Some Aspects of Melanin Formation of Melanocytes Cultured on Collagen-Coated Microcarrier Beads

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.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

The Effects of Hydroxyl Radical Attack on Dopa,Dopamine, 6-Hydroxydopa,and 6-Hydroxydopamine

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

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

Biological Activities of Melanotropic Peptide Fatty Acid Conjugates

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the α-MSH fragment analog, H-Asp⁵-His⁶-D-Phe⁷-Arg⁸-Trp⁹-Lys¹⁰-NH₂. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to α-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a “creeping” potency in the lizard skin bioassay—that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10–100 times more active than α-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Ast⁵,D-Phe⁷,Lys¹⁰]α-MSH₅-₁₀-NH₂, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Insect Melanogenesis. II. Inability of Manduca Phenoloxidase to Act on 5, 6-Dihydroxyindole-2-Carboxylic Acid1

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3, 4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.     

Targeted reduction of complex models with time scale hierarchy--a case study

With the increasing flow of biological data there is a growing demand for mathematical tools whereby essential aspects of complex causal dynamic models can be captured and detected by simpler mathematical models without sacrificing too much of the realism provided by the original ones. Given the presence of a time scale hierarchy, singular perturbation techniques represent an elegant method for making such minimised mathematical representations. Any reduction of a complex model by singular perturbation methods is a targeted reduction by the fact that one has to pick certain mechanisms, processes or aspects thought to be essential in a given explanatory context. Here we illustrate how such a targeted reduction of a complex model of melanogenesis in mammals recently developed by the authors provides a way to improve the understanding of how the melanogenic system may behave in a switch-like manner between production of the two major types of melanins. The reduced model is shown by numerical means to be in good quantitative agreement with the original model. Furthermore, it is shown how the reduced model discloses hidden robustness features of the full model, and how the making of a reduced model represents an efficient analytical sensitivity analysis. In addition to yielding new insights concerning the melanogenic system, the paper provides an illustration of a protocol that could be followed to make validated simplifications of complex biological models possessing time scale hierarchies.     

Melanoma × Macrophage Fusion Hybrids Acquire Increased Melanogenesis and Metastatic Potential: Altered N-Glycosylation as an Underlying Mechanism

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using ³H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.