Tissue-specific geometry and chemistry of modern and fossilized melanosomes reveal internal anatomy of extinct vertebrates.

Recent discoveries of nonintegumentary melanosomes in extant and fossil amphibians offer potential insights into the physiological functions of melanin not directly related to color production, but the phylogenetic distribution and evolutionary history of these internal melanosomes has not been characterized systematically. Here, we present a holistic method to discriminate among melanized tissues by analyzing the anatomical distribution, morphology, and chemistry of melanosomes in various tissues in a phylogenetically broad sample of extant and fossil vertebrates. Our results show that internal melanosomes in all extant vertebrates analyzed have tissue-specific geometries and elemental signatures. Similar distinct populations of preserved melanosomes in phylogenetically diverse vertebrate fossils often map onto specific anatomical features. This approach also reveals the presence of various melanosome-rich internal tissues in fossils, providing a mechanism for the interpretation of the internal anatomy of ancient vertebrates. Collectively, these data indicate that vertebrate melanins share fundamental physiological roles in homeostasis via the scavenging and sequestering of metals and suggest that intimate links between melanin and metal metabolism in vertebrates have deep evolutionary origins.

Hyperthermia sensitizes pigmented cells to laser damage without changing threshold damage temperature.

We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65 W/cm2 at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.

Melanosome transfer evaluation by quantitative measurement of Pmel 17 in human normal melanocyte-keratinocyte co-cultures: effect of an Alaria esculenta extract.

Numerous strategies have been proposed to evaluate melanosome transfer. Methods allowing quantitative measurements of this transfer in human normal cellular models, however, are very few and often require extremely specialized devices that are expensive and difficult to use. As a part of the melanosome-specific membrane-bound glycoprotein, Pmel 17 is released from the melanosome membrane by ectodomain shedding. We reasoned, therefore, that it should be possible to evaluate melanosome transfer by quantifying this "soluble" Pmel 17. The Pmel 17 ELISA assay developed permits a detection of 10 to 1000 ng/ml of this glycoprotein in human normal melanocyte-keratinocyte co-culture media. As expected, niacinamide, a well-known melanosome transfer inhibitor, significantly reduced the Pmel 17 quantities found in the culture media. This validated our experimental design. We then used our model to show that a whitening cosmetic active compound, i.e., an Alaria esculenta extract, can (at least in part) enable a significant decrease in the melanosome transfer to produce a lightening effect without affecting melanin production. This research provides a simple and efficient method to quantify melanosome transfer in a human normal co-culture model. It is a particularly useful tool with which to facilitate the development of new active whitening compounds.

Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content.

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.