Effect of Kojic Acid on the Oxidation of DL-DOPA,Norepinephrine, and Dopamine by Mushroom Tyrosinase

Kojic acid inhibits effectively the rate of formation of pigmented product(s) and of oxygen uptake when DL-DOPA, norepinephrine and dopamine are oxidized by mushroom tyrosinase. In addition to the direct effect of kojic acid on the enzyme, kojic acid also affects the UV-VIS spectrum of the final product(s) formed, this being due to the ability of the o-quinones of these substrates to oxidize kojic acid to a yellow product(s). Kojic acid can thus prevent the conversion of the o-quinones of DL-DOPA, norepinephrine and dopamine to their corresponding melanin.     

A DL-DOPA drop test for the identification of Cryptococcus neoformans

A simple melanin assay using DL.DOPA as the substrate was developed to aid in the identification of Cryptococcus neoformans. The DL-DOPA drop test assay was simple and efficient. The best results (100% of the C. neoformans isolates were positive) occurred when C. neoformans was grown for two days at room temperature on Sabouraud agar modified. One to three loopfuls of yeast cells were then transferred to a starvation medium for 18–24 hours. Two to three drops of 0.3% DL-DOPA solution was applied to the transferred yeast cells. Only C. neoformans produced a brown or blackgrey pigment within 24 hrs, with 85% of the isolates becoming brown or black-grey within thirty minutes.     

Decolorization of synthetic melanins by crude laccases of Lentinus polychrous L��v.

Melanins are complex natural pigments that darken the skin and are difficult to degrade. This study evaluated synthetic melanin decolorization by the crude laccase from fungus Lentinus polychrous in the absence and presence of selected redox mediators. The greatest melanin decolorization activity was 87?% at pH?6.5 within 3?h in the presence of 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), whereas only about 22?% melanin decolorized at pH?5.0 in case of no mediator. The optimum temperatures for melanin decolorization in the absence and presence of ABTS were 55 and 35°C, respectively. Using a natural redox mediator, 1.0?mmol/L vanillin leads to 45?% melanin decolorization. Our results suggest the possibility of applying vanillin for L. polychrous laccase-catalyzed decolorization of melanin.     

Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.     

Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.     

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.     

Melanin precursors prevent premature age-related and noise-induced hearing loss in albino mice

Strial melanocytes are required for normal development and correct functioning of the cochlea. Hearing deficits have been reported in albino individuals from different species, although melanin appears to be not essential for normal auditory function. We have analyzed the auditory brainstem responses (ABR) of two transgenic mice: YRT2, carrying the entire mouse tyrosinase (Tyr) gene expression-domain and undistinguishable from wild-type pigmented animals; and TyrTH, non-pigmented but ectopically expressing tyrosine hydroxylase (Th) in melanocytes, which generate the precursor metabolite, L-DOPA, but not melanin. We show that young albino mice present a higher prevalence of profound sensorineural deafness and a poorer recovery of auditory thresholds after noise-exposure than transgenic mice. Hearing loss was associated with absence of cochlear melanin or its precursor metabolites and latencies of the central auditory pathway were unaltered. In summary, albino mice show impaired hearing responses during ageing and after noise damage when compared to YRT2 and TyrTH transgenic mice, which do not show the albino-associated ABR alterations. These results demonstrate that melanin precursors, such as L-DOPA, have a protective role in the mammalian cochlea in age-related and noise-induced hearing loss.     

Inhibition of melanin biosynthesis by cerulenin in appressoria ofColletotrichum lagenarium

Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation ofColletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin ofC. lagenarium was synthesizedvia the polyketide pathway.     

Melanization of <Emphasis Type="Italic">Fusarium keratoplasticum</Emphasis> (<Emphasis Type="Italic">F. solani</Emphasis> Species Complex) During Disseminated Fusariosis in a Patient with Acute Leukemia

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.     

New biosynthetic step in the melanin pathway of Wangiella (Exophiala) dermatitidis: evidence for 2-acetyl-1,3,6,8-Tetrahydroxynaphthalene as a novel precursor

The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.     

Biocatalytic formation of synthetic melanin: the role of vanadium haloperoxidases, L-DOPA and iodide

The vanadium haloperoxidase (V-HPO) enzyme, extracted from the brown alga Laminaria saccharina, is able to catalyze the formation of a black precipitate, using as precursor the amino acid l-dopa in the presence of hydrogen peroxide and iodide, in one-pot synthesis. The l-dopa oxidation is a multistep reaction with a crucial role played by the iodide in the enzyme catalyzed peroxidative production of dopachrome, a well known intermediate in the synthesis of melanin. Dopachrome is then converted to a synthetic form of melanin through a polymerization reaction. Factors, such as buffer composition and pH, influence significantly the reaction first steps, but further steps of melanin production are hardly influenced. The biosynthetic melanin produced through the combination V-HPO/I/H₂O₂, was characterized by several spectroscopic techniques (UV-vis and FT-IR) as well as XRD. Moreover, this biopolymer is light sensitive, decomposing into oligo- and monomeric units. Scanning electron microscopy (SEM) imaging showed different morphologies when compared with commercial available melanin. The biosynthetic production of melanin can have a wide range of applications from photosensitive cells to biomedicine with the advantage of being produced under eco-friendly and mild conditions.     

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.     

In vitro study on humoral encapsulation of microfilariae: effects of diethyldithiocarbamate and dopachrome on the reaction

The effects of a phenoloxidase inhibitor, diethyldithiocarbamate, and an intermediate of the melanin biosynthetic pathway, dopachrome, on the humoral encapsulation of microfilariae were studied in vitro. Diethyldithiocarbamate inhibited the melanization but did not prevent the humoral encapsulation of microfilariae. In the presence of diethyldithiocarbamate in the in vitro system, transparent material in the form of numerous granules were deposited on the surface of the microfilariae and coalesced to form a consolidated transparent capsule around microfilariae. The thickness of transparent capsule was significantly greater than that of normal melanotic capsule. The transparent capsule material, which was probably the precursor of the melanotic capsule, was demonstrated histochemically as a protein-carbohydrate complex. The ultrastructure of transparent capsule showed that the transparent capsule material was very different from that of melanotic capsule material, being more flocculent and less granular in appearance. When dopachrome was concurrently present with the inhibitor in the in vitro encapsulation system, it failed to restore normal melanotic encapsulation. The data presented lead to the following conclusions: 1. The melanin biosynthetic pathway in the humoral encapsulation of microfilariae is similar to that found in the synthesis of mammalian melanin. 2. The humoral encapsulation of the microfilariae in vitro is a two-step process. The protein-carbohydrate complex is deposited on the surface of microfilariae. Then the tyrosine group of the complex is catalysed by phenoloxidase to melanin.     

Reproductive melanization may protect sperm from harmful solar radiation

Animal pigmentation is incredibly diverse, serving a variety of functions. However, the function of the pigmentation that surrounds the testes of some vertebrates is unknown. Why are the tissues surrounding the testes (scrotum, tunica vaginalis and/or tunica albuginea) melanized in some species but not others? We examined this question in bats, where there is extensive species-specific variation in the presence of darkly pigmented (melanized) tissues surrounding the male gonads, as well as diversity in their ecologies, mating systems, and physiologies. Using data from 136 species of bats, we found that melanin surrounding the testes and epididymis is associated with more exposed roosting sites (presumably with greater sun exposure- and UV radiation), and with the occurrence of long-term male sperm storage. These findings suggest that scrotal melanin may protect mature sperm from UV damage, and from oxidative damage in species with male sperm storage. We found no evidence of an association between group size or mating system with reproductive melanin, or that phylogeny explains the distribution of pigmentation. Although our study suggests that scrotal melanin may protect sperm, the mechanism remains unknown. We outline several avenues for future work on reproductive pigmentation aimed at investigating additional roles of reproductive melanization in male bats.     

Effect of different isomers of dihydroxybenzoic acids (DBA) on the rate of DL-dopa oxidation by mushroom tyrosinase.

Dihydroxybenzoic acids (DBA), such as 3,4-DBA, 3,5-DBA, and 2,4-DBA--at all concentrations tested--inhibited the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) by mushroom tyro0sinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DBA on the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Synergism exerted by 4-methyl catechol, catechol, and their respective quinones on the rate of DL-DOPA oxidation by mushroom tyrosinase.

4-Methyl catechol and catechol, at concentrations ranging from 0.03 to 9 mM and 0.066 to 20 mM, respectively, have a synergistic effect on the rate of DL-DOPA oxidation by mushroom tyrosinase to material absorbing at 475 nm. The synergism results from the ability of 4-methyl catechol-o-quinone (4-methyl-o-benzoquinone) and of catechol-o-quinone (o-benzoquinone) to oxidize DL-DOPA non-enzymatically to dopaquinone, with the later being immediately converted to dopachrome (lambda max = 475 nm).     

Comparative Mössbauer and infrared analysis of iron-containing melanins

A Mössbauer study was carried out on some types of melanin. ⁵⁷Fe³⁺ was bound to melanin from sepia ink and to that obtained by autooxidation of l-DOPA and dopamine. Mössbauer spectra, performed in parallel with infrared measurements, indicated that paracrystalline subunits are present, showing a superparamagnetic behaviour. The preferential binding sites for iron are different in melanins having l-DOPA as precursor from those obtained starting from dopamine. In particular, the linkage to carboxylic groups in predominant in the former, and linkage to phenolic, aminic and/or indolic groups can be observed in the latter. The results also confirm the close similarity between natural sepia melanin and that obtained from an l-DOPA precursor.     

A Potential Benefit of Albinism in Astyanax Cavefish: Downregulation of the oca2 Gene Increases Tyrosine and Catecholamine Levels as an Alternative to Melanin Synthesis

Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.