Fungal melanins differ in planar stacking distances

Melanins are notoriously difficult to study because they are amorphous, insoluble and often associated with other biological materials. Consequently, there is a dearth of structural techniques to study this enigmatic pigment. Current models of melanin structure envision the stacking of planar structures. X ray diffraction has historically been used to deduce stacking parameters. In this study we used X ray diffraction to analyze melanins derived from Cryptococcus neoformans, Aspergillus niger, Wangiella dermatitides and Coprinus comatus. Analysis of melanin in melanized C. neoformans encapsulated cells was precluded by the fortuitous finding that the capsular polysaccharide had a diffraction spectrum that was similar to that of isolated melanin. The capsular polysaccharide spectrum was dominated by a broad non-Bragg feature consistent with origin from a repeating structural motif that may arise from inter-molecular interactions and/or possibly gel organization. Hence, we isolated melanin from each fungal species and compared diffraction parameters. The results show that the inferred stacking distances of fungal melanins differ from that reported for synthetic melanin and neuromelanin, occupying intermediate position between these other melanins. These results suggest that all melanins have a fundamental diffracting unit composed of planar graphitic assemblies that can differ in stacking distance. The stacking peak appears to be a distinguishing universal feature of melanins that may be of use in characterizing these enigmatic pigments.     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

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.     

Melanins and melanosomes from llama (Lama glama L.)

Analysis of melanins and melanosomes in eight hair and skin samples taken of adult pigmented Argentine llamas (Lama glama L.) has been carried out. In each sample, eumelanins, pheomelanins and alkali-soluble melanins were identified. The total amount of melanins and the amount of eumelanins both decreased from black to reddish brown colour, while pheomelanins were found to be present in small quantities in each sample. Eumelanosomes were round and oval-shaped, displaying transverse striations clearly visible at low magnification. Dark brown samples revealed all four melanosomes stages. Stages I and II melanosomes appeared as large, asymmetrical vacuoles containing numerous microvesicles randomly scattered within an amorphous proteinaceous material (vesiculo-globular bodies). Stage III melanosomes had microgranular melanin deposits in the microvesicles and in the matrix. The fully melanized melanosomes (stage IV) were primarily round-shaped, showing an irregular outline and the electron-dense pigment was arranged to form large clusters. In light brown melanocytes, numerous melanosomes at different maturation stages could be found. Premelanosomes appeared ovoid, containing amorphous proteinaceous material and spotty and microgranular deposits. Mature melanosomes were fully melanized, homogeneously electron-dense, ovoid granules.     

Ionizing radiation: how fungi cope, adapt, and exploit with the help of melanin

Life on Earth has always existed in the flux of ionizing radiation. However, fungi seem to interact with the ionizing radiation differently from other inhabitants of the Earth. Recent data show that melanized fungal species like those from Chernobyl's reactor respond to ionizing radiation with enhanced growth. Fungi colonize space stations and adapt morphologically to extreme conditions. Radiation exposure causes upregulation of many key genes, and an inducible microhomology-mediated recombination pathway could be a potential mechanism of adaptive evolution in eukaryotes. The discovery of melanized organisms in high radiation environments, the space stations, Antarctic mountains, and in the reactor cooling water combined with phenomenon of 'radiotropism' raises the tantalizing possibility that melanins have functions analogous to other energy harvesting pigments such as chlorophylls.     

Physico-Chemical Evaluation of Rationally Designed Melanins as Novel Nature-Inspired Radioprotectors

Background

Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.

Methodology/Principal Findings

We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14·10¹⁸, 7.09·10¹⁸, and 9.05·10¹⁷ spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy (¹³⁷Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.

Conclusions/Significance

We propose that due to melanin''s numerous aromatic oligomers containing multiple π-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.     

The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization

Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells.     

Melanin-based plumage coloration and flight displays in plovers and allies

Plovers and their allies exhibit an impressive diversity of melanin-based plumage patterns ranging from non-melanized to completely melanized species. We use phylogenetic comparative methods to test whether melanization has evolved in relation to sexual selection for attracting mates, to selection for signalling territory defence, or to natural selection for camouflage. First, according to sexual-selection theory, melanized plumage has evolved to amplify the courtship displays of males. As predicted by this hypothesis, we found that males with aerial displays had more melanized plumage than males of ground-displaying species. In addition, sexual dimorphism in melanization was greater in species with display flights than in species with ground displays. Second, melanization may have evolved through social interactions to signal competitive ability in territory defence. We did not find evidence for this hypothesis, since breeding density was unrelated to the melanization of either sex. Finally, melanized plumage may camouflage the incubating parent. The latter hypothesis was not supported, since melanization was unrelated either to the darkness of nest substrate or the extent of vegetation cover. Taken together, our results are most consistent with the sexual-selection hypothesis, and suggest that melanized plumage has evolved to enhance the aerial displays of male plovers.     

Bacterial degradability of an intrafeather unmelanized ornament: a role for feather‐degrading bacteria in sexual selection?

The impact of feather‐degrading bacilli on feathers depends on the presence or absence of melanin. In vitro studies have demonstrated that unmelanized (white) feathers are more degradable by bacteria than melanized (dark) ones. However, no previous study has looked at the possible effect of feather‐degrading bacilli on the occurrence of patterns of unmelanized patches on otherwise melanized feathers. The pied flycatcher Ficedula hypoleuca Pallas, 1764 is a sexually dimorphic passerine with white wing bands consisting of unmelanized patches on dark flight feathers. These patches are considered to be a sexually selected trait in Ficedula flycatchers, especially in males, where the patches are more conspicuous (larger and possibly whiter) than in females. Using in vitro tests of feather bacterial degradation, we compared the degradability of unmelanized and melanized areas of the same feather for 127 primaries collected from the same number of individuals in a population breeding in central Spain (58 males and 69 females). In addition, we also looked for sex differences in feather degradability. Based on honest signalling theory and on the fact that there is stronger sexual selection for males to signal feather quality than in females, we predicted that unmelanized areas should be more degradable by bacteria than melanized ones within the same feather, and that these unmelanized areas should also be more degradable in males than in females. We confirmed both predictions. Microstructural differences between cross‐section dimensions of unmelanized and melanized barbs, but not differences in the density of barbs within unmelanized and melanized areas of feathers in males and females, could partly explain differences in degradability. This is the first study to show differences in bacterial degradability among markings on the same feather and among unmelanized feather patches between males and females as predicted by sexual selection theory. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 409–419.     

The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.     

Testicular melanization has evolved in birds with high mtDNA mutation rates

Melanin is mainly found in the integument of animals, but it also appears in several extracutaneous tissues. The presence of melanin in testes has been anecdotally reported in all vertebrate groups, but the causes and functions of this melanin remain unknown. Similar to other extracutaneous melanins, testicular melanin may protect male germ cells from oxidative stress. Given the high respiratory activity of spermatozoa, oxidative stress generated by mitochondrial dysfunction as a consequence of mtDNA mutations directly affects sperm viability. Thus, natural selection may favour testicular melanization in males of species with high historical mutation rates in the mitochondrial genome. Here, we tested this hypothesis using information on occurrence of testicular melanization and mutation accumulation as reflected by cytochrome b mtDNA base pair substitution rates in a large set of 134 species of birds, controlling for the confounding effects of body mass, reproductive activity and phylogeny. We found that testicular melanization has evolved in species with high rates of accumulated mitochondrial mutations and propose that this is an adaptive response related to the protective capacity of melanin against oxidative stress. In support of this hypothesis, testicular melanization was more frequently observed during the breeding season of birds (i.e. when spermatogenesis is likely to occur) than during reproductive inactivity. In contrast to other extracutaneous melanins whose abundance seems to reflect skin and coat colour, we did not find a correlation between the proportion of plumage coloured by melanins and occurrence of testicular melanization. Whereas future experimental studies should test these hypotheses, our study highlights for the first time that melanization patterns in animals may evolve as a response to historical mutation rates.     

Ultrastructural investigation of autophagocytosis of melanosomes and programmed death of melanocytes in White Leghorn feathers: a study of morphogenetic events leading to hypomelanosis

White Leghorn chickens have decreased feather melanin, not because pigment cells are absent, but because of a genetically determined programmed cell death that causes pigment cells to degenerate prematurely before the melanin can be deposited in the feathers. In this paper, we studied the feather germs of this breed and of control Black Minorca chickens by light and electron microscopy to elucidate further the mechanism of cell death.White Leghorn feature-germ melanocytes produced a large number of unmelanized melanosomes which, however, did not become melanized, nor were they transferred into the keratinocytes of the follicles. From day 10 of incubation onward, large autophagosomes appeared in the melanocytes of White Leghorn feather follicles. These autophagosomes were acid phosphatase positive and engulfed incompletely melanized melanosomes. They also contained melanosome degradation products. Finally, degeneration of the whole melanocyte followed. These necrotic melanocytes were engulfed by normal-looking keratinocytes of the same follicle. In Black Minorcas, on the other hand, there was a normal sequence of synthesis, melanization, and transfer of melanosomes. The melanocytes degenerated only at the time of hatching, without the formation of large autophagosomes.     

Inhibition of human immunodeficiency virus type 1 replication and cytopathicity by synthetic soluble catecholamine melanins in vitro

Synthetic soluble melanins were synthesized by spontaneous oxidation of L-dopamine, norepinephrine or 5-hydroxytryptamine (serotonin) in weak alkaline solution. These three melanins inhibited infection of human CD4+ lymphoblastoid cells (MT-2) by cell-free human immunodeficiency virus type 1 (HIV-1), without cell toxicity, at concentrations of 0.15-10 micrograms/ml. Also, syncytium formation and resulting cytopathic effects when uninfected cells were mixed with chronic HIV-1-infected cells were blocked by these melanins. Antisyncytial activity was greater when infected cells were preincubated with melanin than when uninfected cells were preincubated with melanin, thus suggesting that interaction of melanin with viral proteins is an important aspect of the antiviral mechanism. These results make synthetic soluble melanins interesting candidates for further study as possible anti-HIV-1 therapeutics.     

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl-2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post-mortem brain and primary mesencephalic cell cultures treated with MPP(+). We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post-mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax-positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax-positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase-3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP(+) treatment of tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.     

An association of actin isoforms with the expression of motile response in pigmentation variants induced from goldfish erythrophoroma cells

Comparison of actin isoforms in unpigmented goldfish cells (a normal dermal fibroblast-like cell line, and an unpigmented erythrophoroma cell line capable of being induced to undergo melanization) and in normal and neoplastic melanized goldfish cells shows that the melanized phenotype is accompanied by the presence of multiple actin isoforms. In contrast, the unpigmented cells have only beta-actin. The possible significance of this to pigment organelle translocation is discussed.     

Clonal heterogeneity in physiological properties of melanized cells induced from goldfish erythrophoroma cell lines

Abstract. Cells of the uncloned goldfish erythrophoroma lines, GEM-81 and GEM-218, have been induced to melanize by cultivation in autologous serum. The melanized cells continue to proliferate and exhibit clonal heterogeneity in terms of morphology, growth rates, contact behavior and pigment content, and distribution and translocation in response to hormones. Based on these characteristics and those of their normal counterparts, the melanized tumor cells have been categorized as type-I and type-I1 melanocytomas, and melanophoromas. The melanophoroma cells are capable of pigment translocation in response to epinephrine, melatonin, and/or MSH, whereas melanocytoma cells are not. The distinguishing characteristics of each type are apparent at the first appearance of melanized cells and appear to be stable except in some type-II melanocytoma clones which contain cells capable of differentiating into melanophoroma cells in long-term cultures. It appears that the parent erythrophoroma lines contain stem cells, melanoblastomas, which are capable of melanogenesis. These stem cells may themselves be a heterogeneous population with respect to the characteristics of the melanized cells to which they give rise.     

Developmental Aspects of Vertebrate Chromatophores

Melanophores, xanthophores, and iridophores are fundamentallydistinct chromatophores in their appearance, composition, andfunction. All migrate from their neural crest site of originto populate the integument. Their respective pigments, melanins,ptendines, and purines are found in organelles designated respectivelyas melanosomes, pterinosomes and reflecting platelets. Theseorganelles are all derived from an endoplasmic reticular vesicle.This is in keeping with a hypothesis about the common originof pigment cells from a stem cell containing a primordial organellewith the potential of becoming any of the circumscribed pigmentaryorganelles. It is believed that chromatoblasts may not be specificallydetermined until they reach a final destination where they willdifferentiate in accordance with a pattern already specifiedin the integument. In leopard frogs, it appears that the initialinduction of pattern in the skin is general, but later it becomeshighly specific.     

Effects of melanin upon susceptibility of Cryptococcus to antifungals

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.     

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.     

Magnetic Resonance Imaging Characteristics of Nonthermal Irreversible Electroporation in Vegetable Tissue

We introduce and characterize the use of MRI for studying nonthermal irreversible electroporation (NTIRE) in a vegetative tissue model. NTIRE is a new minimally invasive surgical technique for tissue ablation in which microsecond, high electric-field pulses form nanoscale defects in the cell membrane that lead to cell death. Clinical NTIRE sequences were applied to a potato tuber tissue model. The potato is used for NTIRE studies because cell damage is readily visible with optical means through a natural oxidation process of released intracellular enzymes (polyphenol oxidase) and the formation of brown-black melanins. MRI sequences of the treated area were taken at various times before and after NTIRE and compared with photographic images. A comparison was made between T1W, T2W, FLAIR and STIR MRIs of NTIRE and photographic images. Some MRI sequences show changes in areas treated by irreversible electroporation. T1W and FLAIR produce brighter images of the treated areas. In contrast, the signal was lost from the treated area when a suppression technique, STIR, was used. There was similarity between optical photographic images of the treated tissue and MRIs of the same areas. This is the first study to characterize MRI of NTIRE in vegetative tissue. We find that NTIRE produces changes in vegetative tissue that can be imaged by certain MRI sequences. This could make MRI an effective tool to study the fundamentals of NTIRE in nonanimal tissue.