bluuebunny/crossref_metadata_2025 huggingface dataset:
X‐ray Diffraction Studies on Melanins in Lyophylized Melanosomes
Authors: BRIDELLI MARIA GRAZIA , CRIPPA PIER RAIMONDO , UGOZZOLI FRANCO
Published: July 2006

A series of experiments was performed on lyophylized melanosomes in order to analyze the melanin in the natural state as polymerized into these organdies and to verify in such biological amorphous material the possibility of obtaining intensity scattering curves from which Bragg distances can be calculated. The results confirm the feasibility of the method and show that melanins in melanosomes maintain many structural features of the purified form and that the biochemical composition of the organelles can be responsible for the observed differences in the diffractograms. The presence in melanosomes of supramolecular paracrystalline aggregates was also clearly demonstrated.
Further Evidence for Synthesis of Screening Pigment Granules Involved in the Photosensory Membrane Turnover of the Crayfish Photoreceptor
Authors: SCHRAERMEYER ULRICH
Published: July 2006

Photosensory membrane degradation in crayfish occurs at first in multi‐vesicular bodies (MVBs) and then, with the aid of lysosomal enzymes, in lysosome related lamellar bodies. In organ culture experiments with the isolated crayfish retina (Orconectes limosus) small screening pigment‐like granules became visible under the electron microscope in such lamellar bodies and suggested a possible relation of photosensory membrane degradation and screening pigment granule synthesis. Chloroquine, an inhibitor of lysosomal activity, when added to the culture medium reduced the appearance of screening pigment‐like granules in lamellar bodies, but led to the appearance of these granules in mature MVB's, indicating the involvement of lysosomal enzymes in the formation of pigmented lamellar bodies. In a second set of experiments the effect of bright light on the screening pigment granule ultrastructure of crayfish phoreceptors was investigated. It was found that after bright light exposure large numbers of little screening pigment granules (0.15–0.3 μm) were located between or close to rhabdomeral microvilli that were not at these sites in crayfish kept under natural light. MVB's were also reduced in size, and among the little screening pigmentary organelles granules of different electron density and morphology appeared. Additionally, vesicle flux to little screening pigment granules was detected. The screening pigment granules of the little type did not seem to be transported close to or between the microvilli, but appeared to be synthesized at these sites within little MVBs.
Histopathology of Melanocytic Lesions in Goats and Establishment of a Melanoma Cell Line: A Potential Model for Human Melanoma
Authors: PARSONS P.G. , TAKAHASHI H. , CANDY J. , MEYERS B. , VICKERS J. , KELLY W.R. , SMITH I. , SPRADBROW P.
Published: July 2006

Melanocytic cells from white Angora goats were studied in vivo and in vitro. The histopathology of pigmented areas of skin from the most common sites of melanoma (solar‐exposed areas of the ear, face, and perineum) resembled that of the epidermal melanocytes in Hutchinson's melanotic freckle in humans. Seven melanoma biopsies from 6 Angora goats showed histopathological features in common with human melanoma. A melanoma cell line, GM‐1, was established in culture from a lymph node metastastis obtained from an animal that had a primary tumor excised and later developed extensive metastatic disease. GM‐1 cells were mainly diploid, amelanotic, proliferated rapidly, spontaneously formed vacuolated cells, and were tumorigenic in nude mice. The species of origin of the GM‐1 line was confirmed by isozyme profiles. GM‐1 cultured cells and the original biopsy both expressed S‐100 protein and tyrosinase antigen. Using GM‐1 cells as the immunogen, a monoclonal antibody (MoAb 1F1) was derived that reacted strongly with a 116 kDa antigen in 50% of the GM‐1 cells, but had little activity with goat fibroblasts (GM‐F) or with human melanoma cells. GM‐F, on the other hand, yielded more intense staining than GM‐1 with an intermediate filament antibody (IFA), reacting with a 58 kDa antigen in both cell lines. The sensitivity of GM‐1 to anticancer agents was similar to that of human melanoma cells. The pathology of caprine melanoma and its association with sun‐exposed sites in relatively young animals suggest that it may be a suitable model for studying induction of melanoma by natural sunlight.
Melanocyte Growth Factor in Normal Human Skin
Authors: HORIKAWA TATSUYA , NISHINO KEISUKE , MISHIMA YUTAKA
Published: July 2006

Normal human skin is shown to contain melanocyte growth factor (MeGF). We found MeGF activity in extracts of both the epidermal portion of skin and the dermal portion. This activity was completely adsorbed onto heparin beads and eluted by 2.5 M NaCl. In addition, the activity of both extracts was completely blocked by antibodies directed against basic fibroblast growth factor (bFGF). It is suggested that melanocytes in epidermis are supported by bFGF‐like MeGF in normal human skin.
Identification of Two Types of Melanocyte Within the Stria Vascularis of the Mouse Inner Ear
Authors: CABLE J. , STEEL K.P.
Published: July 2006

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron‐lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron‐dense acid phosphatase‐rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron‐dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pyenotie nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.
Dysplastic Melanocytic Nevi Contain High Levels of Pheomelanin: Quantitative Comparison of Pheomelanin/Eumelanin Levels Between Normal Skin, Common Nevi, and Dysplastic Nevi
Authors: SALOPEK THOMAS G. , YAMADA KOJI , ITO SHOSUKE , JIMBOW KOWICHI
Published: July 2006

The degree and type of melanogenesis, i.e., either eumelanin of pheomelanin, has been shown to be a reliable marker for the differentiation of the melanocyte. If exposed to UV light, these two melanins were reported to behave differently; eumelanin was photoprotective whereas pheomelanin was phototoxic to cultured tumor cells. Our previous study indicated that dysplastic melanocytic nevus (DMN) undergoes altered melanogenesis, forming pheomelanosome‐like granules. The present study examined chemically the type and degree of melanin synthesized in 31 melanocytic nevi excised from 27 patients as compared with that occurring in the surrounding normal skin. The tissue content of eumelanin and pheomelanin was expressed by the amounts of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP), respectively. We found that DMN lesions contain significantly higher amounts of pheomelanin than either common melanocytic nevus (CMN) or normal skin. Differences in pheomelanin content between DMN and CMN could not be accounted for by inherently higher levels of pheomelanin within the skin in general from DMN patients. Our present finding substantiates our previous claim that epidermal melanocytes in DMN undergo deranged melanogenesis.
NMR Studies of Melanins: Characterization of a Soluble Melanin Free Acid From Sepia Ink
Authors: AIME SILVIO , FASANO MAURO , TERRENO ENZO , GROOMBRIDGE CHRISTOPHER J.
Published: July 2006

This paper deals with the nuclear magnetic resonance characterization of a soluble derivative (melanin free acid) of Sepia melanin obtained by a peroxidative treatment of the parent (insoluble) species. High resolution 13C and 15N solid state NMR spectroscopies allow the assessment of the chemical changes occurring in the macromolecule upon solubilization.

1H and 13C NMR solution spectra are discussed in light of the results obtained from the solid state spectra.

Furthermore, the coordination properties of melanin have been investigated through 27Al NMR spectroscopy and proton relaxation enhancement studies of the paramagnetic gadolinium complex of melanin free acid. Through these experiments it has been possible to evaluate the molecular reorientational time τR (and from it an estimated molecular weight close to 20 KDa) and the strength of the metal‐macromolecule interaction.
Glucocorticoid Stimulates Melanogenesis and Tyrosinase Gene Expression in B16 Melanoma Cells
Authors: ITO AKIRA , TANAKA CHIKAKO , TAKEUCHI TAKUJI , MISHIMAI YUTAKA
Published: July 2006

Effects of dexamethasone on melanogenesis and tyrosinase mRNA levels were determined in B16/F10 melanoma cells. Melanin content of B16 cells increased in a dosedependent manner by the addition of dexamethasone to the culture medium. After 72 hr exposure, dexamethasone (10−6 M) produced a 2.4‐fold increase in melanin content. Northern blot analysis revealed that tyrosinase mRNA level also increased by the addition of dexamethasone to the culture medium. After 24 hr exposure, dexamethasone (10−6 M) caused a 1.8‐fold increase in tyrosinase mRNA levels. A tumor promoter, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) decreased tyrosinase mRNA level at 30 nM concentration. Dexamethasone antagonized this TPA mediated decrease in tyrosinase mRNA. It is suggested that glucocorticoids are involved in the regulation of tyrosinase activity at the transcriptional level.
Effect of Different Isomers of Dihydroxybenzoic Acids (DBA) on the Rate of DL‐DOPA Oxidation by Mushroom Tyrosinase
Authors: SCHVED FERNANDO , KAHN VARDA
Published: July 2006

Dihydroxybenzoic acids (DBA), such as 3,4‐DRA, 3,5‐DBA, and 2,4‐DBA—at all concentrations tested—inhibited the rate of DL‐DOPA oxidation to dopachrome (λmax = 475 nm) by mushroom tyrosinase. 2,3‐DBA and 2,5‐DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL‐DOPA oxidation. The synergistic effect of 0.6‐13.3 mM 2,3‐DRA on the rate of DL‐DOPA oxidation to dopachrome (λmax = 475 nm) was found to be due to the ability of 2,3‐DBA‐o‐quinone (formed by the oxidation of 2,3‐DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL‐DOPA to dopachrome (via dopaquinone) non‐enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5‐DBA on the rate of DL‐DOPA oxidation by mushroom tyrosinase.
Catecholamines and Vitiligo
Authors: MORRONE ALDO , PICARDO MAURO , LUCA CHIARA DE , TERMINALI ORIETTA , PASSI SIRO , IPPOLITO FERDINANDO
Published: July 2006

The levels of some catecholamine metabolites, namely homovanillic acid (HVA), vanil‐mandelic acid (VMA), 3‐methoxytyramine (MT), normetanephrine (NMN), metanephrine (MN), 3,4‐dihydroxy mandelic acid (DOMAC), and 3,4‐dihydroxy phenylacetic acid (DOPAC), have been evaluated in the 24 hr urines of 150 patients affected with different types of vitiligo and in 50 healthy age‐matched individuals. The patients were divided into three groups according to the different phases of the disease. The first group included subjects affected either with the early active phase or with progressive increase in both number and/or the size of previous lesions. The second group included patients in whom no new lesions had appeared for between 4‐8 months. In the third group the white areas had been stable for 1‐5 years. The first and second groups showed values of HVA and VMA from 4 to 10 times and from ½ to 3 times higher respectively than those of controls, while no significant differences were found between the third group and controls. Our results clearly show that a significant increase of urinary levels of HVA and VMA, deriving respectively from dopamine and from norepinephrine and epinephrine characterizes the onset and the progressive active phases of vitiligo, irrespective of the type of distribution. The increased release of catecholamines from the autonomic nerve endings in the microenvironment of melanocytes in the affected skin areas might be involved in the etiopathogenesis of vitiligo through two main mechanisms: (1) a direct cytotoxic action of catecholamines and/or their o‐diphenol catabolites; (2) an indirect action. Skin and mucosa arterioles possess α receptors, activation of which by catecholamine discharge may cause a severe vasoconstriction, leading to epidermal and dermal hypoxia with hyperproduction of toxic oxyradicals generated by different pathways. In both cases a genetic predisposition due to insufficient radical scavengers in the affected areas should be taken into account.
Synthesis and Characterization of Melanins From Dihydroxyindole‐2‐Carboxylic Acid and Dihydroxyindole
Authors: ORLOW SETH J. , OSBER MICHAEL P. , PAWELEK JOHN M.
Published: July 2006

Several studies have confirmed that a melanocyte‐specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6‐dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI‐melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co‐polymerization of DHICA and DHI. With or without DHI, DHICA‐melanins absorbed throughout the ultraviolet and visible spectra (200‐600 nm). The DHICA‐melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA‐melanins could be passed through 0.22 μm filters but could not be dialyzed through semi‐permeable membranes with exclusion limits of 12,000‐14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100‐1,000 DHICA monomers per molecule of melanin. DHICA‐melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.). In experiments reported here, brown, but not black, melanins from mouse hairs, human melanoma cells, and peacock feathers were soluble in aqueous buffers. Since DHICA‐melanins are both soluble and brown, the results raise the possibility that they are determinants of brown colors in the animal kingdom.
Melanin Standard Method: Particle Description
Authors: ZEISE LISA , MURR BROWN L. , CHEDEKEL MILES R.
Published: July 2006

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin—K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.
Measurements of Light Transmission Through Single Melanophores
Authors: ÖDMAN S. , LUNDSTRÖM I. , KARLSON J.O.G. , SVEIDER P. , SVENSSON S.P.S.
Published: July 2006

A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.). The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 μm2. Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor‐mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.
Melanin Standard Method: Empirical Formula 2
Authors: CHEDEKEL MILES R. , AHENE AGO B. , ZEISE LISA
Published: July 2006

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies.

We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.
Morphological and Biochemical Characterization of the Cream Markings in the Integument of the Female Isopod, Armadillidium vulgare
Authors: NAKAGOSHI MOTOKO , NEGISHI SUMIKO
Published: July 2006

Cream markings aligned along the dorsal region of the female isopod, A. vulgare, were investigated with light and a fluorescence microscope and an electron microscope. Biochemical studies were also carried out. The cream markings were observed in the dorsal integument as a group of cream‐colored chromatophores that emit a yellow fluorescence. These chromatophores, which are distinguishable from ommochrome chromatophores, contained numerous granules in the cytoplasm, and these granules (0.6–3.0 μm in length by 0.4–1.5 μm in width) were electron‐lucent and spheroidal in shape with a concentric arrangement of membranes. Based on various biochemical analyses, the principal component of the yellow pigment isolated from the cream markings was identified as sepiapterin. These facts revealed that the cream markings are the chromatophores that contain pteridine granules. The males have no cream markings like those of the females, since the cream‐colored chromatophores are externally hidden by the ommochrome chromatophore layer. The content of sepiapterin in the males was about two times greater than that in the females. This quantitative difference in sepiapterin content between males and females suggests that the pteridine formation in this pigment cell may be regulated by hormones associated with sex determination.
Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell‐Specific Expression in Transgenic Mice
Authors: BEERMANN FRIEDRICH , SCHMID ERIKA , GANSS RUTH , SCHÜTZ GÜNTHER , RUPPERT SIEGFRIED
Published: July 2006

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest‐derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well‐characterizedc‐locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type‐specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell‐type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type‐specificity and developmental regulation in melanocytes and the pigment epithelium.
Conserved Regulatory Mechanisms of Tyrosinase Genes in Mice and Humans
Authors: TANAKA MIKA , TANAKA SATOSHI , MIURA HIROHITO , YAMAMOTO HIROAKI , KIKUCHI HIDEAKI , TAKEUCHI TAKUJI
Published: July 2006

In vertebrates, melanin production is restricted to pigment cells. This cell type‐specific melanogenesis is considered to involve cell type‐specific expression of the tyrosinase gene. Recently, there have been several reports that sequences in the 5’ flanking region of the mouse tyrosinase gene are responsible for cell type‐specific expression of the transgene in mice. As the first step in the study of the evolution of the regulatory mechanisms for tyrosinase gene function in vertebrates, we constructed a fused gene, hg‐Tyrs‐J which includes a 1.0‐kb 5’ flanking sequence of the human tyrosinase gene fused with mouse tyrosinase cDNA. By introducing the fused gene into fertilized eggs of albino mice, we obtained two mice that exhibited pigmentation in the skin and eyes and established a transgenic line from one of them. Further analyses revealed that the transgene was expressed cell type‐specifically in these transgenic mice. We conclude, therefore, that the 1.0 kb 5’ upstream region of the human tyrosinase gene contains conserved cis‐elements essential for cell type‐specific expression of the tyrosinase genes in mice and humans. Results of our study may provide a clue to elucidate the evolutionary process of regulatory mechanisms of the tyrosinase gene.
The Expression of Mouse Tyrosinase in Chick Cells In Vitro and In Vivo When Controlled by a Constitutive Promoter
Authors: FREW TIMOTHY , WHITAKER BETH , SALTER DONALD , PAYNE WILLIAM , HUGHES STEPHEN , YAMAMOTO HIROAKI , TAKEUCHI TAKUJI , BRUMBAUGH JOHN
Published: July 2006

Virally introduced mouse tyrosinase expression was checked both in vitro and in vivo in chicken cells and tissues. The results indicate that a constitutive promoter is able to express mouse tyrosinase in a variety of cells and tissues both in vitro and in vivo. Tyrosinase expression is marked by pigment production in situ, which is visible at macroscopic as well as microscopic levels without the use of substrates. It is concluded that tyrosinase can be a valuable marker for tracking gene insertion since it is spontaneously expressed. The expression of tyrosinase in some cells and tissues has a detrimental effect, however, and should be controlled by tissue‐specific promoters.
Expression and Transmission of Wild‐Type Pigmentation in the Skin of Transgenic Orange‐Colored Variants of Medaka (Oryzias latipes) Bearing the Gene for Mouse Tyrosinase
Authors: MATSUMOTO JIRO , AKIYAMA TOYOKO , HIROSE EUICHI , NAKAMURA MIZUHO , YAMAMOTO HIROAKI , TAKEUCHI TAKUJI
Published: July 2006

Transgenic fish carrying a reconstructed mouse tyrosinase gene, mg‐Tyrs‐J, were produced by microinjecting the gene into the oocyte nucleus of an orange‐colored variant of medaka (Oryzias latipes). Of 64 oocytes microinjected and subsequently inseminated, 13 embryos developed normally beyond hatching and three of them exhibited brown skin pigmentation in the adult as was commonly observed in the wild type of this species. Light and electron microscopic examination disclosed a ubiquitous distribution of typical melanophores in the skin of these transgenic fish. Judging from their population density and distribution pattern, it was presumed that melanogenesis in these fish was elicited in amelanotic melanophores that resided in the skin of the orange‐colored fish of this variant. Immunofluorescence with use of the anti‐mouse tyrosinase antiserum lacking reactivity to medaka tyrosinase clearly disclosed that the gene introduced was expressed in the melanophores of transgenic fish. Crosses of female transgenic fish and males from an orange‐colored variant yielded offspring exhibiting wild‐type or orange‐colored pigmentation in a ratio of 1:1, thus implying that mg‐Tyrs‐J integrated into the medaka genome behaves like a dominant gene. Little melanogenesis was observed in xanthophores, leucophores and iridophores in transgenic fish, suggesting possible specificity in recognition of teleostean cell types (i.e., melanophores) by the regulatory region of the mouse tyrosinase gene.
Proteinuria of B700, a 67 kD Albumin‐like Melanoma‐Specific Antigen
Authors: WALDEN THOMAS L. , FARZANEH NUSHIN K. , HEARING VINCENT J. , GERSTEN DOUGLAS M.
Published: July 2006

B700 is a murine melanoma antigen that is closely related to, but distinct from, serum albumin. The present study examined the metabolic fate and anatomic distribution of radioiodinated B700 and mouse serum albumin (MSA) administered s.c. to mice. In blood, both proteins were associated with the plasma fraction where the halflife of B700, a glycoprotein, was 0.5 days, compared to 2.7 days for MSA. Of particular interest was the observation that B700, a 67 kD anionic protein, was excreted primarily in urine. The selective B700‐proteinuria did not alter urinary volumes or produce hematuria or edema. SDS‐polyacrylamide gel electrophoresis and western blot analysis using the H‐2‐3‐3 B700‐specific monoclonal antibody revealed that B700 proteinuria occurred in B‐16 murine melanoma bearing animals but not in control mice. These studies demonstrate that the tumor‐bearing host readily distinguishes between very similar normal protein (MSA) and tumor‐associated antigen (B700) molecules and processes them differently.
Response of Transformed and Normal Mouse Cell Lines to Anti‐Melanin Compounds, Hyperthermia, and Radiation
Authors: RAAPHORST G. PETER , AZZAM EDOUARD I.
Published: July 2006

Five cell lines (one parental, two transformed melanin producing, and two transformed non‐melanin producing) were evaluated for the responses to 2‐ and 4‐hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non‐specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin‐producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin‐producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.
Synergism Exerted by 4‐Methyl Catechol, Catechol, and Their Respective Quinones on the Rate of DL‐DOPA Oxidation by Mushroom Tyrosinase
Authors: SCHVED FERNANDO , KAHN VARDA
Published: July 2006

4‐Methyl catechol and catechol, at concentrations ranging from 0.03 to 9 mM and 0.066 to 20 mM, respectively, have a synergistic effect on the rate of DL‐DOPA oxidation by mushroom tyrosinase to material absorbing at 475 nm. The synergism results from the ability of 4‐methyl catechol‐o‐quinone (4‐methyl‐o‐benzoquinone) and of catechol‐o‐quinone (o‐benzoquinone) to oxidize DL‐DOPA non‐enzymatically to dopaquinone, with the latter being immediately converted to dopachrome (λmax = 475 nm).
Smyth Chicken Melanocyte Autoantibodies: Cross‐Species Recognition, In Vivo Binding, and Plasma Membrane Reactivity of the Antiserum
Authors: SEARLE EDWARD A. , AUSTIN LISA M. , BOISSY YING L. , ZHAO HUIQUAN , NORDLUND JAMES J. , BOISSY RAYMOND E.
Published: July 2006

Smyth line (SL) chickens, which develop a depigmenting disorder similar to human vitiligo, produce circulating anti‐melanocyte antibodies (Austin, L.M. et al., (1992) The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo. Clin. Immunol. Immunopathol., 64:112–120). In order to characterize these autoantibodies, we studied the reactivity of cultured chicken, mouse, and human melanocytes, as well as frozen sections of chicken feather follicles and embryonic eyes, against SL serum, employing indirect immunofluorescence. Light Brown Leghorn (LBL) serum was used as a negative control. Chicken (SL and LBL), mouse, and human melanocytes exhibited greater fluorescence with SL serum than with LBL serum (up to a 1:60,000 dilution). The fluorescent pattern was predominant along the perimeter of the cells, suggesting plasma membrane staining. Fluorescence‐activated flow cytometry analysis and immunocytochemical localization at the ultrastructural level using intact chicken cells supported this hypothesis. Melanocytes were readily stained in cryosections of regenerating feather follicles and embryonic eyes incubated with SL, but not LBL, serum. In addition, amelanotic melanocytes in albino chicken feathers reacted with SL serum. SL serum also preferentially stained cells emigrating from cultured avian neural tubes and within the dermis of the proliferative germ of regenerating feather follicles suggesting that melanoblasts express the antigens. We conclude that Smyth line serum contains melanocyte autoantibodies that cross‐react with mouse and human melanocytes, are able to bind to pigment cells within tissues, and recognize antigens expressed in the cytoplasm and on the surface of melanocytes and melanoblasts.
Histological, Biochemical, and Ultrastructural Studies on Hyperpigmented Human Skin Xenografts
Authors: FAROOQUI JAMAL Z. , AUCLAIR BILLYE W. , ROBB EDWARD , SARKISIAN EDWARD , COOPER CAROL , ALEXANDER J. WESLEY , WARDEN GLEN , BOISSY RAYMOND E. , NORLUND JAMES
Published: July 2006

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post‐grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4–6 weeks. The number of Dopa‐positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre‐grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (αTy‐SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa‐positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre‐ and post‐grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw ∼14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.
Actin and Tubulin Arrays in Cultured Xenopus Melanophores Responding to Melatonin
Authors: ROLLAG MARK D. , ADELMAN MARK R.
Published: July 2006

Melatonin induces pigment granule aggregation in amphibian melanophores. In the studies reported here, we have used fluorescence microscopic techniques to test the hypothesis that such melatonin‐induced pigment movement is correlated with alterations in either the actin or tubulin cytoskeletal patterns of cultured Xenopus melanophores. In general, the cytoplasmic domains of the cultured melanophores were flat and thin except in the perinuclear region (especially when the pigment was aggregated). The microtubules and microfilaments were usually found in the same focal plane; however, on occasion, microfilaments were closer to the substratum. Microtubules were arranged in arrays radiating from what are presumed to be cytocenters. A small percentage of the melanophores were very large, had actin‐rich circular perimeters and did not respond as rapidly to melatonin treatment as did the other melanophores. Melanophores with either aggregated or dispersed melanosomes had low intensity rhodamine‐phalloidin staining of actin filaments compared to nonpigmented cells, whereas the FITC anti‐tubulin intensities were comparable in magnitude to that seen in nonpigmented cells. When cells were fixed prior to complete melatonin‐induced pigment granule aggregation there was no abrupt diminution in either the tubulin or actin staining at the boundary between pigment granule‐rich and pigment granule‐poor cytoplasmic domains. Nor could the actin and tubulin patterns in cells with partially aggregated melanosomes be reliably distinguished from those in melanophores in which the melanosomes were either completely dispersed or completely aggregated. These data argue against the hypothesis that melatonin causes consistent large‐scale rearrangements of tubulin and actin polymers as it induces pigment aggregation in Xenopus melanophores.
Further Evidence for Involvement of Both Cell Mediated and Humoral Immunity in Generalized Vitiligo
Authors: ABDEL‐NASER M. BADAWY , KRÜGER‐KRASAGAKES S. , KRASAGAKIS K. , GOLLNICK H. , ORFANOS C.E.
Published: July 2006

Immunohistochemical and immunoserological evidence supports the involvement of both cell‐mediated and humoral mechanisms in the pathogenesis of melanocyte destruction in vitiligo. Punch biopsies from depigmented vitiliginous skin (VS), normal‐looking pigmented skin (PS), and marginal skin (MS) from patients with generalized vitiligo (n = 15) were labeled with K 1.2.58, OKM1 (CD11b), Leu 11b (CD16), Leu 19 (CD56), IFN‐γreceptor, IL‐2 receptor (CD25), IgG, IgM, C3c, and C3d MoAbs. In addition, in vitro effects of vitiligo sera (n = 13) on human newborn melanocytes (HMel) under different culture conditions were studied.

The immunohistochemical findings showed absence of K 1.2.58+ epidermal melanocytes in VS and abnormal morphology in MS. In these areas, a few CD11b + cells in the dermis and epidermis could be detected but no significant numbers of CD16+ or CD56+ cells were seen among the mononuclear cellular infiltrate. IL‐2 and IFN‐γ receptors were clearly expressed by the cellular infiltrate. No significant deposition of complement or immunoglobulin was seen.

The addition of vitiligo sera to HMel cultures induced a significant cellular proliferation. The stimulation of cell proliferation occurred regardless whether the sera were added alone or when preheated (56°C for 1 hr) and then supplemented with a complement source (P < 0.01 at 2%, P < 0.001 at 10%, and P < 0.01 at 20% for sera alone) (P > 0.05 at 2%, P < 0.05 at 10%, and P < 0.01 at 20% for decomplemented sera plus complement). In contrast, incubation of vitiligo sera together with normal lymphocytes with HMel significantly decreased the number of living melanocytes in a dose dependent manner, suggesting an antibody‐dependent cellular cytotoxicity (ADCC) reaction (P < 0.01 at 2% and 10%, P < 0.001 at 20%).

The presence of lymphocytic infiltrate at marginal skin with evidence for IL‐2‐ and IFN‐γ‐receptor expression and the decrease in the number of living cells by ADCC‐like mechanisms provide further support for an autoimmune pathogenesis in vitiligo.
Antioxidant Status in the Blood of Patients With Active Vitiligo
Authors: PICARDO MAURO , PASSI SIRO , MORRONE ALDO , GRANDINETTI MAURO , CARLO ALDO DI , IPPOLITO FERDINANDO
Published: July 2006

We have previously reported that patients with active vitiligo (AVP) have elevated urinary levels of catecholamine metabolites, such as homovanillic and vanilmandelic acids, irrespective of the form of the disease (acrofacial, segmental, generalized). We have suggested that abnormal release of catecholamines from autonomic nerve endings might play an etiological role in the onset and development of vitiligo through an overproduction of toxic (oxy)radicals in the microenvironment of melanocytes in the affected areas.

In the present study we have investigated whether this suggested increase in radicals might be associated with an oxidative stress in the blood of AVP. We have analyzed by gas‐chromatography mass‐spectrometry, by high pressure liquid chromatography, by spectophotometry plasma levels of vitamin E (Vit E), lipoperoxides (LIP), and polyunsaturated fatty acids of phospholipids (PL‐FA), erythrocyte reduced glutathione (GSH), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) activities in 62 patients affected with different forms of active vitiligo (acrofacial, segmental, generalized) and in 60 age‐matched controls. Our results show that blood levels of Vit E, SOD, GSH, GSH‐Px activity, LIP and PL‐FA in AVP were not significantly different from those of healthy age matched controls, indicating that melanocyte damage in vitiligo is not linked with a generalized oxidative stress.
A Comparison Between Melanotic and Amelanotic Retinal Pigment Epithelial Cells In Vitro Concerning the Effects of L‐Dopa and Oxygen on Cell Cycle
Authors: AKEO KIYOSHI , TANAKA YASUHIKO , OKISAKA SIGEKUNI
Published: July 2006

Melanin precursors and free radicals, cytotoxic substances, are produced during melanin synthesis by tyrosinase. We compared these cytotoxic effects of L‐dopa and oxygen on the cell cycle of melanotic retinal pigment epithelial (RPE) cells with amelanotic RPE cells because of the differences of tyrosinase activities between melanotic and amelanotic RPE cells. Flow cytometric DNA analysis of RPE cells exposed to L‐dopa (100 μM and 250 μM) were conducted at several oxygen concentrations (20%, 10%, and 5%). The dose‐dependent effect of L‐dopa to arrest the cell cycle (the S phase) was more pronounced in melanotic than in amelanotic RPE cells, and oxygen caused arrest in the G1 phase.
Expression Studies of Pigmentation and POU‐Domain Genes in Human Melanoma Cells
Authors: STURM RICHARD A. , O'SULLIVAN BRENDAN J. , THOMSON J. ANGUS F. , JAMSHIDI NEGAR , PEDLEY JULIE , PARSONS PETER G.
Published: July 2006

Human melanoma cell lines have been used to examine the regulation of the tyrosinase (TYR) and tyrosinase‐related protein genes TRP‐1 and TRP‐2 in response to differentiating chemicals and UV irradiation. TRP‐1 mRNA levels can be repressed by treatment with the differentiating chemicals DMSO and HMBA. There is little effect of UV irradiation on pigment synthesis by human melanoma cell lines or tyrosinase activity, with variable effects on the levels of the TYR, TRP‐1, and TRP‐2 gene transcripts. The human TRP‐1 gene promoter has been isolated and its activity tested by transient cell transfection to begin an examination of signal transduction mechanisms operating in response to pigmenting and differentiating agents. To identify transcription factors that may be involved in melanocytic gene expression, we studied the N‐Oct‐3 and N‐Oct‐5 octamer‐binding activities normally expressed in the neuroectodermal cell lineage and which are expressed at high levels in melanoma cells. POU‐domain‐containing cDNA have been isolated from the A2058 human melanoma cell line that are homologous to the brn‐2 gene that encodes N‐Oct‐3 and N‐Oct‐5.
p53 and PCNA Expression in Malignant Melanomas of the Head and Neck
Authors: GIROD SABINE C. , GROTH W , JUNK M. , GERLACH K.‐L.
Published: July 2006

Mutation in the p53 tumor suppressor gene is the most common genetic alteration in human cancer. As in mutant p53 the protein is stabilised and the half‐life is extended, it becomes detectable by immunohistological staining. p53 immunoreactivity thus seems to be a potential biomarker for the assessment of the oncogenic potential of malignant melanomas. In 103 tissue sections of primary and metastatic malignant melanomas of the head and neck detectable levels of p53 were only found in 3 of the primary tumors and in none of the metastases. At the same time the proliferation status of the malignant melanoma lesions was determined using the cell cycle specific antibody PCNA. 55 primary and metastatic tumors were stained with a PCNA‐MAb to determine the proliferation activity of the tumors. The results of our immunohistochemical investigation suggest that immunoreactivity of p53 cannot be used to determine the malignant potential of melanomas in the head and neck. PCNA staining showed that the majority of the tumors and metastases were proliferating rapidly.
Biologic, Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB‐MEL‐LES and IIB‐MEL‐IAN
Authors: KAIRIYAMA CLAUDIA , SLAVUTSKY IRMA , LARRIPA IRENE , MORVILLO VERONICA , BRAVO ALICIA I. , BOVER LAURA , PODHAJCER OSVALDO L. , MORDOH JOSE
Published: July 2006

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB‐MEL‐LES and IIB‐MEL‐IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB‐MEL‐LES is composed predominantly of spindle shaped cells, whereas IIB‐MEL‐IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S‐100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA‐DR antigens in variable proportions. The MAGE‐1 gene is expressed only by the IIB‐MEL‐IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB‐MEL‐LES and IIB‐MEL‐IAN were 48 and 81, respectively. IIB‐MEL‐LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB‐MEL‐IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q‐. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.
Influences of Sex, Castration, and Androgens on the Eumelanin and Pheomelanin Contents of Different Feathers in Wild Mallards
Authors: HAASE EBERHARD , ITO SHOSUKE , WAKAMATSU KAZUMASA
Published: July 2006

In mallards the bright nuptial plumage of the drake represents the neutral, sex hormone‐independent coloration of the species that both sexes eventually exhibit after castration. We compared the pheo‐ and eumelanin contents of feathers from the head, breast, flank, and under‐tail coverts in five groups of mallards after the post‐nuptial molt in summer: intact hens, intact drakes, castrated drakes, castrated drakes injected with testosterone during the spring, and castrated drakes injected with 5α‐dihydrotestosterone during the spring. In the head feathers and under‐tail coverts, the gonadal hormones of the intact birds and the testosterone injections into castrates significantly reduced the eumelanin content, tended to increase the pheomelanin content, and, thereby, changed the melanin type from eumelanic in the untreated castrates to mixed melanic in the other three groups. The eumelanin contents of the flank feathers did not differ among the groups, but the pheomelanin contents at this site was significantly elevated in the two intact groups and the testosterone‐treated compared to the uninjected castrates. Again, the melanin type changed from eumelanic in the castrates to mixed melanic in the other three groups. The high pheomelanin content of the breast feathers in the castrated birds was significantly reduced in the hens, intact drakes, and testosterone‐injected castrates with a concomitant tendency for elevated eumelanin contents. At this site, a change occurred from pheomelanic to mixed melanic. 5α‐dihydrotestosterone was clearly less effective than testosterone in affecting the melanin contents in castrates and resulted in an intermediate coloration. The differing effects of the two androgens might be a result of differences in their conversion to estrogens.