bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Characterization of High Molecular Weight Melanogenic Inhibitors Naturally Occurring in Melanoma Cells
Authors: OHYAMA YASUAKI , MISHIMA YUTAKA
Published: July 2006

Intrinsic melanogenic inhibitors with high molecular weights have been isolated from Greene's amelanotic hamster melanoma by DEAE ion‐exchange and gel filtration chromatographies. The native molecular weights of two partially purified inhibitors have been determined to be 15 kDa (β‐type) and 67 kDa (γ‐type), respectively, using HPLC gel filtration. Both types of inhibitors, despite their inability to directly inhibit isolated tyrosinase, have been shown to markedly inhibit melanin formation in cultured B16 cells. In contrast to the β‐type inhibitor, the γ‐type inhibitor can induce depigmentation in B16 cells without abolishing their internal tyrosinase activity. Further, it has been determined that both inhibitors contain various amounts of unsaturated fatty acids, C15:1, C18:1, C18:2, C18:3, C20:3, and C20:4, which exhibit depigmentary activities on cultured B16 cells. C15:1 is low in the β‐type, but high in the γ‐type whereas C18:3 is high in the β‐type but low in the γ‐type. These results suggest that the differential action of these inhibitors is most likely due to the composition of the unsaturated fatty acids.
A Standardized Test for the Identification and Characterization of Melanins Using Electron Paramagnetic Resonance (EPR) Spectroscopy
Authors: ENOCHS W.S. , NILGES M.J. , SWARTZ H.M.
Published: July 2006

Melanins are complex, incompletely understood polymeric pigments that historically have been difficult to investigate with common chemical, histochemical, and physicochemical techniques. Because these pigments uniquely contain a stable population of organic free radicals, electron paramagnetic resonance (EPR) spectroscopy is a particularly effective method for studying them, and a set of qualitative EPR criteria has been established for their identification. However, a number of practical problems have arisen in applying these criteria to identify and characterize unknown pigments in relatively scarce pathological specimens, indicating that a standardized approach is needed. As reported here, a standardized EPR test for melanin based on the EPR criteria has been developed, guided by the requirements that it be sensitive, accurate, simple, and easy to interpret. It has been evaluated using the well‐characterized synthetic melanin prepared by alkaline autooxidation of 5,6‐dihydroxyphenylalanine (Dopa) and initially applied to the identification and characterization of an unknown pigment purified from an unusual malignant lung tumor.
From Gene to Protein: Determination of Melanin Synthesis
Authors: URABE KAZUNORI , AROCA PILAR , HEARING VINCENT J.
Published: July 2006

Melanin production in mammals is regulated at a variety of levels (tissue, cellular, and subcellular), and many gene loci are involved in the determination of color patterns directed by the melanocyte. Many of the genes involved in these complex processes have now been cloned, and even the simplest mutation can lead to dramatic changes in the phenotype of the individual. Many, if not all, of the pigment related genes have pleiotropic effects on the development and differentiation of the organism, and perhaps because of this, the melanocyte is evolving as an important model for the study of gene regulation and action at the functional level. In view of the importance of pigmentation as a photoprotective barrier and as a cosmetic factor affecting appearance and social acceptance, the importance of these studies seems destined to increase significantly in the future.
Melanization Stimulating Activity in the Skin of the Gilthead Porgy, Sparus auratus
Authors: ZUASTI A. , MARTÍNEZ‐LIARTE J.H. , FERRER C. , CAÑIZARES M. , NEWTON J. , BAGNARA J.T.
Published: July 2006

The presence of a melanization‐stimulating factor (MSF) was discovered in dorsal and/or ventral skin of Sparus auratus. Skin from this marine species was used to condition Steinberg's balanced salt solution (BSS), which was subsequently tested with the neural tube assay. BBS conditioned by dorsal and/or ventral skin of S. auratus at 25% and 50% concentrations had a profound stimulatory effect on the percentage of melanization of neural crest cells throughout the 3day assay period. In some cases 90% melanization occurred within the first 24 hr. Such stimulated cells showed a doubling of the number of dendrites per cell. assess the effects of MSF on other indices of melanization, dorsal and/or ventral skin was used to condition MEM used in the culture of B16‐F10 murine melanoma cells. During the first 24 hr, B16‐F10 murine melanoma cells responded to conditioned media by demonstrating a considerable increase in activities of tyrosine hydroxylase, dopa oxidase, and dopachrome tautomerase, but no effect was observed on melanin content. In contrast, melanin content increased after 48 hr of incubation, whereas the enzymatic activities were inhibited during this period. It seems that MSF activity, expressed in several ways, may be present generally among marine species.
Organ‐Specific Metastases in Melanoma: Experimental Animal Models
Authors: GATTONI‐CELLI SEBASTIANO , BYERS RANDOLPH H. , CALORINI LIDO , FERRONE SOLDANO
Published: July 2006

Metastases from certain primary tumors frequently exhibit specific organ preference. Animal models have been developed to induce in a reproducible fashion the formation of organ‐specific metastases by malignant melanoma cells. Some of these models rely on the use of immunodeficient mice, which can support the growth of murine as well as human malignant melanomas. Moreover, immunodeficient mice, because of their diminished ability to mount an effective immune response, allow the expression of malignant properties (e.g., preferential colonization of certain organs), which are intrinsic to transplanted melanoma cells. This review discusses the relevant factors (and limitations) of some of the animal models used to study the in vivo properties of melanoma cells.
Tyrosinase Gene Expression in Human Tissues
Authors: ABATTYANI ZIT , XERRI LUC , HASSOUN JACQUES , BONERANDI JEAN‐JACQUES , GROB JEAN‐JACQUES
Published: July 2006

The occasional occurrence of primary extra‐cutaneous malignant melanomas (MM) has led to the hypothesis that melanocytes derived from the neural crest may be arrested in their migration and may undergo an in situ malignant transformation. However, aggregates of nevus cells have only rarely been identified by histological examination in a few organs other than skin and eye. Tyrosinase is a melanin biosynthetic enzyme that is considered one of the most specific markers of melanocytic differentiation. We have at‐tempted to detect cells committed to the melanocytic lineage, in human tissues, by means of tyrosinase gene expression. Total RNA was extracted from normal and neoplastic tissues and analyzed using a highly sensitive reverse transcription PCR assay with primers specific for the tyrosinase gene. Peripheral blood mononuclear cells (PBMC) from healthy subjects were used as negative controls. Tyrosinase transcripts were identified in a wide range of normal organs such as skin, lymph nodes, antrum, colon, kidney, lung, testis, ovary, breast, and peripheral nerve. Tyrosinase RNA was also detected in neoplastic samples including benign cutaneous nevi, lymph nodes involved by MM, breast carcinoma, liposarcoma, malignant lymphoma, and schwannoma. PBMC from patients with meta‐static MM were also positive, while no positivity was detected in blood specimens from patients with other cancers.

Therefore, it appears likely that cells expressing the tyrosinase gene are present in a wide range of human tissues. Although these cells still have to be accurately identified, one could propose that they might correspond to either fully differentiated melanocytes, melanocytic precursors, or Schwann cells bearing potentialities of melanocytic differentiation. Occurrence of at least some cases of primary extra‐cutaneous MM may be ascribed to any one of these possibilities.
Organotypic Culture of Human Skin to Study Melanocyte Migration
Authors: POOLE I. CAROLINE LE , VAN DEN WIJNGAARD RENÉ M.J.G.J. , WESTERHOF WIETE , DORMANS JAN A.M.A. , VAN DEN BERG FRANK M. , VERKRUISEN RONALD P , DINGEMANS KOERT P. , DAS PRANAB K.
Published: July 2006

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air‐liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7‐day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo‐epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer‐aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.
Melanocytes Fail to Survive in Hair Bulbs of the Shiba Goat (Capra hircus) With the Dominant Black‐Eyed White Phenotype
Authors: TANAKA SATOSHI , TOJO HIDEAKI , KASAI KYOKO , SAWASAKI TORU , TACHI CHIKASHI
Published: July 2006

Dominant black‐eyed white phenotypes are one of the most commonly observed traits in domestic animals. Their genetic control mechanisms, however, have not been elucidated. As the first step to approach the problem, we examined histologically the patterns of the distribution of pigment cells in Shiba goats (two each of day‐73‐postcoitum and day‐112‐postcoitum fetuses, and a 15‐week‐old kid) with the dominant black‐eyed white phenotype. Melanocytes were present and fully pigmented in the choroid and the sclera of eyes, as well as in dorsal skin epidermis of the fetuses and of the kid. Melanocytes were also found in approximately 6% of the hair bulbs in the fetal dorsal skin, while the rest (94%) lacked them. Hair follicles of the kid did not harbor melanocytes except for some in the early anagen stage. The results suggest that the survival of melanocytes was inhibited specifically in the hair follicles of the Shiba goat with the dominant black‐eyed white phenotype and that the ostensibly similar phenotypes in the Shiba goat and in the SI or W mutants of the mouse, where melanocytes die en route to the hair bulbs, are regulated by different mechanisms.
Three Modes of Melanosome Transfers in Caucasian Facial Skin: Hypothesis Based on an Ultrastructural Study
Authors: YAMAMOTO OSAMU , BHAWAN JAG
Published: July 2006

The transfer mechanism of melanosome from the melanocyte into the keratinocyte was investigated in mildly photodamaged Caucasian facial skin by electron microscopy. Three ways of transfer are suggested by our observations. The first mechanism probably occurs through the following process: 1) protrusion and insertion of the thick dendrite of the melanocyte into the basal keratinocyte, 2) formation of sac‐dendrite complex in the subnuclear region, 3) digestion and segregation of the enclosed dendrite, 4) formation of the cistern in the paranuclear region, and 5) pinching‐off of the melanosomes in single or aggregated form from the tip of the cistern. The second mechanism probably takes place through a membrane fusion between the melanocyte and the keratinocyte. Such a membrane fusion possibly forms a passage way for release of the melanosome from the former cell to the latter. The third mechanism is considered to include exocytosis of the single melanosome from the melanocyte followed by the endocytosis through the formation of coated‐pit in the keratinocyte.
Deposition of Basic Fibroblast Growth Factor on Surface of Epidermal Melanocytes Suggesting the Stromal Control of Epidermal Pigmentation
Authors: IMAYAMA SHUHEI , FURUMURA MINAO , HORI YOSHIAKI
Published: July 2006

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.
The Presence of Tyrosinase and Related Proteins in Human Epidermis and Their Relationship to Melanin Type
Authors: TOBIN DEREK , QUINN ANTHONY G. , ITO SHOSUKE , THODY ANTHONY J.
Published: July 2006

The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP‐1 and TRP‐2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP‐1 and TRP‐2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase‐positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.
Regulation of Melanogenesis Induced by 5‐Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells
Authors: MENGEAUD VALÉRIE , ORTONNE JEAN PAUL
Published: July 2006

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte‐specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase‐related protein‐1 (TRP‐1, gp75). To get more insight on the molecular mechanisms involved in psoralens‐induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP‐1 in S91 mouse melanoma cells treated by 5‐MOP. High concentration of 5‐MOP (5 × 10‐5 M) induced a time‐dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5‐MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5‐MOP stimulated TRP‐1 synthesis and induced a dose‐dependent decrease of DCT activity without any modification in the expression of the protein.

We explored then the signalling pathways involved in 5‐MOP‐induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5‐MOP. Furthermore, 1‐oleoyl‐2‐acetylglycerol (OAG), a PKC activator, potentiated the 5‐MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5‐MOP induction of tyrosinase activity. Phorbol‐myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5‐MOP when used at long term.

Taken together, these results demonstrate that in murine melanoma cells 5‐MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP‐1 expression are coordinately regulated by 5‐MOP Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5‐MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5‐MOP.
X‐Ray Characterization of Melanins—II
Authors: CHENG JIN , MOSS SIMON C. , EISNER MELVIN
Published: July 2006

Structural modeling of amorphous eumelanin has been carried out by comparing calculated data, S(q) and RDF(r), in reciprocal and real space, respectively, for limited random network models with the experimental X‐ray scattering data of tyrosine melanin (Cheng et al., 1994). A basic picture of the atomic arrangements in amorphous eumelanin, which accounts for the short and intermediate range order, has been formulated. This reveals domains of a fundamental “particle” dimension of R∼ 15 Å, consisting of a paracrystalline array of disordered planar networks polymerized by 4‐8 DHI monomers with a graphite‐like stacking spacing of ∼ 3.45 Å, 4‐5 layers thick.
A Cladistic Analysis of the Evolutionary Relationships of the Members of the Tyrosinase Gene Family Using Sequence Data
Authors: MORRISON RANDALL , MASON KENNETH , FROST‐MASON SALLY
Published: July 2006

Recently, DNA sequence data have been published on tyrosinase and tyrosinase‐related proteins (TRPs) in a wide variety of vertebrates ranging from Rana to Homo. These proteins are in turn members of a larger family of binuclear copper‐binding proteins, which all contain two highly conserved copper‐binding domains. This gene family also includes tyrosinases from fungi and bacteria as well as arthropodan and molluscan hemocyanins. Parsimony‐based alignment and tree construction algorithms (Malign, vl.85 and PAUP, 3.1.1) were used to analyze the diversification of both the evolutionarily conserved copper‐binding domains i6n copper‐binding proteins in general as well as the diversification of the vertebrate tyrosinase gene family more specifically. These analyses show that the diversification of the vertebrate tyrosinase gene family minimally predates the diversification of vertebrates. Vertebrate tyrosinases proper first diverged from an ancestral tyrosinase‐related protein (TRP) that then subsequently diverged to form tyrosinase‐related protein‐Is (TRP‐1s) and tyrosinase‐related protein‐2s (TRP‐2s).
Characterization of Mouse Pmel 17 Gene and Silver Locus
Authors: KWON BYOUNG S. , KIM KACK K. , HALABAN RUTH , PICKARD RICHARD T.
Published: July 2006

Pmel 17 cDNA clones, isolated from wild‐type and si/si murine melanocytes, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the si/si Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C‐terminus and to extend the Pmel 17 protein by 12 residues. The mutation was confirmed by the sequence of the PCR‐amplified genomic region including the mutation site. Silver Pmel 17 was not recognized by antibodies directed toward the C‐terminal amino acids of wild‐type Pmel 17, indicating a defect in this region. These results indicate that silver Pmel 17 protein has a major defect at the carboxyl terminus.
The Mouse Pink‐Eyed Dilution Gene: Association with Hypopigmentation in Prader‐Willi and Angelman Syndromes and with Human OCA2
Authors: BRILLIANT MURRAY H. , KING RICHARD , FRANCKE UTA , SCHUFFENHAUER SIMONE , MEITINGER THOMAS , GARDNER JOHN M. , DURHAM‐PIERRE DONNA , NAKATSU YOSHIMICHI
Published: July 2006

Mutations at the mouse pink‐eyed dilution locus, p, cause hypopigmentation. We have cloned the mouse p gene cDNA and the cDNA of its human counterpart, P. The region of mouse chromosome 7 containing the p locus is syntenic with human chromosome 15q11‐q13, a region associated with Prader‐Willi syndrome (PWS) and Angelman syndrome (AS), both of which involve profound imprinting effects. PWS patients lack sequences of paternal origin from 15q, whereas AS patients lack a maternal copy of an essential region from 15q. However, the critical regions for these syndromes are much smaller than the chromosomal region commonly deleted that often includes the P gene. Hypopigmentation in PWS and AS patients is correlated with deletions of one copy of the human P gene that is highly homologous with its mouse counterpart. A subset of PWS and AS patients also have OCA2. These patients lack one copy of the P gene in the context of a PWS or AS deletion, with a mutation in the remaining chromosomal homologue of the P gene. Mutations in both homologues of the P gene of OCA2 patients who do not have PWS or AS have also been detected.
Evolutionary Origin and Molecular Biology of the Melanoma‐Inducing Oncogene of Xiphophorus
Authors: SCHARTL ANGELIKA , DIMITRIJEVIC NICOLA , SCHARTL MANFRED
Published: July 2006

Melanoma formation in platyfish/swordtail hybrids of genus Xiphophorus is due to overexpression of the receptor tyrosine kinase oncogene Xmrk. This gene is the molecular equivalent to the Tu‐locus of platyfish, formerly identified by Mendelian genetics. The supposed evolutionary origin of the Xmrk oncogene is a nonhomologous recombination event in the 5’region of the corresponding Xmrk protooncogene with an anonymous sequence, D. This event led to a gene duplication of Xmrk, whereby the new copy obtained a novel promoter derived from D. Inactivity of this promoter in parental fish warrants lack of tumorigenicity of the Xmrk oncogene in wild playfish. In hybrids, however, the promoter is active. This leads to the pigment cell transforming overexpression of Xmrk.
The Role of 80K/MARCKS, a Specific Substrate of Protein Kinase C, in Cell Growth and Tumour Progression
Authors: BROOKS GAVIN
Published: July 2006

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80‐87 kDa myristoylated a lanine‐rich C‐kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.
Selective Expression of PNA‐Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential
Authors: DORÉ JEAN‐FRANÇOIS , BERTHIER‐VERGNES ODILE , ZEBDA NOUREDDINE , BAILLY MARYSE , THOMAS LUC , BAILLY CHRISTIANE , COCHRAN ALISTAIR J.
Published: July 2006

Alterations of cell‐surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III‐V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.
Analysis of the Mouse Tyrosinase Promoter In Vitro and In Vivo
Authors: GANSS RUTH , SCHMIDT ANDREA , SCHÜTZ GÜNTHER , BEERMANN FRIEDRICH
Published: July 2006

The restricted expression of the tyrosinase gene in cells producing pigment suggests the presence of cis‐regulatory elements and trans‐acting tissue‐specific factors. Since 270 bp upstream of the transcriptional start site contain sufficient information for tissue‐specific and developmentally regulated expression, we confined our analyses to this region. In this article, we discuss the recent results we have obtained on the regulation of the mouse tyrosinase gene expression demonstrating the existence of one negative and two positive‐acting elements in vitro. We have evidence that the positive elements do not determine pigment production in vivo but rather modulate transcription of the mouse tyrosinase gene.
Analysis of Tyrosinase Mutations Associated With Tyrosinase‐Related Oculocutaneous Albinism (OCAI)
Authors: OETTING WILLIAM S. , KING RICHARD A.
Published: July 2006

Mutations of the tyrosinase gene associated with a partial or complete loss of enzymatic activity are responsible for tyrosinase related oculocutaneous albinism (OCA1). A large number of mutations have been identified and their analysis has provided in‐sight into the biology of tyrosinase and the pathogenesis of these different mutations. Missense mutations produce their effect on the activity of an enzyme by altering an amino acid at a specific site. The location of these mutations in the peptide can be used to indicate potential domains important for enzymatic activity. Missense mutations of the tyrosinase polypeptide cluster in four regions, suggesting that these are important functional domains. Two of the potential domains involve the copper binding sites while the others are likely involved in substrate binding. More critical analysis of the copper binding domain of tyrosinase can be gained by analyzing the structure of hemocyanin, a copper‐binding protein with a high degree of homology to tyrosinase in the copper binding region. This analysis indicates a single catalytic site in tyrosinase for all enzymatic activities.
Coordinated mRNA and Protein Expression of Human LAMP‐1 in Induction of Melanogenesis After UV‐B Exposure and Co‐Transfection of Human Tyrosinase and TRP‐1 cDNAs
Authors: JIMBOW KOWICHI , LUO DONG , CHEN HUA , HARA HIROYUKI , LEE MU HYOUNG
Published: July 2006

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome‐associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV ‐B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase‐related protein‐1 (TRP‐1). Repeated exposure of UV B (2.5–5.0 mJ/cm2) caused not only upregulation of tyrosinase and TRP‐1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp‐1 (lysosome‐associated membrane protein‐1). When COS‐7 kidney cells and amelanotic melanoma (C32 and SKMEL‐24) and melanotic melanoma (G361 and SK‐MEL‐23) cells were exposed to cotransfection of human tyrosinase and TRP‐1 cDNAs, there was also an increased expression of Lamp‐1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP‐1 cDNA or cotransfectants of human tyrosinase and TRP‐1 cDNAs, indicating that TRP‐1 prevented, along with Lamp‐1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP‐1 and Lamp‐1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp‐1 gene, showing the decreased expression of TRP‐1 as identified by three different types of anti‐TRP‐1 monoclonal antibodies. We propose therefore that human tyrosinase and TRP‐l, when activated or expressed together, will coordinate to upregulate the mRNA expression and protein synthesis of Lamp‐1. The Lamp‐1 molecules will, in turn, cover the inner surface of melanosomal membrane, together with TRP‐1 molecules, thus protecting the melanosomal membrane from toxic melanin intermediates generated during melanogenesis in the presence of active tyrosinase. In contrast, the expression of other lysosome‐related proteins, e.g., β‐galactosidase and CD63 is not stimulated in new melanogenesis.
P53 Mutation and c‐fos Overexpression Are Associated With Detection of the Antigen VLA‐2 in Human Melanoma Cell Lines
Authors: KROUMPOUZOS GEORGIOS , EBERLE JÜRGEN , GARBE CLAUS , ORFANOS CONSTANTIN E.
Published: July 2006

In this study, we examined the expression of c‐fos, c‐myc, mutant c‐Ha‐ras and mutant p53 proteins in three normal human melanocyte cell lines and the following 12 melanoma cell lines: M5, Mewo, A375, Bro, Mel 2a, O‐Mel II, IgR 39, SkMel‐13, ‐19, ‐28 Mel‐57 and NKI‐4, using an immunohistochemical assay (APAAP). An effort was made to correlate oncogene expression with growth parameters, differentiation antigens (HMB‐45, vla‐2, k.1.2.58, HLA‐DR, HLA‐I), and pigmentation. All melanocyte cell lines were negative for the oncogenes examined, whereas six of the melanoma cell lines were found also positive (three for c‐fos, two for c‐myc, one for c‐Ha‐ras, and four for p53). Three melanoma cell lines expressed one oncogene and three the combination c‐fos/p53. These three melanoma cell lines were positive for the “late” tumor progression marker A. 1. 43 (vla‐2 adhesion molecule) and negative for the differentiation marker k. 1. 2. 58. Positivity for A. 1. 43 combined with negative staining for k. 1. 2. 58 was found in six out of the 12 cell lines. The observed oncogene expression correlated neither with growth parameters nor melanin content. The present findings revealed a coexpression of mutant p53 and c‐fos proteins being associated with a highly malignant phenotype in melanoma cell lines. Further studies are necessary to clarify the significance of the above findings
Some Aspects of Melanin Formation of Melanocytes Cultured on Collagen‐Coated Microcarrier Beads
Authors: SMIT NICO P.M. , WESTERHOF WIETE , SMIT WILMA , NANNINGA BART , PAVEL STAN
Published: July 2006

Melanocytes cultured on collagen‐coated Cytodex 3 microcarrier Sephadex beads caused remarkable pigmentation of the beads during the period of culture when optimal density was reached. Electron microscopy of melanocytes on the microcarriers revealed that the cells and their dendrites invaginate into the microcarrier surface layer. Removal of the cells by trypsinization showed that some pigment granules were left on the carrier surface and within the cavities present on the microcarrier surface. In order to investigate whether the pigmentation of the microcarriers could be a result of indole intermediates of melanogenesis present in the culture medium, extracts were studied by gas chromatography/mass spectrometry for the presence of these compounds. Two compounds (5,6‐dihydroxyindole‐2‐carboxylic acid and 6‐hydroxy‐5‐methoxyindole‐2‐carboxylic acid) so far have been identified in the medium extracts. Results indicate that microcarrier culture of melanocytes can serve as an interesting model for electron microscopy studies of melanocytes with regard to pigmentation and cell attachment.
Comparative Analysis of Melanins and Melanosomes Produced by Various Coat Color Mutants
Authors: PROTA GIUSEPPE , LAMOREUX M. LYNN , MULLER JACQUELINE , KOBAYASHI TAKESHI , NAPOLITANO ALESSANDRA , VINCENSI M. ROSARIA , SAKAI CHIE , HEARING VINCENT J.
Published: July 2006

Pigmentation in the skin, hair, and eyes of animals is influenced by a number of genes that modulate the activity of melanocytes, the intervention of enzymatic controls at different stages of the melanogenic process, and the physico‐chemical properties of the final pigment. The results of combined phenotypic, ultrastructural, biochemical, and chemical analyses of hairs of a variety of defined genotypes on a common genetic background performed in this study are consistent with the view that pigmentation of dark to black hairs results from the incorporation of eumelanin pigments whereas that of yellow hairs results from the incorporation of eu‐ and pheomelanins. It is also clear that relatively minor differences in melanin content can have dramatic effects on visible hair color. A good correlation was found for expression of (and enzyme activities associated with) TRP1 and TRP2 with eumelanin synthesis and eumelanosome production.
Comparative Study of Experimental Ocular Melanoma Using Two Implantation Techniques of B16‐F10 Melanocytes
Authors: GONZALEZ JOSE GARCIA , ORTEGA VICENTE VICENTE , REDONDO MANUEL , VICENTE JOSE RODRIGUEZ
Published: July 2006

Objective: The aim of this work was to evaluate the behaviour of B16‐F10 melanoma cell cultures implanted in the anterior chamber of the eye of New Zealand white rabbits by studying the clinical‐pathological and ultrastructural characteristics of the lesions. Methods: One group (A) (consisting of 30 rabbits) was transclerally inoculated (1 mm from sclero‐corneal limbus) with 4×106 melanocytes and another group (B) (also 30 animals) was inoculated once per week for 3 consecutive weeks with 5×106 cells (total 15×106); 30 animals acted as the control group (C). All the lesions were processed for optic and electronic microscopy. Results: Tumoral growth in group A was 43% (13/30) and in group B 80% (24/30). All lesions were pigmented and none perforated the eyeball. Microscopically, they were a mixture of epithelioid and fusiform cells disposed around the blood vessels. Ultrastructurally, the presence of melanosomes in different stages of maturation and aberrant melanosomes were characteristic. Conclusion: We suggest that the transcleral inoculation of 15×106 B16‐F10 melanocytes into the anterior chamber of the eye of New Zealand white rabbits may be a valid and reproducible method for obtaining an experimental ocular melanoma model.
Complex Formation Between Mushroom Tyrosinase and Manduca Dopachrome Isomerase
Authors: SUGUMARAN MANICKAM , NELLAIAPPAN KALIAPPAN , SCOTT TIMOTHY , AMARATUNGA CHAMANIE
Published: July 2006

Melanin biosynthesis in animals is initiated by the ubiquitously present tyrosinase and is aided by dopachrome isomerase. We have characterized a novel dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta that generates a new quinone methide intermediate during melanogenesis (Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073–6078). This enzyme has the ability to form a complex with mushroom tyrosinase as judged by a number of physicochemical studies. The isomerase exhibited a marked inhibitory effect on tyrosinase and tyrosinase reciprocated by inhibiting the isomerase. While the isomerase showed no activity toward preformed dopaminechrome, it readily influenced the stability of dopaminechrome generated in situ by tyrosinase. Moreover, mushroom tyrosinase, which lacked specific binding to Concanavalin A Sepharose column, after complexing with the isomerase exhibited binding to this column. The complex formation also affected the pi value as well as mobility on a size exclusion column of these enzymes. Enzymes executing sequential metabolic transformation are known to form complexes called metabolons. Based on these above studies, it is concluded that both the enzymes involved in insect melanogenic pathway—phenoloxidase and dopachrome isomerase—are able to form a metabolon complex.
Transport of Endocytosed Material Into Melanin Granules in Cultured Choroidal Melanocytes of Cattle—New Insights Into the Relationship of Melanosomes With Lysosomes
Authors: SCHRAERMEYER ULRICH
Published: July 2006

Cultured choroidal melanocytes from cattle were incubated with gold labeled albumin. After phagocytosis of the labeled protein, the label appeared inside the melanin granules, as was observed under the electron microscope. Melanin granules associated with gold particles were also exocytosed into the culture medium by the melanocytes. The results of this study show that endosomes or phagosomes are transported from the cell surface of a melanocyte to the melanin granules. Therefore, melanin granules are part of the lysosomal degradation pathway. The possibility that albumin is degraded by proteases present in lysosomes and melanosomes and that the tyrosine released during degradation is used as substrate by tyrosinase and thereby converted to melanin is discussed. The present study additionally shows that the choroidea of cattle can be used as a source for cell culture of melanocytes.
N‐CAM and N‐Cadherin Are Specifically Expressed in Xanthophores, but not in the Other Types of Pigment Cells, Melanophores, and Iridiphores
Authors: FUKUZAWA TOSHIHIKO , OBIKA MASATAKA
Published: July 2006

Little is known about cell‐cell communication in pigment cells, whereas a number of signalling molecules have been implicated to control their migration, differentiation, and proliferation. We set out to investigate the expression of cell adhesion molecules (CAMs) in the three different types of pigment cells in poikilotherms, Oryzias latipes and Xenopus laevis. In the present experiments, the expression of N‐CAM and N‐cadherin in the pigment cells in vitro was examined by immunocytochemistry. Melanophores and xanthophores were isolated and cultured from scales or skins, while iridophores were harvested from skins or peritoneum. The results showed that N‐CAM and N‐cadherin were specifically expressed in xanthophores, but not in melanophores or iridophores in both O.latipes and X.laevis. N‐CAM and N‐cadherin basically colocalized in the restricted regions of xanthophores, although the N‐cadherin‐expressed region was broader than the N‐CAM‐expressed region in the same cell. The incidence of N‐cadherin expression was higher than that of N‐CAM expression. N‐CAM and N‐cadherin were expressed at the tip or the base of dendrites, or at the edge between dendrites in dendritic xanthophores. N‐CAM and N‐cadherin usually localized in small and narrow regions of xanthophores. This distribution pattern was essentially similar in xanthophores with round morphology, which exhibited spot, band, or semicircular immunoreactive regions on the peripheral edge of the cells. The difference in the distribution of pigment granules within the cells, culture period, fixatives, or immunofluorescent markers used in the experiments did not alter the immunostaining pattern.
Keratinocyte Extracellular Matrix‐Mediated Regulation of Normal Human Melanocyte Functions
Authors: NAKAZAWA KEIKO , NAKAZAWA HISAYOSHI , COLLOMBEL CHRISTIAN , DAMOUR ODILE
Published: July 2006

Active roles of cell‐cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell‐cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation‐associated protein (MEL‐5) and proliferation‐associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell‐matrix interactions with those of direct melanocyte‐keratinocyte, cell‐cell contact in co‐culture on melanocyte functions.

Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation‐associated protein detected by monoclonal antibody, MEL‐5, was not changed on the kECM, while it was increased in melanocytes in co‐culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co‐culture compared to that in a control culture.

We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca++) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.
Changes in Adrenergic Innervation to Chromatophores During Prolonged Background Adaptation in the Medaka, Oryzias latipes
Authors: SUGIMOTO MASAZUMI , OSHIMA NORIKO
Published: July 2006

The pattern of adrenergic innervation to scale chromatophores of the wild‐type medaka, Oryzias latipes, was examined by autoradiography with 3H‐norepinephrine and found for the first time to be changed reversibly during prolonged background adaptation. In scales of the medaka, which was adapted to a black background for 10‐15 days, a great number of melanophores and dense networks of varicose fibers were observed: many fibers built up a radial plexus around each melanophore. However, the dense distribution of varicose fibers disappeared with a decrease in the number of melanophores during long‐term adaptation to a white background. As to the changes in the innervation pattern to amelanotic melanophores of the medaka, orange‐red variety, a similar result was obtained. Although the increase in the number of leucophores was observed in the medaka adapted to a white background, no exact plexuses of labeled fibers were confirmed around leucophores. From these results, it is concluded that the density of chromatic nerve fibers changes in parallel with the variation of the number of melanophores during prolonged background adaptation.
Characterization of Vitiligo Antigens
Authors: CUI JIAN , ARITA YUKO , BYSTRYN JEAN‐CLAUDE
Published: July 2006

Patients with vitiligo have circulating antibodies directed in part to pigment cell antigens with MWs of approximately 90, 75, and 40‐45 kDs. These antigens are denominated VIT 90, VIT 75, and VIT 40, respectively. To further characterize these “vitiligo” antigens, we examined their relation to antigens defined by a panel of 25 monoclonal antibodies (moab) to pigment cell antigens. We found by immunoprecipitation and SDS‐PAGE analysis of 125I labelled, detergent soluble, human melanocyte macromolecules, that 24 (83%) of 29 patients with vitiligo had antibodies to one or more vitiligo antigens vs. 2 (7%) of 28 control individuals. Seventeen of the 25 moabs did not react with any labelled antigen in the same lysate. Of the remaining eight moabs, only four precipitated an antigen that co‐migrated with one of the vitiligo antigens. Moab TA99, HMSA‐5, and TMH‐1 (all directed to the 75 kD tyrosinase‐related protein [TRP1]) co‐migrated with VIT 75. Moab W6/32 (directed to class I HLA antigen) co‐migrated with VIT 40. Immunodepletion studies with vitiligo antibodies selectively depleted the antigen defined by W6/32 but not the antigen defined by TA99 and HMSA‐5, indicating that VIT 75 was not the 75 kD tyrosinase‐related protein. The vitiligo antigens were easily labelled by the lactoperoxidase technique but poorly labelled with 35S‐methionine, suggesting they are expressed on the cell surface.

These studies indicate that VIT 90 and VIT 75 differ from antigens defined by currently available moabs to pigment cell antigens. VIT 40 appears to share a cross‐reactive epitope, or be tightly bound to, class I HLA antigen.
Genomic Organization and FISH Mapping of Human Pmel 17, the Putative Silver Locus
Authors: KIM K‐K. , YOUN B‐S. , HENG H.H.Q. , SHI X.M. , TSUI L‐C. , LEE Z.H. , PICKARD R.T. , KWON B.S.
Published: July 2006

The Pmel 17 gene is expressed preferentially in pigment cells. It has been mapped to human chromosome 12 pter‐q21 and mouse chromosome 10, near the silver locus. The Pmel 17 gene contains an insertional mutation at its carboxyl terminus in the silver mouse, suggesting that the silver locus might correspond to the gene. In the current studies, we have isolated and characterized human Pmel 17 genomic clones and employed FISH mapping for a precise localization of this gene in the human chromosome. The FISH mapping placed the Pmel 17 gene at human chromosome 12 q12‐q13. The human gene consists of nine exons and eight introns, and the entire coding region of the gene spans approximately 7.9 kb of the human chromosome 12. The putative functional domains, such as the signal sequence, histidine‐rich, 26‐amino acid repeats, cysteine‐rich, transmembrane and cytoplasmic domains, were encoded by separate exons. Cis‐transcription elements such as a TATA, a CAT and other potential elements for pigment cell‐specific gene expression were found within 1100 base pairs of the 5’flanking region.
Activation of Tyrosinase Reduces the Cytotoxic Effects of the Superoxide Anion in B16 Mouse Melanoma Cells
Authors: VALVERDE PALOMA , MANNING PHILIP , McNEIL CALUM J. , THODY ANTHONY J.
Published: July 2006

Tyrosinase may protect against oxidative stress by using the superoxide anion (O−2) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10‐differential deficient (‐DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O−2 and cytotoxicity assessed by measuring cell survival. XO increased O−2 concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10‐DD cells. [Nle4,DPhe7]MSH increased tyrosinase activity and melanin content, reduced O−2 concentration and increased the resistance of F10 cells to the cytotoxic effects of O−2. No such effects were seen in F10‐DD cells. The effect of [Nle4,DPhe7]MSH on the resistance of the F10 cells was time‐dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O−2. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle4,DPhe7]MSH. Moreover, although melanin was capable of scavenging O−2 it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10‐DD cells. We conclude that tyrosinase is able to utilise O−2 to produce melanin and this provides pigment cells with a unique anti‐oxidant mechanism.