bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural Immunogold Localization of Some Organelle‐Transport Relevant Proteins in Wholemounted Permeabilized Nonextracted Goldfish Xanthophores
Authors: KIMLER VICTORIA A. , TAYLOR JOHN D.
Published: July 2006

By whole‐cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against β‐tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with antiactin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti‐p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti‐PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti‐MAP 1C (dynein) immunolabeling was generally seen on club‐shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti‐MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.
Effect of Penicillin‐Streptomycin and Other Antibiotics on Melanogenic Parameters in Cultured B16/F10 Melanoma Cells
Authors: MARTÍNEZ‐LIARTE JOSÉ H. , SOLANO FRANCISCO , LOZANO JOSÉ A.
Published: July 2006

Penicillin and streptomycin, the most widely used antibiotics in mammalian cell cultures, caused a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity of B16/F10 melanoma cells at the routine concentration (100 units/ml penicillin and 100 μg/ml streptomycin) used for preventing bacterial growth in cultured animal cells. At these concentrations, tyrosinase activities and melanin content augmented with time during the first 24–48 hr. The opposite effect acted on cell viability. After withdrawal of the antibiotics from the culture medium, the recovery of melanogenic parameters to normal values was fully reached after few hours (around 10), and it was already noticeable as soon as 4 hr after removal. Other antibiotics used in cell culture, like kanamycin, gentamicin, and the antimicotic nystatin, exerted similar low effects at the recommended concentrations, always lower than two‐fold and thus lower than those reported for amphotericin B. Taking into account these relatively low effects, and the high risk of contamination of mammalian cells culture without antibiotics, penicillin and streptomycin may still be routinely used in experiments leading to explore the melanogenic activity of malignant melanocytes in culture, unless very precise studies and strict conditions were needed.
Turtle Lung Cells Produce a Melanization‐Stimulating Activity That Promotes Melanocytic Differentiation of Avian Neural Crest Cells
Authors: HOU LING , KWON BYOUNG S.
Published: July 2006

We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC‐2, was found to produce a putative melanization‐stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC‐2‐derived MSA was different from that of basic fibroblast growth factor (bFGF), α‐melanocyte stimulating hormone (α‐MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation.
Atypical Androgen Receptor in the Human Melanoma Cell Line IIB‐MEL‐J
Authors: MORVILLO VERÓNICA , LUTHY ISABEL A. , BRAVO ALICIA I. , CAPURRO MARIANA I. , DONALDSON MÓNICA , QUINTANS CARLOS , CALANDRA RICARDO S. , MORDOH JOSÉ
Published: July 2006

To evaluate the presence of androgen receptors in the human melanoma cell line IIBMEL‐J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (KD) at 37°C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [3H]‐R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy‐flutamide [OH‐FLU]) competed for [3H]‐R1881 binding, but also estradiol, progesterone, and cortisol at 500‐fold excess concentration. Binding of [3H]‐estradiol and [3H]‐R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained.

Different doses of flutamide (FLU) and OH‐FLU tested on cultured IIB‐MEL‐J cells in the presence of serum inhibited significantly cell proliferation in a dose‐dependent manner. When cells were incubated with 10 nM DHT and 1%charcoal‐adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 μM OH‐FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen.

In addition, male nude mice transplanted with IIB‐MEL‐J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals.

As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross‐reactivity and competition studies.
Oxidation of 3,4‐Dihydroxybenzylamine Affords 3,4‐Dihydroxybenzaldehyde via the Quinone Methide Intermediate
Authors: SUGUMARAN MANICKAM
Published: July 2006

Dopamine and related compounds are known to be toxic to melanoma cells. Some of their toxicity may be related, in part, to the oxidation products generated from them upon their interaction with melanogenic enzymes. In this paper, we present our studies on the oxidation chemistry of 3,4‐dihydroxybenzylamine, the lower homolog of dopamine. Mushroom tyrosinase catalyzed oxidation of 3,4‐dihydroxybenzylamine rapidly generated the corresponding quinone. However, aminomethyl‐o‐benzoquinone thus formed did not accumulate in the reaction mixture, but readily transformed to another product that exhibited absorbance maxima at 280 and 310 nm. This compound was identified to be 3,4‐dihydroxybenzaldehyde based on its HPLC elution profile, cochromatography with authentic sample and UV spectral properties. Possible mechanism for the formation 3,4‐dihydroxybenzaldehyde from 3,4‐dihydroxybenzylamine and the nature of cytotoxic quinonoid intermediates formed are discussed.
Melanins in Physiological Conditions Protect Against Lipoperoxidation. A Study on Albino and Pigmented Xenopus
Authors: CORSARO CONCETTA , SCALIA MARINA , BLANCO ANNA RITA , AIELLO IVANA , SICHEL GIOVANNI
Published: July 2006

The susceptibility to lipoperoxidation in liver of albino and pigmented Xenopus laevis Daudin, has been studied. Albino Xenopus liver was richer in polyunsaturated fatty acids (PUFAs) than the pigmented one; moreover, it was also richer in mitochondrial superoxide dismutase (MnSOD) and in reduced glutathione (GSH). The thiobarbituric acid‐reactive substances (TBARS) values were more abundant in the albino tissue compared to the pigmented tissue both during spontaneous and Fe++ induced lipoperoxidation. Therefore, when isolated and purified melanin, in physiological quantities, was added to albino tissue, the TBARS values drastically decreased. Thus, melanin, in our experimental conditions, protects the albino tissue even more than SOD and GSH do. Melanin, in our opinion, acts as an antioxidant, because it is able to scavenge O2−.
Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser‐G
Authors: SMIT NICO PM. , WESTERHOF WIETE , MENKO WILMA J. , VERBEEK NETTY M. , PAVEL STAN
Published: July 2006

Melanocyte cultures were established and maintained routinely in Ham's F‐10 medium containing 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser‐G, Nutridoma‐Hu and Nutricyte‐H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long‐term culture experiments by in situ cell counts at different periods of time. Only with Ultroser‐G (1‐2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser‐G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser‐G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α‐melanocyte stimulating hormone). The culture medium with 1% Ultroser‐G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α‐MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.
Antimelanoma Antibodies in Swine With Spontaneously Regressing Melanoma
Authors: CUI JIAN , CHEN DUNLU , MISFELDT MICHAEL L. , SWINFARD RONALD W. , BYSTRYN JEAN‐CLAUDE
Published: July 2006

Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth. To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS‐PAGE analysis of 125I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68–75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68–75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P<0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation. These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo‐like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.
Effects of Calphostin C, Specific PKC Inhibitor on TPA‐Induced Normal Human Melanocyte Growth, Morphology and Adhesion
Authors: NAKAZAWA KEIKO , NAKAZAWA HISAYOSHI , SAHUC FLORENT , DAMOUR ODILE , COLLOMBEL CHRISTIAN
Published: July 2006

Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth‐stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate‐stimulating agents or 12‐0‐tetrade‐canoylphorbol 13‐acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)‐activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC‐mediated pathway in the TPA‐dependent melanocyte functions—i.e., proliferation, morphology, and adhesion—using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 ± 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long‐term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA‐dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.
Monoclonal Antibody MAT‐1 Against Human Tyrosinase Can Detect Melanogenic Cells on Formalin‐Fixed Paraffin‐Embedded Sections
Authors: SATO NORIKO , SUZUKI SATOSHI , TAKIMOTO HIROYUKI , MASUI SHIGEKI , SHIBATA KOUSHI , NAKANO HIROYUKI , TOMITA YASUSHI
Published: July 2006

Immunohistochemical localization of tyrosinase was examined with a monoclonal antibody (MoAb MAT‐1) against human tyrosinase on routine formalin‐fixed paraffin‐embedded sections of 3 normal skin specimens, 15 melanocytic tumors (6 pigmented nevi, 3 juvenile melanomas and 6 malignant melanomas) and 3 non‐melanocytic tumors. In the melanotic melanomas, almost all tumor cells were clearly stained with the antibody. In the nevocytic nevi, the nevus cells in lower epidermis and upper dermis were positive for MoAb MAT‐1, but negative in middle and lower dermis. All three juvenile melanomas, one amelanotic melanoma, and three non‐melanocytic tumors were entirely negative for MoAb MAT‐1. Thus, MoAb MAT‐1 could recognize the cells with melanogenic activity on routine formalin‐fixed paraffin‐embedded sections. However, the staining quality was not adequate for normal epidermal melanocytes, indicating that small technical innovations in the immunostaining process such as formalin fixation after PBS washing are required. Nevertheless, MoAb MAT‐1 can be expected to be very useful for identifying melanogenic cells on paraffin‐embedded sections, because we have to date no other antibody available for it.
Tumor Suppressor p53 Down‐Regulates Tissue‐Specific Expression of Tyrosinase Gene in Human Melanoma Cell Lines
Authors: KICHINA JULIA , GREEN ALBERT , RAUTH SIKHA
Published: July 2006

Tyrosinase, the key gene in melanin pigment synthesis, is tissue‐specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein on tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue‐specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down‐regulates the tissue‐specific expression of tyrosinase gene and subsequent melanin synthesis.
Melanins From Opioid Peptides
Authors: ROSEI MARIA ANNA
Published: July 2006

Opioid peptides and other Tyr‐NH2‐terminal peptides are substrates in vitro for mushroom and sepia tyrosinase, giving rise to synthetic melanins retaining the peptide moiety (opiomelanins). The melanopeptides are characterized by a total solubility in hydrophylic solvents at neutral and basic pH. Opioid peptides (enkephalins, endorphins, and esorphins), if oxidized by tyrosinase in the presence of Dopa, are easily incorporated into Dopa‐melanin, producing mixed‐type pigments that can also be solubilized in hydrophylic solvents. Melanins derived from opioid peptides exhibit paramagnetism, as evidenced by an EPR spectrum identical to that of Dopa‐melanin. However, the presence of the linked peptide chain is able to influence dramatically the electron transfer properties and the oxidizing behaviour of the melanopeptides, so that whereas Tyr‐Gly‐melanin appears to behave as Dopa‐melanin, Enk‐melanin does not exhibit any oxidizing activity. Opiomelanins are characterized by a peculiar UV‐VIS spectrum; that is, by the presence of a distinct peak (330 nm) that disappears upon chemical treatment by acid hydrolysis. Opiomelanins are stable pigments at neutral and basic pH in the dark, whereas the addition of H2O2 leads to a 15% degradation. Under simulated solar illumination, opiomelanins are more easily destroyed with respect to Dopa‐melanin, with increasing degradation when exposed to increased hydrogen peroxide concentrations and more alkaline pH. Some speculations on the possible existence and role of opiomelanins have been outlined.
Insights Into Pigmentary Phenomena Provided by Grafting and Chimera Formation in the Axolotl
Authors: HOUILLON CHARLES , BAGNARA JOSEPH T.
Published: July 2006

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, and grafts of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.
Quantitative Analysis of Striped Coat‐Color Patterns in Large White→Duroc Chimeric Pigs With Special Reference to the Genetic Control Mechanisms of the Dominant Black‐Eyed White Phenotype
Authors: INOUÉ KAORI , TANAKA SATOSHI , KASHIWAZAKI NAOMI , NAKAO HARUHIKO , NAKATSUJI NORIO , SAKAKI NOBUO , TOJO HIDEAKI , TACHI CHIKASHI
Published: July 2006

Coat colors of four chimeric pigs produced by the microinjection of dissociated blastomeres of (Landrace × Large White) blastocysts to the blastocyst cavity of (Duroc × Duroc) blastocysts (Kashiwazaki et al., 1992) exhibited characteristic horizontal stripe‐patterns. We carried out quantitative analysis of those patterns in order to derive information concerning the genetic regulatory mechanisms of the dominant black‐eyed white phenotypes in the pig. In the four chimeras, the theoretical mean widths of the single‐clone stripe calculated from the estimated widths of minimal recognizable stripe (MRS) (Tachi, 1988) were 2.1 ± 0.1, 2.23 ± 0.15,1.89 ± 0.06, and 1.93 ± 0.28 cm respectively. The estimated number of single‐clone stripes in the thoracico‐lumbar region of those animals were 42.3, 40.7, 46.3, 44.2, and about twice the mean number of vertebrae in the same region (Duroc, 20 or 21; Large White, 21 or 22). Furthermore, the mean length of thoracico‐lumbar vertebrate in two of the chimeric pigs, as measured on X‐ray radiographs, was approximately twice the mean single‐clone stripe width. It was concluded that the stripe‐patterns of the chimeric pigs probably represented the dermatome patterns of epidermis; and in the pig, a single somite was likely to be derived from the clones of two primordial cells, as originally proposed by Gearhart & Mintz (1972) in the mouse. It was suggested, furthermore, that in the Large White→Duroc chimeric pigs, melanocytes that migrated into the region of skin formed by a Large White dermatome could not survive, thus creating a clearly demarcated white stripe. Possible involvement of KL or c‐kit in the dominant black‐eyed white phenotype of the pig is discussed.
Proposed Reclassification of Melanoma: A Meeting Held at the XVIth International Pigment Cell Congress, 3rd November 1996
Authors: COCHRAN ALISTAIR J.
Published: July 2006

The current classification of cutaneous melanoma was developed in 1972 and revised in 1982. Since that time, new concepts and terminology have evolved that require consideration of a further revision. Regional meetings of interested parties have been held to review the Classification and there will be an open meeting on the topic at the 1997, 4th World Conference on Melanoma in Sydney, Australia. This paper reports on a meeting to discuss some of the concepts that will form part of that process, held on November 3, 1996 as part of the XVIth International Pigment Cell Conference. A questionnaire is included that will allow the interested reader to provide comments on the topic.
The Regulation of Tyrosinase Gene Transcription
Authors: FERGUSON CHRISTINE A. , KIDSON SUSAN H.
Published: July 2006

Tyrosinase is one of the key enzymes essential for melanogenesis. The control of its activity rests in part at the level of transcriptional regulation. The 5’ promoter regions of the human, mouse, chicken, quail, snapping turtle, and frog tyrosinase sequences have been isolated and the mechanisms regulating the activity of these sequences are beginning to be elucidated. This review provides an update on the following aspects of tyrosinase gene regulation: basal promoter elements that determine the site of transcription initiation for RNA polymerase II; the cis‐acting elements and DNA‐binding factors that mediate melanocyte‐specific expression of the tyrosinase gene; promoter elements involved in the temporal control of tyrosinase gene expression; additional elements that may be required to achieve wild‐type levels of gene expression; and specific elements that may be required for modulation of tyrosinase gene expression in response to humoral factors or external stimuli that are known to influence the amounts of melanin synthesized by fully differentiated melanocytes. The wild type expression of tyrosinase is the result of the interaction of many different factors and it is becoming evident that certain elements and factors play more than one role in this process.
Regulation of the Tyrosinase Promoter in Transgenic Mice: Expression of a TyrosinaselacZ Fusion Gene in Embryonic and Adult Brain
Authors: TIER KIRSTEN , SCHMIDT ANDREA , BEERMANN FRIEDRICH
Published: July 2006

The enzyme tyrosinase is indispensable for pigmentation and the gene is expressed mainly in pigment cells. Regulatorg elements, at ‐12 to ‐15 kb (enhancer) and within the 270 bp directly upstream of the transcription start site, have been described recently and their importance demonstrated in transgenic experiments. We were interested in tyrosinase promoter activity during development and used β‐galactosidase as reporter gene. Transgenic mice were generated carrying a tyrosinase‐lacZ fusion gene, containing 6.1 kb of tyrosinase 5’ sequences. In transgenic embryos, β‐galactosidase activity was detected along the entire neural tube, with the most prominent expression in the developing telencephalon, and also in the adult brain. Equivalent expression was observed in the developing retina. Tyrosinase protein was identified in embryonic and adult brain, but no DOPAoxidase or tyrosine hydroxylase activity was detected. From our results we conclude that 1) tyrosinase protein is present in embryonic and adult mouse brain and 2) the tyrosinase promoter can direct expression of a reporter gene to pigment cells and neural tissues.
Survival of Cloudman Mouse Melanoma Cells After Irradiation by Solar Wavelengths of Light
Authors: CIESZKA KRYSTYNA , HILL HELENE Z. , XIN PATRICK , AZURE MICHAEL , HILL GEORGE J. , MEYENHOFER MARKUS F. , BOISSY RAYMOND E. , MITCHELL DAVID L.
Published: July 2006

A number of variants of Cloudman S91 mouse melanoma cells that differ with respect to the amount of pigment produced are available for study. In this report, we compare the photobiological responses of S91/amel, which contains about 1 pg of melanin per cell, with S91/I3, which contains about 3 pg/cell. Earlier studies had shown that UVC induced more oxidative damage (in the form of thymine glycols) in cell line S91/I3 than in S91/amel and that cell line S91/amel was more resistant to killing by UVC than S91/I3. The present study finds that S91/amel cells are also relatively resistant to killing by near monochromatic UVB from a Philips TLO1 fluorescent lamp and by near monochromatic UVA from a Philips HPW125 lamp. However when the cells are irradiated with a Westinghouse FS20 polychromatic lamp, the S91/I3 cells are more resistant than the S91/amel cells. These findings cannot be explained on the basis of pigment induction because in S91/I3 this is about the same after UVB and FS20, although the maximum is reached earlier after UVB. Nor can our findings be explained on the basis of pyrimidine dimer formation, which is comparable in the two cell lines regardless of the type of irradiation. These results suggest that, with a pigment such as melanin, which absorbs light across the visible and ultraviolet ranges of the spectrum, cellular responses to monochromatic light do not necessarily predict responses to polychromatic mixtures.
Biological Role of Tyrosinase Related Protein and its Biosynthesis and Transport From TGN to Stage I Melanosome, Late Endosome, Through Gene Transfection Study
Authors: JIMBOW KOWICHI , GOMEZ PAUL FABIAN , TOYOFUKU KAZUTOMO , CHANG DANIEL , MIURA SHUNSUKE , TSUJIYA HIDENOBU , PARK JONG SUNG
Published: July 2006

Tyrosinase‐related protein (TRP)‐1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP‐1 and its transport from TGN (trans‐Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP‐1 cDNA co‐transfection studies indicated that human TRP‐1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP‐1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome‐associated membrane protein)‐1 that would directly prevent the tyrosinase‐mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP‐1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP‐1 will be transported from TGN to the stage I melanosome. At this stage, TRP‐1 will have its own target signal, in particular, tyrosine‐rich leucine residues in cytoplasmic tail. Our TRP‐1 cDNA transfection and immunoelectron microscopy study shows that TRP‐1 will be transported through small vesicles, probably non‐clathrin‐coated type, to large vacuoles, identical to the MPR (mannose‐6‐phosphate receptor)‐positive, late endosomes. In this transport process, a low molecular weight G‐protein, rab‐7, was isolated from the purified melanosomal protein on 2D‐PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co‐localization of rab‐7 and TRP‐1 in the melanosomes with early stages of maturation (I‐III). Furthermore, this process will also be regulated by phosphatidylinositol 3‐kinase (PI‐3 kinase).
Selective and Synergistic Activity of L‐S,R‐Buthionine Sulfoximine on Malignant Melanoma Is Accompanied by Decreased Expression of Glutathione‐S‐Transferase
Authors: FRUEHAUF JOHN P. , ZONIS SVETLANA , AL‐BASSAM MOHAMMAD , KYSHTOOBAYEVA AINURA , DASGUPTA CHIRANJIB , MILOVANOVIC TATJANA , PARKER RICARDO J. , BUZAID ANTONIO C.
Published: July 2006

L‐buthionine‐S,R‐sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S‐transferase (GST) and GSH‐peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (μM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 μM, a level 20‐fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 μM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST‐μ. protein and mRNA levels were significantly reduced in both cell lines. GST expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.
X‐Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia
Authors: AKEO KIYOSHI , FUJIWARA TATSUSHI , YORIFUJI HIROSHI , OKISAKA SHIGEKUNI
Published: July 2006

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia.

Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X‐ray microanalysis by scanning electron microscope after being fixed, freeze‐dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra‐structural changes inside cell.

Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen.

Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.
Genetic and Biochemical Studies on Ommochrome Genesis in an Albino Strain of a Terrestrial Isopod, Armadillidium vulgare
Authors: HASEGAWA Y. , NEGISHI S. , NAITO J. , ISHIGURO I. , MARTIN G. , JUCHAULT E , KATAKURA Y.
Published: July 2006

Genetic studies and quantitative determination of levels of 3‐hydroxykynurenine and kynurenine were performed in an albino strain of a terrestrial isopod Armadillidium vulgare. From the results of matings between the albino and the albino, the red, the dark red, or the wild type individuals, the albino A. vulgare seems to be regulated by an autosomal gene(s) recessive to its wild allele. Litter mating of F1 progenies obtained by crossing the albino and the red mutant or the albino and the dark red mutant yielded progenies at a ratio of 3:6:3:4 for the red, the dark red, the wild, and the albino phenotypes, respectively. The albino gene(s) seems not to be allelic but to be epistatic to the red gene(s) with respect to ommochrome biosynthesis. Quantitative determination of 3‐hydroxykynurenine carried out by high‐performance liquid chromatography with electrochemical detection revealed that the 3‐hydroxykynurenine content in the albino was significantly lower than that in the wild or the red type. The whole content of 3‐hydroxykynurenine after enzymatic conversion of kynurenine to 3‐hydroxykynurenine was still considerably lower than that found in the wild type, even though it increased after the conversion. The albino gene(s) seems to be associated with a blockage at distinct level(s) of ommochrome biosynthesis.
The Amphibian Kupffer Cells Build and Demolish Melanosomes: An Ultrastructural Point of View
Authors: SICHEL GIOVANNI , SCALIA MARINA , MONDIO FILIPPO , CORSARO CONCETTA
Published: July 2006

This ultrastructural research was carried out to investigate the nature of the liver pigment cells of anuran and caudate amphibians, the pattern of melanosome ontogenesis, and the demolition processes of old melanosomes. We demonstrate that these liver pigment cells are able to internalize zymosan particles and latex beads; therefore, being professional phagocytes, they, as liver resident macrophages, can be classified as Kupffer cells (KCs). They show “melanosomogenesis centers” in which several maturation stages of premelanosomes are visible; the premelanosomes are formed by two principal components: a filamentous structure that will constitute the “inner” area of the melanosome and a vesicular component, budding from the Trans Golgi Network and that carries enzymes, which will constitute the “cortical area” of the melanosome. Thus the KCs, thanks to the presence of the “melanosomogenesis centers,” are also melanosome producing cells. They are also able to demolish melanosomes by heterophagocytosis and, probably, also by autophagocytosis. In conclusion, we propose a classification of vertebrate pigment cells.
The Site and Time of Expression of MIF in Frog Development
Authors: FUKUZAWA TOSHIHIKO , OKUMOTO HITOSHI , NISHIOKA MIDORI
Published: July 2006

A ventrally localized melanization‐inhibiting factor (MIF) has been suggested to play a role in the expression of dorsal‐ventral pigment patterns in amphibia. Here we investigate the onset and localization of MIF appearance in frog development. The expression of MIF was analyzed in the wild‐type and gray‐eyed mutant (g/g) of Rana japonica by immunoblotting and immunohistochemistry using an anti‐MIF neutralizing monoclonal antibody. Western blot analysis revealed that the anti‐MIF antibody recognized ∼51 kDa and ∼58 kDa bands. The 51 kDa band first appeared at the external gill stage, while 58 kDa band was additionally detected at the hindlimb bud stage. With the use of immunohistochemistry, it was found that the anti‐MIF antibody stained the whole epidermis of the embryos at the external gill stage; however, the staining was stronger in lateral and ventral epidermis than in dorsal. Staining with the anti‐MIF antibody was observed only in the outer epidermis of the ventral skin, but not in the dorsal skin during and after metamorphosis. The spatial expression of MIF in the wild‐type was the same as that in the gray‐eyed mutant. The same immunohistochemical result was obtained in the adults of R. nigromaculata. These results suggest that MIF is involved in the formation of the dorsal‐ventral pigment pattern.
β‐Lactoglobulin Suppresses Melanogenesis in Cultured Human Melanocytes
Authors: NAKAJIMA MITSUNARI , SHINODA ICHIZO , MIKOGAMI TAKASHI , IWAMOTO HIROSHI , HASHIMOTO SHIN‐ICHI , MIYAUCHI HIROFUMI , FUKUWATARI YASUO , HAYASAWA HIROTOSHI
Published: July 2006

The effects of whey proteins from bovine milk on melanogenesis in cultured human melanocytes were examined. Among the major protein components of milk whey including β‐lactoglobulin (BLG), α‐lactalbumin, serum albumin, and IgG, only BLG exhibited the depigmenting effect at a concentration of 1 mg/ml. Also, BLG suppressed the activity of tyrosinase in these cells. Retinol, to which BLG is known to bind, slightly increased the pigmentation of the cells at concentrations in the range of 1–100 nM, and retinoic acid, a metabolite of retinol, exhibited a strong pigmentation‐promoting effect within the same concentration range. Treatment of the cells with 1 mg/ml BLG completely abrogated the pigmentation induced by these A vitamins. These results demonstrate a novel biological activity of BLG and suggest that this activity is dependent on its ability to bind retinol.
Light‐Induced Pigment Aggregation in Xanthophores of the Medaka, Oryzias latipes
Authors: OSHIMA NORIKO , NAKATA ETSUKO , KAMAGATA MIYUKIOHTA SADATOSHI
Published: July 2006

The response mechanism of medaka xanthophores to light was examined at the cellular level. Innervated and denervated xanthophores of adult medakas responded to light (9,000 lux) within 30 sec by pigment aggregation, and this aggregation was not mediated through α‐adrenoceptors on the cell membrane. Maximum sensitivity to light was at wavelengths of 410–420 nm, and the direct effect of light was reversible. Xanthophore responsiveness to light in summer was higher than that in winter. Ca2+ and calmodulin were not involved in the response, but rather, an important role for cAMP and phos‐phodiesterase (PDE) was suggested. It seems likely that photoreception by visual pigment which is sensitive to light at wavelengths of 410–420 nm increases PDE activity, probably via a G‐protein, such as occurs with visual cells in the retina, which causes a decrease in levels of cytosolic cAMP, in turn leading to pigment aggregation within medaka xanthophores.
Involvement of Pteridines in the Body Coloration of the Isopod Armadillidium vulgare
Authors: NEGISHI SUMIKO , HASEGAWA YURIKO , KATOH SETSUKO
Published: July 2006

The pteridine content was measured as a function of age in Armadillidium vulgare, and the fine structure of the pteridine‐containing granules in the integument was examined in relation to pteridine content. Yellow chromatophores are an essential component of the cream‐markings, which are a defining feature of the female A. vulgare. Four kinds of pteridines in the integument including a yellow pigment (sepiapterin) were determined by HPLC.

The body color of the red phenotype of A. vulgare varies from dark red to yellowish red and was formerly thought to be due to the quality and quantity of ommochrome pigment. Our analysis of the pteridine content in the integument of this phenotype revealed a significant change in sepiapterin content per body weight with age. Sepiapterin content per body weight decreased gradually with age, while that of biopterin tended to increase with age. Ultrastructural observations of the pigment granules in the yellow chromatophores revealed a corresponding change in the fine structure of pigment granules. In the older adults, some of the electron‐dense fibrous materials in the pteridine‐containing granules was concentrically arranged, and in the younger adults, most of pteridine‐containing granules were electron‐lucent. The role of pteridine quality in determining the structure of pteridine‐containing granules is discussed.
Electron Microscopy and Spectroscopical Studies on the Coloured Patches on the Wing of a Butterfly, Graphium sarpedon (Lepidoptera: Papillionidae) With Reference to Their Photobiological and Electrical Properties
Authors: DEY S. , HOOROO R.N.K. , BHATTACHARJEE C.R.
Published: July 2006

The surface ultra‐structural features of the coloured patches on the wing of a butterfly Graphium sarpedon have been studied with the help of scanning electron microscopy. Comparisons have been made between the dark brown area and the light green patches of the wing. A diffraction grating pattern with 15 lines per μm2 with a uniform spacing of about 1 μm is present in the light green patches. A slightly coarser grating is present on the dark brown area, which constitutes the major portion of the wing. Sensilla chaetica was found on both the light green and dark brown area. A special type of sensilla trichodea with a big socket and some elongated projections were localized only on the light green patches. This region of the wing also contains some spherical structures with a diameter of 0.5 to 0.6 μm.

The infra‐red spectroscopy has revealed some differences in the nature and position of the peaks in the low‐energy region in the dark brown area and the light green patches. The atomic absorption spectroscopy also shows qualitative as well as quantitative differences in the inorganic set up of the two regions. The electron spin resonance spectroscopy reveals the presence of a peak in the dark brown region only, indicating the presence of free radical in it. The differences observed in the ultra‐structural and spectroscopical features, and also in the inorganic components of the two regions, are discussed in relation to their physical and physiological properties.
α‐MSH and Melanogenesis in Normal Human Adult Melanocytes
Authors: HEDLEY SUSAN J. , GAWKRODGER DAVID J. , WEETMAN ANTHONY P , MACNEIL SHEILA
Published: July 2006

Normal human skin melanocytes do not pigment consistently to α‐melanocyte stimulating hormone (α‐MSH) in culture. The aim of this study was to establish media conditions in which to obtain a reproducible melanogenic response to α‐MSH in these cells. Twenty‐five media of varying mitogen composition were examined. As previously noted by other workers, melanocyte morphology and proliferation are greatly affected by media composition. However, under the majority of media conditions that supported melanocyte survival and proliferation, cells did not respond to α‐MSH with any consistent increase in dopa oxidase activity or melanin content. In only one medium condition, where basic fibroblast growth factor (bFGF) was the sole mitogen present, α‐MSH induced both an increase in dopa oxidase activity (at 48%) and in melanin content (of 283%).
Epidermal Oxidative Stress in Vitiligo
Authors: PASSI SIRO , GRANDINETTI MAURO , MAGGIO FRANCESCO , STANCATO ANDREA , DE LUCA CHIARA
Published: July 2006

Epidermal levels of enzymatic and non‐enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px), vitamin E (Vit E), ubiquinol (CoQ10H2), and reduced glutathione (GSH), as well as polyunsaturated fatty acids of phospholipids (PL‐PUFA), were evaluated in the affected epidermis of 15 patients with active vitiligo (AVP) and in the corresponding epidermis of 15 healthy phototype matched controls. The epidermal levels of CoQ10H2, Vit E, GSH, and CAT activity were significantly reduced in AVP and were associated with a marked increase of oxidized glutathione, whereas SODs and GSH‐Px activities and ubiquinone concentration remained similar to control values. Antioxidant deficiency, in particular the decline of lipophilic antioxidants, i. e., CoQ10H2 and Vit E, accounts well for PL‐PUFA reduction observed in vitiligo epidermis, mainly affecting C18: 3 n‐3, C20: 3 n‐6, C20: 4 n‐6, and C22: 6 n‐3 fatty acids and suggesting the occurrence of a lipoperoxidative process.

In conclusion, both an imbalance of the intracellular redox status and a significant depletion of enzymatic and non‐enzymatic antioxidants feature the epidermis of AVP, and represent a fingerprint of an abnormal oxidative stress leading to epidermal cell injury.
Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*
Authors: DONOIS ERIC , MARMOL VÉRONIQUE DEL , WAKAMATSU KAZUMASA , ITO SHOSUKE , GHANEM GHANEM , SURLÈVE‐BAZEILLE JEAN‐ÉTIENNE
Published: July 2006

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.