Wakamatsu Kazumasa

<jats:p>The iridescent colors of birds originate in nanoscale feather structures that interact with light. Although the physical principles governing avian iridescence are well established, the molecular mechanisms assembling these nanostructures into photonic materials remain unknown. Here, we integrated genomic, cellular, and optical approaches to investigate the molecular basis of iridescent color formation in birds. We leveraged mutations that arose in the domesticated Indian peafowl and natural color variation in wild birds. In peafowl, we identified eight genes underlying gains, losses, and shifts of iridescence, whereas in two wild species, we used single-cell transcriptomics to profile asymmetric feathers in which iridescent and noniridescent barbules develop on opposite sides of the same feather. We found that all peafowl mutations mapped to melanogenesis genes, showing that variation in this pathway can modify diverse geometric features of feather photonic nanostructures. Mutations altering melanin composition collapsed multilayered photonic systems and yielded noniridescent tissues, whereas changes in melanosome abundance, elongation, or deposition timing generated multilayer architectures with variable periodicity and color. We further show in peafowl that transitions from noniridescent to iridescent plumage were associated with single-nucleotide mutations in a melanogenesis gene, suggesting that, in species already capable of forming multilayered nanostructures, iridescence can be gained by relatively small genetic changes. Single-cell transcriptomes from wild species also supported extensive melanocyte-centered regulatory rewiring associated with iridescence, including changes in melanosome maturation, trafficking, and intercellular signaling. Together, these results show that the nanoscale order underlying iridescence is developmentally plastic and highly responsive to the melanogenic environment.</jats:p>

Melanin pigments play a critical role in physiological processes and shaping animal behaviour. Fossil melanin is a unique resource for understanding the functional evolution of melanin but the impact of fossilisation on molecular signatures for eumelanin and, especially, phaeomelanin is not fully understood. Here we present a model for the chemical taphonomy of fossil eumelanin and phaeomelanin based on thermal maturation experiments using feathers from extant birds. Our results reveal which molecular signatures are authentic signals for thermally matured eumelanin and phaeomelanin, which signatures are artefacts derived from the maturation of non-melanin molecules, and how these chemical data are impacted by sample preparation. Our model correctly predicts the molecular composition of eumelanins in diverse vertebrate fossils from the Miocene and Cretaceous and, critically, identifies direct molecular evidence for phaeomelanin in these fossils. This taphonomic framework adds to the geochemical toolbox that underpins reconstructions of melanin evolution and of melanin-based coloration in fossil vertebrates.

The melanin pigments eumelanin (EM) and pheomelanin (PM), which are dark brown to black and yellow to reddish-brown, respectively, are widely found among vertebrates. They are produced in melanocytes in the epidermis, hair follicles, the choroid, the iris, the inner ear, and other tissues. The diversity of colors in animals is mainly caused by the quantity and quality of their melanin, such as by the ratios of EM versus PM. We have developed micro-analytical methods to simultaneously measure EM and PM and used these to study the biochemical and genetic fundamentals of pigmentation. The photoreactivity of melanin has become a major focus of research because of the postulated relevance of EM and PM for the risk of UVA-induced melanoma. Our biochemical methods have found application in many clinical studies on genetic conditions associated with alterations in pigmentation. Recently, besides chemical degradative methods, other methods have been developed for the characterization of melanin, and these are also discussed here.

Abstract The dark pigment neuromelanin (NM) is abundant in cell bodies of dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in the human brain. During the progression of Parkinson’s disease (PD), together with the degeneration of the respective catecholamine (CA) neurons, the NM levels in the SN and LC markedly decrease. However, questions remain among others on how NM is associated with PD and how it is synthesized. The biosynthesis pathway of NM in the human brain has been controversial because the presence of tyrosinase in CA neurons in the SN and LC has been elusive. We propose the following NM synthesis pathway in these CA neurons: (1) Tyrosine is converted by tyrosine hydroxylase (TH) to L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted by aromatic L-amino acid decarboxylase to DA, which in LC neurons is converted by dopamine β-hydroxylase to NE; (2) DA or NE is autoxidized to dopamine quinone (DAQ) or norepinephrine quinone (NEQ); and (3) DAQ or NEQ is converted to eumelanic NM (euNM) and pheomelanic NM (pheoNM) in the absence and presence of cysteine, respectively. This process involves proteins as cysteine source and iron. We also discuss whether the NM amounts per neuromelanin-positive (NM⁺) CA neuron are higher in PD brain, whether NM quantitatively correlates with neurodegeneration, and whether an active lifestyle may reduce NM formation.

Neuromelanin (NM) in dopaminergic neurons of human substantia nigra (SN) has a melanic component that consists of pheomelanin and eumelanin moieties and has been proposed as a key factor contributing to dopaminergic neuron vulnerability in Parkinson's disease (PD). While eumelanin is considered as an antioxidant, pheomelanin and related oxidative stress are associated with compromised drug and metal ion binding and melanoma risk. Using postmortem SN from patients with PD or Alzheimer's disease (AD) and unaffected controls, we identified increased L-3,4-dihydroxyphenylalanine (DOPA) pheomelanin and increased ratios of dopamine (DA) pheomelanin markers to DA in PD SN compared to controls. Eumelanins derived from both DOPA and DA were reduced in PD group. In addition, we report an increase in DOPA pheomelanin relative to DA pheomelanin in PD SN. In AD SN, we observed unaltered melanin markers despite reduced DOPA compared to controls. Furthermore, synthetic DOPA pheomelanin induced neuronal cell death in vitro while synthetic DOPA eumelanin showed no significant effect on cell viability. Our findings provide insights into the different roles of pheomelanin and eumelanin in PD pathophysiology. We anticipate our study will lead to further investigations on pheomelanin and eumelanin individually as biomarkers and possibly therapeutic targets for PD.

Chemical leukoderma is an acquired depigmentation of the skin caused by repeated exposure to specific agents damaging to epidermal melanocytes. Case reports of chemical leukoderma have been associated with some consumer products. To date, there are no well-accepted approaches for evaluating and minimizing this risk. To this end, a framework is presented that evaluates the physical and chemical characteristics of compounds associated with chemical leukoderma and employs structure-activity relationship (SAR) read-across and predictive metabolism tools to determine whether a compound is at increased risk of evoking chemical leukoderma. In addition to in silico approaches, the testing strategy includes in chemico quinone formation and in vitro melanocyte cytotoxicity assays to dimension the risk as part of an overall weight of evidence approach to risk assessment. Cosmetic ingredients raspberry ketone, undecylenoyl phenylalanine, tocopheryl succinate, p-coumaric acid, resveratrol, resveratrol dimethyl ether, sucrose dilaurate, tranexamic acid, niacinamide and caffeic acid are evaluated in this framework and compared to positive controls rhododendrol and hydroquinone. Overall, this framework is considered an important step toward mitigating the risk of chemical leukoderma for compounds used in consumer products.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Melanoma is one of the most lethal and malignant cancers and its incidence is increasing worldwide, and Japan is not an exception. Although there are numerous therapeutic options for melanoma, the prognosis is still poor once it has metastasized. The main concern after removal of a primary melanoma is whether it has metastasized, and early detection of metastatic melanoma would be effective in improving the prognosis of patients. Thus, it is very important to identify reliable methods to detect metastases as early as possible. Although many prognostic biomarkers (mainly for metastases) of melanoma have been reported, there are very few effective for an early diagnosis. Serum and urinary biomarkers for melanoma diagnosis have especially received great interest because of the relative ease of sample collection and handling. Several serum and urinary biomarkers appear to have significant potential both as prognostic indicators and as targets for future therapeutic methods, but still there are no efficient serum and urinary biomarkers for early detection, accurate diagnosis and prognosis, efficient monitoring of the disease and reliable prediction of survival and recurrence. Levels of 5-S-cysteinyldopa (5SCD) in the serum or urine as biomarkers of melanoma have been found to be significantly elevated earlier and to reflect melanoma progression better than physical examinations, laboratory tests and imaging techniques, such as scintigraphy and echography. With recent developments in the treatment of melanoma, studies reporting combinations of 5SCD levels and new applications for the treatment of melanoma are gradually increasing. This review summarizes the usefulness of 5SCD, the most widely used and well-known melanoma marker in the serum and urine, compares 5SCD and other useful markers, and finally its application to other fields.

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1-3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4-7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Neuromelanin (NM) is a dark brown pigment found in dopaminergic neurons of the substantia nigra (SN) and in norepinephrinergic neurons of the locus coeruleus (LC). Although NM is thought to be involved in the etiology of Parkinson's disease (PD) because its content decreases in neurodegenerative diseases such as PD, details are still unknown. In this study, we characterized the biosynthetic pathway of the oxidation of dopamine (DA) by tyrosinase in the presence of thiol peptides and proteins using spectroscopic and high-performance liquid chromatography (HPLC) methods and we assessed the binding of DA via cysteine residues in proteins by oxidation catalyzed by redox-active metal ions. To examine whether the protein-bound DA conjugates exhibit pro-oxidant activities, we measured the depletion of glutathione (GSH) with the concomitant production of hydrogen peroxide. The results suggest that the fate of protein-bound DA conjugates depends on the structural features of the proteins and that DA-protein conjugates produced in the brain possess pro-oxidant activities, which may cause neurodegeneration due to the generation of reactive oxygen species (ROS) and the depletion of antioxidants.

Recent progress has been made in paleontology with respect to resolving pigmentation in fossil material. Morphological identification of fossilized melanosomes has been one approach, while a second methodology using chemical imaging and spectroscopy has also provided critical information particularly concerning eumelanin (black pigment) residue. In this work we develop the chemical imaging methodology to show that organosulfur-Zn complexes are indicators of pheomelanin (red pigment) in extant and fossil soft tissue and that the mapping of these residual biochemical compounds can be used to restore melanin pigment distribution in a 3 million year old extinct mammal species (Apodemus atavus). Synchotron Rapid Scanning X-ray Fluorescence imaging showed that the distributions of Zn and organic S are correlated within this fossil fur just as in pheomelanin-rich modern integument. Furthermore, Zn coordination chemistry within this fossil fur is closely comparable to that determined from pheomelanin-rich fur and hair standards. The non-destructive methods presented here provide a protocol for detecting residual pheomelanin in precious specimens.

Adaptive evolution in new or changing environments can be difficult to predict because the functional connections between genotype, phenotype, and fitness are complex. Here, we make these explicit connections by combining field and laboratory experiments in wild mice. We first directly estimate natural selection on pigmentation traits and an underlying pigment locus, Agouti, by using experimental enclosures of mice on different soil colors. Next, we show how a mutation in Agouti associated with survival causes lighter coat color through changes in its protein binding properties. Together, our findings demonstrate how a sequence variant alters phenotype and then reveal the ensuing ecological consequences that drive changes in population allele frequency, thereby illuminating the process of evolution by natural selection.