentrations of 4‐bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10−9 to 10−8 M) of the phorbol ester TPA exhibited MCH‐like activity, eliciting a dose‐dependent melanosome aggregation. Higher doses, however, displaced to the right the dose‐response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH.

Both MCH and norepinephrine lightening actions were abolished by beta‐glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.
Does Melanin Affect the Low LET Radiation Response of Cloudman S91 Mouse Melanoma Cell Lines?
Authors: HILL HELENE Z. , CATHCART KATHLEEN NELL , BARGELLINI JOSEPH , TRIZNA ZOLTAN , HILL GEORGE J. , SCHALLREUTER KARIN U. , WOOD JOHN M.
Published: July 2006

Melanin contains melanin‐free radicals and can both absorb and produce additional free radicals and active oxygen species on exposure to various stimuli. Yet its role in the radiation responses of malignant melanoma has been little studied. In this report, three subclones of Cloudman S91 mouse melanoma clone PC1A varying in constitutive melanin content were compared with respect to killing by gamma irradiation. Radiation responses correlated with melanin content. The least melanotic line, S91/amel, was most sensitive and the most melanotic line, S91/I3, was most resistant. Curve fitting using the linear‐quadratic model suggests that S91/amel is killed only by single event inactivations; S91/I3, only by double event inactivations; and S91/M1B, with intermediate melanin and radiation response, by both types of inactivations. Split dose experiments confirmed a lack of immediate split dose recovery in S91/amel and its existence in S91/I3. Potentially lethal damage and its repair could be demonstrated in both S91/amel and S91/I3. Double strand break (DSB) induction was evaluated as a function of gamma ray dose in DNA of S91/I3 and S91/amel, as well as in EMT6, a mouse mammary cancer line that lacks tyrosinase and melanin. The rates of induction were proportional to cellular melanization, i.e., the rate of DSB induction was greatest in S91/I3, least in EMT6. Levels of thioredoxin reductase (TR), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) were determined in S91/amel and S91/I3. TR was the same in both cell lines, while the other three enzymes were 3‐ to 4‐fold lower in S91/amel. The results suggest that 1) melanin can act as a radiosensitizer of DNA damage and, 2) the presence of moderate constitutive levels of melanin in cells may effect radiation resistance by indirect means, possibly by causing cells to maintain high levels of antioxidant defenses and/or DNA repair systems.
Primary Structure and Relative Potency of an Analog of β‐PDH (Pigment‐Dispersing Hormone) From the Crayfish Procambarus clarkii
Authors: McCALLUM MARY LOU , RAO K. RANGA , RIEHM JOHN P. , MOHRHERR CARL J. , MORGAN WILLIAM T.
Published: July 2006

A pigment‐dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation‐exchange chromatography, partition chromatography, and reversed‐phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn‐Ser‐Glu‐Leu‐Ile‐Asn‐Ser‐Ile‐Leu‐Gly‐Leu‐Pro‐Lys‐Val‐Met‐Asn‐Glu‐Ala‐NH2. This peptide differs from β‐PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7‐fold less potent than β‐PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4‐fold more potent than β‐PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light‐adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH‐receptor interactions vary with the species and with the target cell type within a given species.
Ionic Basis for Color Changes in the Iridescent Cornea of the Sand Goby (Pomatoschistus minutus)
Authors: ASHCROFT NEVILLE R. , LYTHGOE JOHN N.
Published: July 2006

The iridescence from the cornea of the sand goby (Pomatoschistus minutus) occurs because of thin layer interference from the platelet‐like cells in the stroma. It is suggested that ionic pumps across the epithelium control the water content in the stroma and thus the spectral reflection. A saline was perfused over goby eyes and simple ion manipulation was carried out to observe any changes in the iridescent characteristics. It was found that removal of CI−and K+ions reduced the peak reflected wavelength to the blue end of the spectrum, whereas Na+ had little effect. The removal of K+ also caused a dramatic change to the normal shift in reflected spectral intensity. The iridescence was also found to be sensitive to pH, and the buffer HEPES was detrimental to the cornea compared to controls. These results suggest similarities to amphibian and mammalian corneal hydration control.
Differential Sensitivities of Murine Melanocytes and Melanoma Cells to Buthionine Sulfoximine and Anticancer Drugs
Authors: THRALL BRIAN D. , RAHA GEETHA A. , SPRINGER DAVID L. , MEADOWS GARY G.
Published: July 2006

High levels of intracellular glutathione (GSH) may result in resistance of tumor cells to cytotoxic drugs. Because of the innate refractory nature of melanoma cells to chemotherapy, we have used a syngeneic murine system consisting of nontumorigenic Mel‐ab melanocytes, tumorigenic H‐ras‐transformed melanocytes (C9.1), and the highly meta‐static BL6 melanoma cells to examine the GSH content, glutathione S‐transferase (GST) activity, and sensitivity to buthionine sulfoximine (BSO) and other cytotoxic drugs. Compared to the nontumorigenic melanocytes, both C9.1 and BL6 melanoma cells have nearly fivefold higher GSH content, and BL6 cells have increased GST activity. C9.1 and BL6 cells are more resistant to the cytotoxic effects of BCNU and adriamycin; however, the degrees of resistance do not reflect the increased GSH content in these cells. Pretreatment of BL6 melanoma cells with 50 μM BSO depleted over 90% of their GSH content and enhanced the growth‐inhibitory effects of l‐dopa methylester, BCNU, bleomycin, and dacarbazine. Exposure to BSO alone was not toxic to the tumor cells for up to 24 hr, but was significantly cytotoxic in the melanocytes after 9 hr. The sensitivity of these cells to BSO appears to depend on a critical level of GSH depletion which is not related to the initial GSH content. These studies suggest that the resistance of melanoma cells to cytotoxic drugs is only partially attributed to changes in the GSH system caused during cellular transformation.
Pigments and Ultrastructures of Pigment Cells in Xanthic Sailfin Mollies (Poecilia latipinna)
Authors: BLANCHARD PAUL D. , ANGUS ROBERT A. , MORRISON RANDALL L. , FROST‐MASON SALLY K. , SHEETZ JAMES H.
Published: July 2006

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulumderived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi‐derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to β‐carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.
Reciprocal Changes in Sensitivity to MCH and Noradrenaline After Denervation of Teleost Melanophores
Authors: SVENSSON SAMUEL P.S. , ANDERSSON ROLF G.G. , KARLSSON JAN OLOF G.
Published: July 2006

Melanophores of isolated fish scales survive for weeks in a culture medium. During this isolation period a progressive increase in sensitivity to noradrenaline (NA) takes place. In the present study, a 100‐fold increase in sensitivity to NA was found after 9 days. However, at the same time, a 12‐fold decrease in sensitivity to MCH was detected.
A cis‐Acting Element Involved in Mouse Tyrosinase Gene Expression and Partial Purification of its Binding Protein
Authors: PONNAZHAGAN S. , KWON BYOUNG S.
Published: July 2006

The tyrosinase gene is specifically expressed in melanocytes. Understanding the molecular basis of tissue‐specific expression of the tyrosinase gene will greatly explain the mechanisms controlling pigmentation. We report a nucleotide sequence, TGATGTATTC, located ‐236 base pairs upstream of the transcription start site, that enhances tyrosinase gene expression in mouse melanoma cells. The sequence is referred to as the tyrosinase element‐1 (TE‐1). TE‐1 was protected from DNase I cleavage by pigment cell nuclear extracts but was not protected by non‐pigment cell nuclear extract. Partial purification of TE‐1 binding protein (TEBP‐1) was performed from the B16 mouse melanoma cell nuclear extract using biotin‐cellulose affinity chromatography. The affinity‐purified fraction exhibited binding to the DNA fragment containing TE‐1, and to a synthetic oligomer representing TE‐1. UV‐cross‐linking indicated that the size of TEBP‐1 is approximately 49 kD. TE‐1 also directed enhanced CAT activity in the B16 melanoma cells but not in non‐pigment cells. These data indicate that TE‐1 may be an enhancer element that is responsible for pigment cell specific expression of the tyrosinase gene.
Extracellular Matrix Constituents and Pigment Cell Expression in Primary Cell Culture
Authors: FUKUZAWA TOSHIHIKO , HAMER STEPHANIE J. , BAGNARA JOSEPH T.
Published: July 2006

Three types of pigment cells were isolated and cultured from larval Rana pipiens, and their attachment, maintenance, and proliferation were examined in the presence of extra‐cellular matrix constituents (ECMs) in primary cell culture. The initial profile of pigment cell types present on day 2 of culture reflects the relative attachment of the cells to the dishes. Changes in the numbers of cells present after day 2 reflects the influence of factors present in the culture media on the maintenance, proliferation, or detachment of each type of pigment cell. Fetal bovine serum (FBS) promoted melanophore expression, but inhibited iridophore expression. FBS had no effect on xanthophores. In contrast, ventral skin conditioned medium (VCM), which contains melanization inhibiting factor, strongly stimulated iridophore expression, while it markedly inhibited melanophore expression. VCM had little effect on xanthophores. Of the ECMs tested, collagen type I had no effect on pigment cells. Fibronectin slightly inhibited melanophore expression, while it moderately stimulated iridophores and xanthophores. The stimulatory effect of fibronectin was not as strong as that of FBS or VCM. Laminin was also tested; however, it did not allow pigment cells to attach to the dishes, at least under the culture conditions utilized. The results of these experiments are discussed in terms of the general mechanisms of pigment pattern formation.
Analysis of Mutations in the Copper B Binding Region Associated With Type I (Tyrosinase‐Related) Oculocutaneous Albinism
Authors: OETTING WILLIAM S. , KING RICHARD A.
Published: July 2006

Mutations of the tyrosinase gene are responsible for type I (tyrosinase‐related) oculocutaneous albinism (OCA), an autosomal recessive genetic syndrome with a broad phenotypic spectrum. Mutant tyrosinase alleles can be associated with no melanin synthesis (I‐A, tyrosinase‐negative OCA), small to moderate amounts of melanin (I‐B, yellow OCA) or unusual pigment patterns (I‐TS, temperature‐sensitive OCA). A total of 26 mutations of this gene have been described in type I OCA. Analysis of all known missense mutations (n = 17) shows that most cluster in three areas of the coding region. Two clusters involve the copper A or copper B binding sites and may disrupt the metal ion‐protein interaction necessary for enzyme function and the third cluster is located in exon I. Computer modeling of the secondary structure of the copper binding regions based on homology with the known crystal structure of hemocyanin show that they both consist of two a helicies containing three histidine ligands that complex to a single copper atom. Mutations in the copper B binding region lie in the region between the two a helices that consists of a loop structure. These mutations may affect tyrosinase activity by either altering the position of the a helical domains and thus preventing proper copper binding to the histidine ligands, or affecting a catalytic or substrate binding site located between the two a helical domains.
Mutations of the Tyrosinase Gene in Oculocutaneous Albinism
Authors: SHIBAHARA SHIGEKI
Published: July 2006

Since our first report showing that the phenotype of tyrosinase‐negative or type IA oculocutaneous albinism (OCA) is a consequence of a mutation in the tyrosinase gene (Tomita et al., Biochem. Biophys. Res. Commun., 164:990–996, 1989), a number of mutations were found in the tyrosinase gene of OCA patients. However, to establish the molecular basis of OCA in each patient, we must carry out several important experiments as summarized here. First, we should confirm that the cloned or amplified genomic DNA segments are not derived from the pseudogene or related gene. It should be noted that the putative tyrosinase pseudogene contains the sequence almost identical to exons 4 and 5, including their exon/intron boundaries of the authentic tyrosinase gene. Thus, the mutations, detected in exon 4 or 5 amplified from genomic DNA, must be carefully analyzed to exclude a possibility that the mutation is located in the pseudogene. Second, it is of significance to confirm the promoter activity of the patients’ tyrosinase gene. Accordingly, we established the cell‐free transcription system derived from melanoma cells where the cloned tyrosinase gene is faithfully transcribed. Finally, transient expression assay of mutant tyrosinase is invaluable to conclude that OCA phenotypes are associated with the mutant tyrosinase alleles. I also discuss the implications of a cluster of mutation sites in exon 1 coding for the amino‐terminus of tyrosinase.
Phylogeny of Regulatory Regions of Vertebrate Tyrosinase Genes
Authors: YAMAMOTO HIROAKI , KUDO TOSHIYUKI , MASUKO NORIO , MIURA HIROHITO , SATO SHIGERU , TANAKA MIKA , TANAKA SATOSHI , TAKEUCHI SAKAE , SHIBAHARA SHIGEKI , TAKEUCHI TAKUJI
Published: July 2006

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP‐1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5’ flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5’ upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5’ upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence‐coding regions.
L‐DOPA Inhibits In Vitro Phosphorylation of Melanoma Glycoproteins
Authors: SLOMINSKI ANDRZEJ , FRIEDRICH THOMAS
Published: July 2006

L‐DOPA had no effect on the endogenous phosphorylation of proteins after extraction with 1% Triton X‐100 from hamster melanoma. When proteins were purified further by wheat germ‐agglutinin chromatography, however, a dramatic and dose‐dependent inhibitory effect of DOPA on glycoprotein phosphorylation was observed in the presence of Mn +2.
Hyperpigmentation of Chinchilla Stria Vascularis Following Acoustic Trauma
Authors: GRATTON MICHAEL ANNE , WRIGHT CHARLES G.
Published: July 2006

This report describes morphological alterations of the chinchilla stria vascularis seen 30 days after exposure to impulse noise. The observed changes included a dramatic increase in strial melanin content which occurred in 7 of 36 animals exposed to electronically synthesized impulses presented in various temporal patterns at either 135 or 150 dB peak SPL. In these animals, densely pigmented areas of stria 1.5 to 3 mm in length were found in the basal cochlear turn. Light and electron microscopic study revealed that these areas contained large numbers of melanin granules situated primarily in pale‐staining cells of the middle layer of the stria. Unlike the pigment granules present in normal chinchilla stria, the melanosomes found in the noise‐exposed material clearly showed ultrastructural features characteristic of eumelanin. Melanin granules were also observed in marginal and basal cells of the noise‐exposed stria. In some cases, pigment granules which had apparently been expelled from the marginal cells were present in the endolymphatic space beneath Reissner's membrane and on the strial surface. These findings support the view that the melanin‐bearing cells of the inner ear are capable of markedly increased activity in response to stressful conditions.
Experimental Autoimmune Anterior Uveitis (EAAU): Induction by Melanin Antigen and Suppression by Various Treatments
Authors: BROEKHUYSE R.M. , KUHLMANN E.D. , WINKENS H.J.
Published: July 2006

The uveitogenicity of melanin has been a controversial subject for a long time, presumably as a result of the use of ill‐defined preparations in the experiments. We have developed procedures for the preparation of purified uveitogenic melanins from the retinal pigment epithelium and choroid that are free from pathogenic retinal photoreceptor proteins. The active melano‐antigen is located at the surface of the melanin granules and is probably identical in both tissues. It retains its pathogenicity in hot polar detergent and during in vitro proteolysis, but it is inactivated by macrophage phagocytosis and hydrolysis in hot hydrochloric acid. Lewis rats immunized with microgram doses of bovine retinal pigment epithelial or choroidal melanin develop severe experimental autoimmune anterior uveitis (EAAU) about 10 days later. Retinitis and pinealitis are not observed. Skin melanin prepared in a similar way evokes EAAU as well, but it is only weakly pathogenic. EAAU cannot be transferred by serum, and its development can effectively be inhibited by antibodies to the inciting antigen and by cyclosporin. Vitamin E treatment of the animals causes a delay in its onset. The results indicate that cell‐mediated immunity plays a dominant role in the pathogenesis of EAAU. This is the first time it has been shown that purified ocular and skin melanins are able to induce an autoimmune disease. The relevance of this finding for the study of melanin‐related immunopathology in man is discussed.
Deficiency of the Gene B Impairs Differentiation of Melanophores in the Medaka Fish, Oryzias latipes: Fine Structure Studies
Authors: HIROSE EUICHI , MATSUMOTO JIRO
Published: July 2006

In an orange‐colored variant of the medaka fish, Oryzias latipes, which is homozygous for b allele, the melanophores represent a tissue‐specific differentiation, manifesting an amelanotic appearance in the skin, an incomplete melanogenesis in the choroid and the peritoneum, and mosaic phenotype‐like melano‐iridophores in the peritoneum. In a wild‐type strain of this species carrying the B gene, all melanophores are terminally differentiated irrespective of the tissues in which they are located. This indicates that the deficiency of B gene impairs the differentiation of melanophores in the medaka. Electron microscopy disclosed that the deficiency of B gene causes deterioration of melanogenesis to occur inside the melanosomes and that the manner of deterioration in the melanophores in the skin, the choroid and the peritoneum is different. The ubiquitous occurrence of reflecting platelet‐laden melanophores in the peritoneum of this variant and the total absence of a mosaicism in pigment cells of the wild‐type strain indicate that the deficiency of B gene predestines melanoblasts distributed in this tissue to an ambiguous state with regard to their differentiation. Little difference is observed between melanosomes maturation in pigment epithelial cells of the orange‐colored variant and the wild‐type strain, indicating an innocent role of the B gene in their differentiation.
Production of [14C]‐Labeled 3‐Hydroxy‐L‐Kynurenine in a Butterfly, Heliconius charitonia L. (Heliconidae), and Precursor Studies in Butterfly Wing Ommatins
Authors: KOCH P. BERNHARDT
Published: July 2006

A method was developed to produce radiolabeled 3‐hydroxy‐L‐kynurenine by injection of [14C]‐L‐tryptophan into pupae of the heliconid butterfly, Heliconius charitonia, which was converted into [14C]‐3‐hydroxy‐L‐kynurenine and deposited as a wing pigment. Extractions of 3‐hydroxykynurenine (3‐OHK) with 60% methanol from wings yielded in 14.4 μg per mg dry weight. In extracts from yellow wing areas, 3‐OHK represented 100% of detectable amino acids. Resulting specific radioactivity of [14C]‐3‐OHK was between 0.05 and 0.07 mCi/mmol when 0.5 μCi [14C]‐tryptophan was injected into pupae 1 or 2 days before emergence of the butterfly.

Incorporation of [14C]‐3‐OHK into wing ommochromes was studied in nymphalid butterflies, Araschnia levana and Precis coenia. After injection into pupae [I4C]‐3‐OHK as well as [14C]‐tryptophan were specifically incorporated into red and red‐brown wing scales as shown by autoradiography. The same incorporation occurred in isolated wings after incubation in Grace's medium containing [14C]‐3‐OHK. In Araschnia levana, [14C]‐3‐OHK offered to left wing pairs was incorporated into dihydroxanthommatin six times more effectively than [14C]‐tryptophan offered to right wing pairs from the same specimen. Therefore, 3‐OHK seems to be the ultimate precursor of wing ommatins.
Free Radicals in Melanin‐Cationic Porphyrins Complexes in the Dark and Under Light Irradiation
Authors: RIDENTE YARA , AZZELLINI GIANLUCA CAMILLO , ITO AMANDO SIUITI
Published: July 2006

Electron Paramagnetic Resonance (EPR) spectroscopy was employed in the study of the interaction between L‐3,4‐dihydroxyphenylalanine (L‐Dopa) melanin and the cat‐ionic porphyrins meso‐tetrakis(1‐methylpyridinium‐4yl)‐porphyrin (TMPyP), meso‐tetrakis‐(1‐benzylpyridinium‐4‐yl)‐porphyrin (TBzPyP), and their respectives complexes ZnTMPyP and ZnTBzPyP. By monitoring signal intensities and progressive microwave power saturation it was shown that the interaction increases the equilibrium concentration of free radicals in L‐Dopa melanin in the dark. The extent of increase is dependent on the presence of molecular oxygen and on the type of porphyrin. Not all interacting sites available for complexation in L‐Dopa melanin are involved in the formation of free radicals. It was also observed that the interaction with porphyrins promotes an increase in the number of photoinduced free radicals in L‐Dopa melanin during illumination with visible light.
Does Melanin Turnover Occur in the Eyes of Adult Vertebrates?
Authors: SCHRAERMEYER ULRICH
Published: July 2006

This paper is a review of what is known about the turnover of melanin in iris, choroid, and retinal pigment epithelium (RPE) of the adult vertebrate eye. Differences in size and structure of choroideal and retinal pigment epithelial melanin granules are shown by electron micrographs. The classical stages of melanin synthesis, including the premelanosome, are shown in the RPE of adult hamsters that had been exposed to intense light. Degradation or synthesis of melanin also seem to occur in the melanocytes of the choroid in these animals.

It is postulated that all three pigmented eye tissues (iris, RPE, and choroid) of adult vertebrates form melanin granules in vivo. However, nothing is known about the amount of this turnover.
Improved Tyrosinase Activity Stains in Polyacrylamide Electrophoresis Gels
Authors: JIMENEZ‐CERVANTES CELIA , VALVERDE PALOMA , GARCÍA‐BORRÓN JOSÉ C. , SOLANO FRANCISCO , LOZANO JOSÉ A.
Published: July 2006

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post‐translational processing with possible regulatory implications. So far, SDS‐PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L‐dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluoro‐graphic detection of radioactive melanin after incubation of the gels in the presence of L‐[3‐14C]‐dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84–89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH. Its sensitivity is also more than one order of magnitude higher than the one obtained with L‐dopa alone, and comparable to the one of the fluorographic method, but, as opposed to this latter, the complete staining can be performed in less than 1 hr.
Effects of Mutations at the W Locus (c‐kit) on Inner Ear Pigmentation and Function in the Mouse
Authors: CABLE J. , HUSZAR D. , JAENISCH R. , STEEL K.P.
Published: July 2006

The W locus encodes a tyrosine kinase receptor, c‐kit, which affects survivial of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (Wv/Wv) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (Wx, Wsh, and W41) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of Wsh/Wx, 25% of Wsh/Wsh, 60% of W41/Wx, and 69.2% of W41/W41 ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W41/Wx, Wsh/Wsh, and W41/W41 mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5‐day‐old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.
Stem Cell Factor Regulates Human Melanocyte‐Matrix Interactions
Authors: SCOTT GLYNIS , EWING JAMES , RYAN DANIEL , ABBOUD CAMILLE
Published: July 2006

Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c‐kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down‐regulated the expression of the α2 receptor, and up‐regulated the expression of the α3, α5 and β1 integrin receptors. SCF down‐regulated expression of α2, α5 and β1 integrins by fetal melanocytes, and up‐regulated expression of the αv and α3 integrin receptors. Analysis of melanocyte migration using time‐lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin.
Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis
Authors: SCHRAERMEYER ULRICH
Published: July 2006

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane‐bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.
Effects of Odorants on Pigment Aggregation and cAMP in Fish Melanophores
Authors: KARLSSON JAN OLOF G. , SVENSSON SAMUEL P.S. , MÅRTENSSON LENA G.E. , ÖDMAN SVANTE , ELWING HANS , LUNDSTRÖM K. INGEMAR
Published: July 2006

Odor perception within olfactory neuroepithelium and pigment translocation within melanophores both seem to rely on a cAMP‐based second messenger system.

From studies on cultured frog melanophores, Lerner et al. (Proc. Natl. Acad. Sci. USA 85:261‐264, 1988) suggested that some aspect of odor perception may be mediated by a nonspecific mechanism whose signal is transduced by a cAMP‐based second messenger system.

In the present study, odorants (β‐ionone, benzylaldehyde, cineole, cinnamaldehyde, and octanol), which previously have been shown to stimulate formation of cAMP in the olfactory neuroepithelium, were investigated for possible pigment dispersing and cAMP‐increasing effects. Pretreatment of fish melanophores with the adenylate cyclase activator forskolin (1 μM) resulted in an approximately 300% increase in cAMP and an almost complete blockage of noradrenaline‐induced pigment aggregation. However, none of the tested odorants were able to increase the cAMP level and only cinnaldehyde and β‐ionone were found to have any pigment dispersing activity.
The Pathology of Melanoma as a Basis for Prognostic Models: The UCSF Experience
Authors: SAGEBIEL RICHARD W.
Published: July 2006

A patient survival model is proposed which allows visualization of a data base, and which includes only routine and commonly recorded attributes in most melanoma clinics. It is proposed that a network of such data be collected for meta‐analysis (MELNET), which could make stratification within the individual subsets more significant by virtue of the larger numbers. Such a network could then be fully tested in various melanoma clinics for clinical usefulness.
Human TRP‐1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity
Authors: ZHAO HUIQUAN , ZHAO YANG , NORDLUND JAMES J. , BOISSY RAYMOND E.
Published: July 2006

Human TRP‐1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP‐1 for the two substrates, L‐tyrosine and L‐DOPA. Immunopurified TRP‐1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP‐1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP‐1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of 3H‐tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L‐DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase‐negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP‐1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L‐DOPA to the assay. These results suggested that TRP‐1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP‐1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.
Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare
Authors: NEGISHI S. , HASEGAWA Y. , MARTIN G. , JUCHAULT P. , KATAKURA Y.
Published: July 2006

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black‐grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron‐dense central region surrounded by an electron‐lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large‐sized vesicles containing a small amount of electron‐dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.
Growth of Neural Crest Cells In Vitro Is Enhanced By Extracts From Silky Fowl Embryonic Tissues
Authors: LECOIN LAURE , MERCIER PASCALE , DOUARIN NICOLE M. LE
Published: July 2006

In the Silky Fowl (SF) breed of chicken, most of the internal organs are infiltrated with melanocytes. Previous studies have shown that this generalized mesodermal pigmentation is not due to a cell autonomous abnormality of the melanocytes but to environmental factors able to promote both the homing of pigment cell precursors in abnormal embryonic sites and their proliferation and differentiation.

To analyse the mode of these environmental cues, we tested the effect of SF embryo extract (SFEE) on cultured quail neural crest cells as compared with that of EE from normal chickens of the JA57 strain (JA57EE). We found that SFEE enhances crest cell proliferation as judged by 3H‐TdR incorporation and cell counting. In contrast, no effect of SFEE was observed either on the proportion of cultured cells that are engaged into the melanocytic differentiation pathway or on the amount of melanin produced by each differentiated pigment cell. The simple observation, however, reveals that SFEE has a significant effect on pigmentation of the cultured quail neural crest cells. This effect has therefore to be accounted for by the general increase in cell number induced by SFEE. The question is raised as to whether the in vivo SF phenotype is generated exclusively by this mechanism.
Tamoxifen Inhibition of Ocular Melanoma Cell Attachment to Matrix Proteins
Authors: MACNEIL S. , WAGNER M. , RENNIE I.G.
Published: July 2006

Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti‐estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen‐receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.
X‐Ray Characterization of Melanins—I
Authors: CHENG JIN , MOSS SIMON C. , EISNER MELVIN , ZSCHACK PAUL
Published: July 2006

The intrinsic local structure characterization of natural sepia melanin and L‐dopa and tyrosine synthetic melanin powder has been carried out by X‐ray diffraction using synchrotron radiation. The derived structure factor, S(q), shows six significant diffuse peaks within the q‐range from 0.3 Å‐1 to 16 Å‐1 in the reciprocal space (q= (4π sin θ)/λ, 2θ is the scattering angle). The Fourier transform of S(q), which yields the radial distribution function (RDF), gives us information in real space of a 1.42 Å distance averaged over the C‐C, C‐O and C‐N bond lengths as well as peaks at 2.40‐2.41 Å, 3.67‐3.71 Å and 4.67‐4.70 Å discrete neighbor distances. There is a great similarity in the scattering intensity profiles of the natural and synthetic melanins indicating that the synthetically prepared material may be essentially similar to “real” melanin in its local atomic arrangements. An evidence of a prepeak at q = 0.45 Å‐1 has been confirmed which indicates a preferred length scale of ∼ 13‐20 Å that corresponds to the initial particle size in colloidal melanin solutions.
The Mouse Brown (b) Locus Protein Functions as a Dopachrome Tautomerase
Authors: WINDER ALISON J. , WITTBJER ANNA , ODH GERD , ROSENGREN EVALD , RORSMAN HANS
Published: July 2006

The mouse b locus controls black/brown coat coloration. Its product, the b‐protein or TRP‐1, has significant homology to tyrosinase, and this has led to suggestions that the b‐protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b‐protein. We were unable to con‐firm previous reports that the b‐protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b‐protein‐expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A‐Sepharose, and the major product of its action on L‐dopachrome is 5,6‐dihydroxyindole‐2‐carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b‐protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b‐protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6‐hydroxy‐5‐methoxyindole‐2‐carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20‐fold lower, and not significantly above background. Hence, it appears that the b‐protein acts as a dopachrome tautomerase in vivo as well as in vitro.