bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of a Melanotropic Peptide on Melanoma Cell Growth, Metastasis, and Invasion
Authors: GEHLSEN K.R. , HADLEY M.E. , LEVINE N. , RAY C.G. , HENDRIX M.J.C.
Published: July 2006

Melanocyte stimulating hormone (α‐MSH, α‐melanotropin),Ac‐Ser‐Tyr‐Ser‐Met‐Glu‐His‐Phe‐Arg‐Trp‐Gly‐Ly‐Pro‐Val‐NH2, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D‐Phe7]α‐MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S‐91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin‐treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.
Melanin Standard Method: Titrimetric Analysis
Authors: ZEISE LISA , 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, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48–53, 1992).
Intrinsic Pigment‐Cell Stimulating Activity in the Catfish Integument
Authors: ZUASTI ADELINA , JOHNSON WARREN C. , SAMARAWEERA PREMINDA , BAGNARA JOSEPH T.
Published: July 2006

In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus. This species has a dark dorsal surface in marked contrast to an almost white midventral surface. Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev. Biol. 129:25). A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS). When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed. The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis. Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis. A similarly conditioned medium based upon Leibovitz's L‐15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens. Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores. A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results. The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument. It is suggested that the light‐colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF. Thus, the expression of melanophores is inhibited while that of iridophores is enhanced. In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.
Tyrosinase and the Regulation of Coat Color Changes in C3H‐HeAvy Mice
Authors: THODY ANTHONY J. , BURCHILL SUSAN A.
Published: July 2006

Hair follicular tyrosinase activity was measured during hair growth in neonatal, pubertal, and adult C3H‐HeAvy mice that show differences in coat color as a result of changes in the synthesis of eumelanin and pheomelanin. Tyrosinase activity increased during hair growth in all mice but higher levels were found at puberty, when the mice grow a dark, eumelanin coat of hair, than during early and adult life, when the hair follicular melanocytes produce mainly pheomelanin. This suggests that tyrosinase is more important for the synthesis of eumelanin than that of pheomelanin. The increased tyrosinase activity associated with eumelanogenesis in the pubertal mice could not be explained by enhanced transcription of the tyrosinase gene or enzyme synthesis and appeared to be the result of a post‐translational activation. Such an activation of tyrosinase was lacking in the neonatal and adult mice; in the latter this was accompanied by a reduction in the glycosylation of tyrosinase and the proportion of enzyme associated with the melanosomal fraction. Our findings suggest that post‐translational mechanisms are important control points in the regulation of tyrosinase and that differences in their level of activation are responsible for determining the patterns of melanogenesis in the C3H‐HeAvy mice, but it is still not clear how these mechanisms are regulated. Although cyclic AMP increased tyrosinase synthesis it had no post‐translation activating effect. The neonatal mice, unlike their pubertal and adult counterparts, also lacked dopachrome converting activity and TRP tyrosinase‐related protein‐1 expression. Since these factors may have a role in regulating eumelanin synthesis their absence could also explain why the first coat of hair in C3H‐HeAvy mice is of the phaeomelanin type.
Proliferation and Neoplastic Transformation of Pigment Cells in Metallothionein/ret Transgenic Mice
Authors: TAKAHASHI MASAHIDE , IWAMOTO TAKASHI , NAKASHIMA IZUMI
Published: July 2006

Although melanoma is a common human disease, there were few animal models in which melanoma developed at high incidence. To date, the Xiphophorus fish has been used as a model system to study melanoma formation. Studies on this fish showed the presence of a dominant oncogene, Tu, which encodes a transmembrane, tyrosine kinase of epidermal growth factor receptor type (Wittbrodt et al., Nature, 341:415–421, 1989). Recently, we succeeded in establishing novel transgenic mouse lines in which melanosis and melanocytic tumors developed stepwise by introducing another transmembrane tyrosine kinase oncogene, ret (Iwamoto et al., EMBO J., 10:3167–3175, 1991). In our transgenic mice, high levels of expression of the ret transgene induced proliferation and neoplastic transformation of melanin‐producing cells. In addition, crossbreeding experiments between transgenic mice and Wv mice showed that the ret oncogene can also induce melanogenesis and melanocyte development in Wv/Wv mice.
A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line
Authors: POTENZA MARC N. , LERNER MICHAEL R.
Published: July 2006

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.
The Effect of Sodium Iodate and Melanin on the Formation of Glyoxylate
Authors: BAICH ANNETTE , ZIEGLER MICHELLE
Published: July 2006

Sodium iodate damages retinal pigment epithelium specifically, but the reason for this specificity is not well understood. The work reported here describes an effect of sodium iodate on melanin, a major component of the retinal pigment epithelium. Sodium iodate increases the ability of melanin to convert glycine to glyoxylate. Almost ten times as much glyoxylate is formed when sodium iodate is present compared to the amount formed with melanin alone, although iodate alone does not convert glycine to glyoxylate. A chemical reaction between sodium iodate and melanin is suggested as a partial explanation of the specificity of iodate toxicity towards retinal pigment epithelium.
Penile Lentiginosis. An Ultrastructural Study
Authors: BREATHNACH A.S. , BALUS L. , AMANTEA A.
Published: July 2006

This study on five patients has revealed more extensive alterations to melanocytes than previously reported, and emphasizes the fact that depigmentation is an essential element of the condition. In hyperpigmented areas, melanocytes were increased in number along the basal layer of the epithelium, were hyperactive, and in some cases contained bizarre melanosomes. In two cases there was suggestion of a defect in melanosome transfer to keratinocytes. Lymphocytes were closely apposed to melanocytes, and, in hypopigmented areas, were clearly involved in their disintegration. In depigmented areas, there was complete absence of melanocytes and of melanosomes in keratinocytes, and lymphocytes were present in the basal layer. In general, the appearances did not resemble melanoma in situ with spontaneous regression, although a second biopsy of one patient after one year did reveal features of melanocytes suggestive of an early stage of this condition. The study has provided no clear information as to the initial cause of the condition, but the manner of destruction of melanocytes suggests an immune reaction. Neither has it been of assistance in suggesting a more precise name for it.
Circadian Motile Activity of Erythrophores in the Red Abdominal Skin of Tetra Fishes and Its Possible Significance in Chromatic Adaptation
Authors: HAYASHI HIROSHI , SUGIMOTO MASAZUMI , OSHIMA NORIKO , FUJII RYOZO
Published: July 2006

The red abdominal skin of the neon tetra Paracheirodon innesi and the cardinal tetra P. axelrodi was found to blanch at night or in the dark. Melatonin added to the bathing medium caused blanching of the red skin. Microscopic observations of the erythrophores indicated that the erythrosomes aggregated in response to norepinephrine, melanin‐concentrating hormone (MCH), and melatonin. Of these compounds, melatonin was the most effective. By contrast, many erythrophores were refractory to MCH. Alpha‐melanophore‐stimulating hormone, isoproterenol, adenosine, and ATP each caused dispersal of the pigment to some extent. Isoproterenol dispersed the pigment only when an alpha‐adrenergic blocker, tolazoline, was present. It appears that the change in color of the abdominal skin is primarily due to increased secretion during the night of the pineal hormone melatonin, while other hormonal and nervous factors may modify the distribution of the pigment in the erythrophores.
Electrophoretic Heterogeneity of Pigmented Hamster Melanoma Cells
Authors: HYRC KRZYSZTOF , WILCZEK ADAM , CIESZKA KRYSTYNA
Published: July 2006

Pigmented hamster melanoma tumors growing in situ contain two subpopulations of melanoma cells that have different electrophoretic mobilities (EPM). A mild neuraminidase treatment, which removes sialic acid residues from the cell surface glycoproteins, reduces the EPM of both groups of melanoma cells yielding an electrophoretically uniform population. This shows that the differences in the EPM between the subpopulations of pigmented melanoma cells stem from the different content of sialic acid residues on the cell surface. The relationship between the different EPM melanoma cell subpopulations was, therefore, examined during tumor growth, development, and formation of metastases.

The relative content of cells having high electrophoretic mobility, the “fast moving” cells, increases as the tumors grow larger. However, tumors of the same diameter contain nearly the same fraction of “fast moving” cells despite their age. The proportion of the “fast moving” cells is significantly higher in the central part than in the outermost layer of pigmented melanoma tumors. These data suggest that the development of “fast moving” cells is promoted by some size‐dependent changes in the intratumor environment.

In vivo selection of melanoma cells for their ability to colonize lungs renders tumors that reveal elevated metastatic potential and contain a significantly higher fraction of cells possessing high electrophoretic mobility than the parent tumor. Moreover, the metastatic nodules contain a remarkably elevated fraction of the “fast moving” cells. The reported correlation between the “fast moving” cell fraction and the metastatic potential suggests that the relative content of cells having high electrophoretic mobility may determine the metastaticity of pigmented hamster melanoma.
Interaction Between Chemicals and Melanin
Authors: LARSSON BENGT S.
Published: July 2006

Various drugs and other chemicals, such as organic amines, metals, polycyclic aromatic hydrocarbons, etc., are bound to melanin and retained in pigmented tissues for long periods. The physiological significance of the binding is not evident, but it has been suggested that the melanin protects the pigmented cells and adjacent tissues by adsorbing potentially harmful substances, which then are slowly released in nontoxic concentrations. Long‐term exposure, on the other hand, may build up high levels of noxious chemicals, stored on the melanin, which ultimately may cause degeneration in the melanin‐containing cells, and secondary lesions in surrounding tissues. In the eye, e.g., and in the inner ear, the pigmented cells are located close to the receptor cells, and melanin binding may be an important factor in the development of some ocular and inner ear lesions. In the brain, neuromelanin is present in nerve cells in the extrapyramidal system, and the melanin affinity of certain neurotoxic agents may be involved in the development of parkinsonism, and possibly tardive dyskinesia. In recent years, various carcinogenic compounds have been found to accumulate selectively in the pigment cells of experimental animals, and there are many indications of a connection between the melanin affinity of these agents and the induction of malignant melanoma.
Cultured Human Melanocytes Respond to MSH Peptides and ACTH
Authors: HUNT GILLIAN , DONATIEN PHILIPPE D. , LUNEC JOHN , TODD CAROLE , KYNE SYLVIA , THODY ANTHONY J.
Published: July 2006

Although the administration of melanocyte‐stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α‐MSH and its synthetic analogue Nle4DPhe7α‐MSH (NDP‐MSH) induce dose‐related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP‐MSH induces increases in tyrosinase mRNA and tyrosinase‐related protein‐1 (TRP‐1) mRNA. The dose‐response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose‐response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro‐opiomelanocortin peptides in human skin pigmentation.
O‐Methylation of L‐dopa in Melanin Metabolism and the Presence of Catechol‐O‐Methyltransferase in Melanocytes
Authors: SMIT NICO , TILGMANN CAROLA , KARHUNEN TUULA , SLINGERLAND ROBBERT , ULMANEN ISMO , WESTERHOF WIETE , PAVEL STAN
Published: July 2006

O‐Methylation of L‐dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L‐dopa, 3‐O‐methoxytvrosine was detected in extracts of cultured human melanocytes. The enzyme catechol‐O‐methyltransferase is responsible for this O‐methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane‐bound isoforms. Immuno‐electron microscopy suggests the presence of the membrane‐bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol‐O‐methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O‐methylation of L‐dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum.
Melanins in IGR 1 Melanoma Cells
Authors: ODH GERD , HINDEMITH ANNIKA , CARSTAM RAGNAR , PAULSON JAN , ROSENGREN EVALD , RORSMAN HANS
Published: July 2006

Information on the composition of melanins is obtained by analysis both of 4‐amino‐3‐hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole‐4,5‐dicarboxylic acid (TDCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5‐S‐cysteinyldopa (5‐S‐CD) was similar in all cultures, while 6‐hydroxy‐5‐methoxyindole‐2‐carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.
Effects of Ultraviolet Irradiation on the Cell Cycle
Authors: BOLOGNIA JEAN L. , SODI STEFANO A. , CHAKRABORTY ASHOK K. , FARGNOLI MARIA CONCETTA , PAWELEK JOHN M.
Published: July 2006

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20–30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid‐1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M “injury responses” of other cell types might be expected, consistent with their differentiated phenotypes.
Confrontation of Developing Melanophore Bars of Dark and White Axolotls With Endogenous Dark‐Embryo Mannose‐Binding Lectin Correlates With Melanophore Bar Disruption
Authors: MARTHA GALE N. , MILOS NADINE C.
Published: July 2006

Lectins are carbohydrate‐binding proteins suggested to be important in embryonic cell adhesion/differentiation. Dark and white axolotls contain an endogenous mannosebinding lectin that is especially prevalent during larval melanophore pattern formation (Martha et al., 1990). To determine if this lectin can alter melanophore patterning, lectin extracts have been isolated from Dark embryos by affinity chromatography. The main protein band is 44K on SDS‐PAGE. Dark and white embryos at the early chromatophore migration stage have been confronted with Dark lectin or its nonmetabolized inhibitor, 2‐deoxyglucose (2‐DG). The barred melanophore pattern of both genotypes is disrupted by lectin or 2‐DG treatment suggesting that endogenous mannose‐binding lectin and its receptor participate in bar formation.
Melanosome Formation in Cultured Amelanotic Melanophores of Rana brevipoda by a Frog Tyrosinase cDNA Transfection
Authors: OKUMOTO HITOSHI , NISHIOKA MIDORI , MIURA IKUO , OBIKA MASATAKA
Published: July 2006

A cDNA encoding tyrosinase of Rana nigromaculata was introduced into cultured, tyrosinase‐negative amelanotic melanophores of R. brevipoda by a calcium phosphate precipitation method. Within a few days following transfection, dark pigmentation became visible in a small number of cells. Light microscopic observation revealed that the morphology of these transformed cells was comparable to that of normal melanophores in culture, and their proliferative activity was lower than that of amelanotic cells. Ultrastructural examination verified that amelanotic melanophores possessed a relatively small number of premelanosomes while the transformants contained numerous melanosomes at various stages of pigment deposition. The result indicated that tyrosinase cDNA of R. nigromaculata was expressed in amelanotic melanophores of R. brevipoda inducing the maturation of premelanosomes. It was also suggested that the expression of transfected tyrosinase cDNA had promoted differentiation of the amelanotic cells into fully developed melanophores.
Developmental Regulation of Focal Contact Protein Expression in Human Melanocytes
Authors: SCOTT GLYNIS A. , LIANG HONG , CASSIDY LINDA L.
Published: July 2006

Focal contacts are transmembrane links between the extracellular matrix and the actin cytoskeleton that play a critical role in directed cell migration, adhesion, and normal growth. Several different component proteins of the focal contact show develop‐mentally dependent changes in expression, suggesting that this is an important mechanism by which focal contact formation is controlled during embryogenesis. In this report we examine the expression of focal contact‐associated proteins in human fetal and neonatal melanocytes using Western blotting. We show that expression of paxillin, a 69‐kDa vinculin binding protein, is fourfold higher in neonatal melanocytes than in fetal melanocytes. Further, we show that talin, a high molecular weight structural protein that links integrins to the actin cytoskeleton, is proteolytically cleaved in fetal, but not in neonatal melanocytes. Immunofluorescence microscopy of cells grown on fibronectin confirmed the presence of paxillin, talin, and vinculin at the ends of actin stress fibers at presumptive focal contacts in melanocytes. Adhesion experiments to extracellular matrix ligands revealed significant differences in adhesion of fetal and neonatal melanocytes to fibronectin. The developmentally specific changes in focal contact protein expression observed suggest that this may be an important mechanism by which focal contact assembly is controlled in human melanocytes during development.
Melanosomal Tyrosine Transport in Normal and Pink‐eyed Dilution Murine Melanocytes
Authors: GAHL WILLIAM A. , POTTERF BRIAN , DURHAM‐PIERRE DONNA , BRILLIANT MURRAY H. , HEARING VINCENT J.
Published: July 2006

Tyrosine is the endogenous substrate for melanin production within melanosomes, but the method of tyrosine transport into the melanosome has not been investigated. In the mouse, melanogenesis is disrupted by mutations in the p gene resulting in the pink‐eyed dilution phenotype; it has been suggested that the p gene codes for a tyrosine transport protein. We determined that normal (melan‐a) melanosome‐rich granular fractions take up 10 μm [3H]tyrosine at 21.1 ± 6.1 (SEM, standard error of the mean) pmol/min/mg protein (N=7) compared with 21.3 ± 5.8 SEM pmol/min/mg protein (N=5) for pink‐eyed dilution, whose plasma membrane tyrosine transport was also normal (Km 89 μM; Vmax 302 pmol/min/mg cell protein). We also demonstrated that pink‐eyed dilution melanosomes are immature by virtue of their low density, high hexosaminidase activity, and lack of pigment. These data indicate that a tyrosine transport system exists in the melanosomal membrane and that the p gene does not encode a tyrosine transporter of critical importance.
The Effects of Hydroxyl Radical Attack on Dopa, Dopamine, 6‐Hydroxydopa, and 6‐Hydroxydopamine
Authors: NAPPI ANTHONY J. , VASS EMILY , PROTA GIUSEPPE , MEMOLI SOFIA
Published: July 2006

High pressure liquid chromatography with electrochemical detection (HPLC‐ED) was employed in conjugation with a sensitive and specific salicylate hydroxylation assay to evaluate the immediate effects of hydroxyl radical (·OH) attack on four catechol intermediates of eumelanin, dopamine (3,4‐dihydroxyphenylethylamine), its precursor dopa (3,4‐dihydroxyphenylalanine), and their respective neurotoxic trihydroxyphenyl derivatives, 6‐hydroxydopamine (2,4,5‐trihydroxyphenylethylamine,6‐OHDA) and 6‐hydroxydopa(2,4,5‐trihydroxyphenylalanine, TOPA). Semiquinone and quinone species were identified as the initial products of the oxidation of these four catechol substrates. The enhanced oxidations of the catechols when exposed to ·OH attack was accompanied by marked decreases in the level of each semiquinone species. Quinone levels were elevated in reactions involving ·OH attack on dopamine and 6‐OHDA, but absent in reactions involving radical attack on dopa or TOPA, suggesting that dopaquinone (DOQ) and TOPA p‐quinone (TOPA p‐Q) are oxidized more rapidly by‘OH than are the quinones of dopamine and 6‐OHDA. The formation of 6‐OHDA p‐quinone (6‐OHDA p‐Q) in incubations involving DA and ·OH suggest that the ·OH‐mediated hydroxylation of DA may be a mechanism for generating this potentially cytotoxic trihydroxyphenyl. The results of this study demonstrate for the first time that semiquinone and quinone intermediates of eumelanin are the initial products derived from the ·OH‐mediated oxidations of dopa, DA, TOPA, and 6‐OHDA. These observations suggest that if ·OH is generated beyond the capabilities of cytoprotective mechanisms, the radical can rapidly oxidize catechol precursors, augment melanogenesis, and generate additional cytotoxic quinoid intermediates of eumelanin.
In Vivo and In Vitro Morphological Analysis of Melanocytes Homozygous for the misp Allele at the Murine Microphthalmia Locus
Authors: BOISSY RAYMOND E. , LAMOREUX M. LYNN
Published: July 2006

The mi8p allele (microphthalmia‐spotted), a mutant allele at the murine microphthalmia (mi) locus, when homozygous, results in a normal phenotype in which there is no apparent alteration in pelage pigmentation or ocular development. However, when heterozygous with other mi locus alleles, specifically Miwh (microphthalmia‐white) the mi8p allele exerts an affect on the phenotype. We examined the ultrastructure of melanocytes in the anagen hair bulb and the choroid plus the retinal pigmented epithelium of C57BL/6J‐mi8p/mi8p mice, C57BL/6J‐Miwh/Miwh mice, C57BL/6J‐Miwh/mi8p mice, and C57BL/6J‐+/+ control mice. Melanocytes of the mi8p/mi8p mice appeared normal in situ. However, melanocyte cultures derived from neonatal skins of mi8p/mi8p mice exhibited small primary colonies that did not dramatically expand in size. Occasionally, abnormalities in the structure of the Golgi apparatus were observed in primary cultures of mi8p/mi8p melanocytes. These results demonstrate that while the mi8p allele has no obvious effect on the phenotype of the mouse, it does dramatically suppress the survival of melanocytes in normal culture conditions.
Distribution of Keratan Sulphate and Chondroitin Sulphate in Wild Type and White Mutant Axolotl Embryos During Neural Crest Cell Migration
Authors: OLSSON LENNART , STIGSON MICHAEL , PERRIS ROBERTO , SORRELL J. MICHAEL , LÖFBERG JAN
Published: July 2006

In embryos of the white mutant axolotl, prospective pigment cells are unable to migrate from the neural crest (NC) due to a deficiency in the subepidermal extracellular matrix (ECM). This raises the question of the molecular nature of this functional defect. Some PGs can inhibit cell migration on ECM molecules in vitro, and an excess of this class of molecules in the migratory pathways of neural crest cells might cause the restricted migration of prospective pigment cells seen in the white mutant embryo. In the present study, we use several monoclonal antibodies against epitopes on keratan sulphate (KS) and chondroitin sulphate (CS) and LM immunofluorescence to examine the distribution of these glycosaminoglycans at initial (stage 30) and advanced (stage 35) stages of neural crest cell migration. Most KS epitopes are more widely distributed in the white mutant than in the wild type embryo, whereas CS epitopes show very similar distributions in mutant and wild type embryos. This is confirmed quantitatively by immunoblotting: certain KS epitopes are more abundant in the white mutant. TEM immunogold staining reveals that KS as well as CS are present both in the basal lamina and in the interstitial ECM in both types of embryos. It remains to be investigated whether the abundance of certain KS epitopes in the white mutant embryo might contribute to the deficiency in supporting pigment cell migration shown by its ECM.
Chemical Characterization of Melanins in Sheep Wool and Human Hair
Authors: OZEKI HIROYUKI , ITO SHOSUKE , WAKAMATSU KAZUMASA
Published: July 2006

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA‐derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.
Effective Elimination of Fibroblasts in Cultures of Melanocytes by Lowering Calcium Concentration in TPA Depleted Medium Following Geneticin Treatment
Authors: HORIKAWA TATSUYA , NORRIS DAVID A. , ZEKMAN TAMARA , MORELLI JOSEPH G.
Published: July 2006

Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method for eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin‐1 and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.
Stem Cell Factor Regulates the Melanocyte Cytoskeleton
Authors: SCOTT GLYNIS , LIANG HONG , LUTHRA DIPIKA
Published: July 2006

Stem cell factor is a growth factor for normal human melanocytes, that acts through the tyrosine kinase receptor c‐kit. We have previously demonstrated that stem cell factor increases melanocyte adhesion and migration on fibronectin, and regulates integrin protein expression. In this report, we have characterized the effect of stem cell factor on the organization of the actin cytoskeleton in human melanocytes attached to fibronectin, and have examined the effect of stem cell factor on the phosphorylation of the focal contact protein paxillin and on the expression of the focal contact proteins talin, paxillin, vinculin, and α‐actinin. Paxillin is a vinculin‐binding protein that is a substrate of focal adhesion kinase, a nonreceptor tyrosine kinase, and in its phosphorylated form is believed to stabilize focal contacts. We show that stem cell factor induces a rapid increase in actin stress fiber formation in melanocytes, which can be abrogated by genistein, a tyrosine kinase inhibitor, and that stem cell factor induces phosphorylation of paxillin on tyrosine residues. In contrast, stem cell factor did not regulate expression of any of the four focal contact proteins tested. These findings have implications for the models describing the mechanisms of action of stem cell factor on melanocyte adhesion and migration, and suggest that reorganization of the cytoskeleton is a primary effect of stem cell factor on human melanocytes.
Increase in Shedding of Intercellular Adhesion Molecule‐1 in Human Malignant Melanoma Cell Lines Treated With Hyperthermia In Vitro
Authors: NAKAYAMA JUICHIRO , KAGESHITA TOSHIRO , NAKASHIMA MANABU , TSUJISAKI MASAYUKI , IMAI KOZO , HORI YOSHIAKI
Published: July 2006

Human malignant melanoma cell lines were found to increase shedding of soluble intercellular adhesion molecule‐1 (sICAM‐1) into the culture medium when the cells were treated with hyperthermia at 41–43°C for 3–6 hr in vitro. The content of ICAM‐1 in the cell lysate was also found to be increased after hyperthermia. The increased rate of ICAM‐1 concentration in the cells was at maximum when they were incubated at 41°C for 3 hr. Also, the melanoma cell lines heat‐treated at 41°C showed more intense immuno‐fluorescence in the ICAM‐1 expression on the cell surface. It remains to be investigated further whether the effects of hyperthermia on the ICAM‐1 expression in melanoma cells is to augment membrane ICAM‐1 expression, which in turn leads to shedding of soluble ICAM‐1 or only to acceleration of shedding of sICAM‐1 by unknown mechanisms.
Pigment Cell Research: What Directions?
Authors: PROTA GIUSEPPE
Published: July 2006

Over the past few years, Pigment Cell Research has experienced unprecedented impetus in practically all areas. However, as is usually the case in periods of rapid development, several critical issues buried under the glitz of recent success are more or less the same as they have been for many years. Persisting misconceptions and scientific prejudices also contribute to cloud many issues. It appears, for example, that the common perception of melanocyte function is still biased by the traditional concept of a pigment producing cell. In fact, in addition to melanin, epidermal melanocytes are known to produce and excrete a number of melanin‐related metabolites, such as DHI and DHICA, which appear to play a critical role in protection of epidermal tissues against toxic oxygen radical species as well as in inflammatory and immune reactions.

Another major gap concerns the pheomelanin pigmentary system. This has traditionally been a neglected area of research in spite of compelling evidence for the abnormal susceptibility of red heads to sun damage and skin cancer. An increased attention to the peculiar biological and biochemical features of the pheomelanin forming cells will expectedly open new vistas on the UV susceptibility trait and the etiology of melanoma.
Molecular Basis of Congenital Hypopigmentary Disorders in Humans: A Review
Authors: BOISSY RAYMOND E. , NORDLUND JAMES J.
Published: July 2006

Many specific gene products are sequentially made and utilized by the melanocyte as it emigrates from its embryonic origin, migrates into specific target sites, synthesizes melanin(s) within a specialized organelle, transfers pigment granules to neighboring cells, and responds to various exogenous cues. A mutation in many of the respective encoding genes can disrupt this process of melanogenesis and can result in hypopigmentary disorders. Following are examples highlighting this scenario. A subset of neural crest derived cells emigrate from the dorsal surface of the neural tube, become committed to the melanoblast lineage, and are targeted along the dorsal lateral pathway. The specific transcription factors PAX3 and MITF (microphthalmia transcription factor) appear to play a regulatory role in early embryonic development of the pigment system and in associated diseases (the Waardenburg syndromes). During the subsequent development and commitment of the melanoblast, concomitant expression of the receptors for fibroblasts growth factor (FGFR2), endothelin‐B (EDNRB), and steel factor (cKIT) also appears essential for the continued survival of migrating melanoblasts. Lack or dysfunction of these receptors result in Apert syndrome, Hirschsprung syndrome and piebaldism, respectively. Once the melanocyte resides in its target tissue, a plethora of melanocyte specific enzymes and structural proteins are coordinately expressed to form the melanosome and to convert tyrosine to melanin within it. Mutations in the genes encoding these proteins results in a family of congenital hypopigmentary diseases called oculocutaneous albinism (OCA). The tyrosinase gene family of proteins (tyrosinase, TRP1, and TRP2) regulate the type of eumelanin synthesized and mutations affecting them result in OCA1, OCA3, and slaty (in the murine system), respectively. The P protein, with 12 transmembrane domains localized to the melanosome, has no assigned function as of yet but is responsible for OCA2 when dysfunctional. There are other genetically based syndromes, phenotypically resembling albinism, in which the synthesis of pigmented melanosomes, as well as specialized organelles of other cell types, is compromised. The Hermansky‐Pudlak syndrome (HPS) and the Chediak‐Higashi syndrome (CHS) are two such disorders. Eventually, the functional melanocyte must be maintained in the tissue throughout life. In some cases it is lost either normally or pre‐maturely. White hair results in the absence of melanocytes repopulating the germinative hair follicle during subsequent anagen stages. Vitiligo, in contrast, results from the destruction and removal of the melanocyte in the epidermis and mucous membranes.
The Role of Alpha‐MSH, Its Agonists, and C‐AMP in In Vitro Avian Melanocytes
Authors: BOWERS ROGER R. , BIBOSO ARCADIO , CHAVEZ OFELIA
Published: July 2006

Little is known about the effect of alpha‐MSH and other melanogenic stimulators on avian melanocytes. Tissue cultures of Barred Plymouth Rock regenerating feather melanocytes were established and the culture medium contained selected concentrations of alpha‐MSH and other melanogenic stimulators in Ham's F‐10 medium supplemented with antibiotics and 10% new born calf serum. Cultures were maintained at 37°C in 95% air/5% CO2.

No increase in melanogenesis over control levels due to the addition of 10‐5 M Forskolin, 10‐4 M IBMX, 10‐3 M c‐GMP, and 10‐3 M db‐c‐AMP was observed in the cultures on days 5 and 7. However, 2.5 (optimum), 5, and 10 μg/ml alpha‐MSH and 10‐3 M 8‐bromo‐c‐AMP significantly increased melanogenesis over control levels on days 5 and 7. The stimulation of melanogenesis was detectable by a significantly increased number of melanocytes containing numerous stage IV melanosomes. No increase in melanocyte cell number was observed in any of the experimental cultures. The addition of 1, 2 (optimum), or 3 mM calcium did enhance the increased pigmentation effect of 2.5 μg/ml alpha‐MSH.

Two very convincing experiments showed that c‐AMP was the second messenger for alpha‐MSH in these birds. First, the c‐AMP inhibitor, 10‐3 M Rp‐c‐AMPS, completely inhibited the stimulatory effect of alpha‐MSH in these in vitro melanocytes. Second, direct measurements of c‐AMP levels in feather tissue showed a significant increase in c‐AMP levels 10 min after alpha‐MSH treatment. Controls received no alpha‐MSH.

The results showed that these avian melanocytes have alpha‐MSH receptors and were able to respond to the hormone. C‐AMP was the second messenger in this system. Apparently db‐c‐AMP was not able to enter these mature, highly‐differentiated cells and c‐AMP agonists, Forskolin and IBMX, were also either unable to enter these older cells or if they did enter the cells, were unable to stimulate c‐AMP production. Evidently the more lipophilic 8‐bromo‐c‐AMP was able to enter these cells and stimulate melanogenesis.
Melanogenesis, Tyrosinase Expression, and Reproductive Differentiation in Black and White Truffles (Ascomycotina)
Authors: MIRANDA MICHELE , ZARIVI OSVALDO , BONFIGLI ANTONELLA , AMICARELLI FERNANDA , AIMOLA PIERPAOLO , RAGNELLI ANNA MARIA , PACIONI GIOVANNI
Published: July 2006

White and black truffles of the genus Tuber are Ascomycotina as well as Neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. Tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. We present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. L‐tyrosine 3‐monooxygenase and L‐DOPA oxidase activities colocalize as histochemically detected and are similarly located in white and black truffles, from the hypothecium through the sporogenic hyphae to asci and spores. Sulfur components of truffle flavours reversibly inhibit tyrosinase. The respiratory phenotype of truffle mitochondria is discussed in relation to reproductive differentiation and melanogenesis.
Melanin: A Two Edged Sword?
Authors: HILL HELENE Z. , LI WEIXJIONG , XIN PATRICK , MITCHELL DAVID L.
Published: July 2006

Melanin is both photosensitizer and photoprotector. Skin cancer rates decrease with increasing constitutive pigmentation, yet the pigment has been shown to be photoreactive and capable of producing damaging reactive oxygen species. We utilized model systems of related cells or similar cell type that vary in constitutive and in induced pigment. Induction of eumelanin in Cloudman S91 mouse melanoma cells leads to less UV‐induced killing and to less mutation induction at the ouabain locus (Na+, K+‐ATPase). Pigmented mouse melanocytes, melan‐b (brown) and melan‐a (black) were slightly less sensitive than melan‐c (albino) melanocytes to killing after UVC and UVA but were more sensitive to killing after UVB and UVB + UVA. Pigment had a small sensitizing effect on pyrimidine dimer DNA damage in both the melanoma cells and the melanocytes. The lack of consistency in these results suggests that intracellular pigment may disregulate the milieu intérieur resulting in end effects that are unrelated to the original genomic damage.
Prediction of Outcome for Patients with Cutaneous Melanoma
Authors: COCHRAN ALISTAIR J.
Published: July 2006

Most patients with primary melanoma are cured by local surgery, but a significant minority develop fatal metastases. The ability to identify patients with progressive disease is central to efficient management: permitting optimal deployment of adjunctive therapy and sparing the non‐progressing majority the morbidity of aggressive therapy. Accurate prediction on an individual patient basis is the ideal, but the best current prognosticators permit only assignment to risk categories. Formulaic combinations of well tried correlates of outcome (gender, ulceration, depth, thickness, and mitotic rate increase accuracy of prediction, but not to personalised level. The use of large data bases against which the attributes of individual patients may be compared is useful and amalgamation of data bases will increase the availability of this approach. The development of markers of proliferation fraction (PCNA and MIB‐1) and of the metastatic phenotype (PNA‐receptor status) will further refine the process. Staging of disease is critical. Ac‐curacy of staging is improved by mapping the (sentinel) lymph nodes likely to contain early tumor by lymphoscintigraphy and dye/radiomarker localisation. The application of exquisitely sensitive immunohistochemical and molecular biological techniques to biopsies from tissues likely to be the site of metastases permit assessment of clinical stage with a previously impossible degree of accuracy (ultrastaging).