Experimental study of the radioprotective action of melanin on the somatic development during irradiation in the antenatal period of ontogenesis

Daily introduction per os of the exogenous melanin in a doze of weight of 10 mg/kg pregnant female rats Wistar eliminated the functional deficiency of somatic development revealed at posterity at chronic gamma-irradiation in a doze 1.00-1.25 Gy for all period of pregnancy. The irradiation or introduction melanin antenatal to a phase ontogenesis resulted in stimulation of the immune answer, which was determined at offspring on 3rd week after birth. On the basis of the received data it is concluded presence radioembryoprotective actions melanin in the relation embryotoxic effects of small dozes of ionizing radiation and its participation in regulation immunogenes.     

Fluorescent quantification of melanin

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non‐melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

Applications of affinity chromatography to the study of drug-melanin binding interactions

This short review reports on progresses in the study of drug-melanin interactions using the technique of affinity chromatography. Melanins are natural or synthetic pigments derived from the oxidation and polymerization of various precursors including L-dopa, tyrosine and cystein. Accumulation of toxic compounds, drugs, and metal ions in pigmented tissues through reversible binding to melanin has been linked to chronic toxicity. Affinity chromatography using chromatographic stationary phases based on physically adsorbed or chemically bonded melanin provides a useful tool for studying the interactions of small molecules and metal ions with melanin     

Interference of melanin in protein determination

Using several assay methods, synthetic eumelanin prepared by autooxidation of L-beta-(3,4-dihydroxyphenyl)alanine and a natural melanin isolated from dog hair melanosomes were tested in model experiments to assess their possible interference in protein determination. The degree of interference was assessed by comparing the data obtained with the melanin samples with those derived from measurements of bovine serum albumin. In the common biuret and Lowry methods melanin interferes by falsely increasing the values obtained; the addition of Folin reagent only after melanin removal, as suggested by Doezema, decreased but did not eliminate melanin interference. Methods working at acid pH such as those according to Salo and Honkavaara with Ponceau S or Sedmak and Grossberg or Spector using Coomassie blue G-250 proved much better. Although melanins adsorbed a small amount of dye from the reaction systems in these procedures, their sensitivity to proteins makes the melanin interference negligible. Such procedures can therefore be recommended for protein determination in the presence of melanin.     

Abnormally high variability in the uncrossed retinofugal pathway of mice with albino mosaicism

Female mice showing albino mosaicism due to an X-autosome translocation [Is(In7;X)Ct] have been studied in order to investigate the relationship between the distribution of melanin and the formation, early in development, of the abnormally small uncrossed retinofugal pathway characteristically found in all albino mammals. Earlier evidence indicates that cells normally bearing melanin play a role in producing the abnormality. In the mosaic mice, the albino gene is expressed in only about half of the cells due to random X-inactivation and the patches of normal and albino cells are extremely small relative to total retinal size (less than 1/50). We argued that if all the cells that would normally bear melanin play a role in producing the albino abnormality then the mosaic mice would have a pathway abnormality, about half the size of that in the albino mice. If, however, only a small patch of these cells plays a role, as has been proposed in earlier studies, then one would expect the size of the uncrossed pathway to be highly variable in the mosaic mice. The size of the uncrossed pathway was assessed by placing horseradish peroxidase in the region of the optic tract and lateral geniculate nucleus unilaterally and then counting the number of retrogradely labelled retinal ganglion cells on the same side. The mosaic mice showed a highly variable uncrossed pathway. In some of the mosaic mice, it was the same size as in the albinos and, in others, it was the same size as in normally pigmented mice. Surprisingly, in a small number of mosaic mice, the uncrossed pathway was larger than normal. Whether this relatively rare occurrence of a supernormal uncrossed pathway is due to the higher gene dosage or to the translocation itself remains an open question.     

Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus

An optical, ultrastructural, and biochemical study of the melanin accumulation nodules found in the kidney of the teleost fish Sparus auratus is presented. These nodules are randomly distributed in the interstitium of the renal tissue. They are formed by large aggregates of cells replete with melanin granules. The melanin granules occur singly or also in aggregates inside the cells. Most of the granules are electron-dense, but sometimes small electron-lucent spaces within them can be seen. Some secondary lysosomes and dendritic processes can also be observed. Biochemical studies have proved for the first time the existence of measurable tyrosinase activity in those nodules. That activity was assayed using three methods: tyrosine hydroxylation, dopa oxidation, and melanin formation. Furthermore, inhibitors of well-characterized plant and animal skin tyrosinases were effective agents for inhibiting those activities in fish kidney preparations. This finding supports the notion of the existence of true tyrosinase in the melanin accumulation nodules of this tissue. Taking into account the results obtained, the origin and functions of the melanin-containing cells found in the teleost fish kidney are discussed.     

Fluorescence spectrophotometric analysis of melanins in the house mouse

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (A ^y /a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.This work was supported by Grant 244004 from the Ministry of Education. Part of this work was presented at the X International Pigment Cell Conference.     

Primary malignant melanoma of the duodenum without visible melanin pigment: a mimicker of lymphoma or carcinoma

ABSTRACT: Primary malignant melanoma of the duodenum is an unusual oncologic entity. Patients usually present the similar clinical symptoms like other common tumors in this site. And there are no specific radiological features either. The cases with only little melanin pigment or without notable melanin pigment are very misleading, especially in small biopsies or frozen sections. Definite diagnosis depends on both careful histologic examination and the use of proper immunohistochemical stains. Moreover, detailed history and thorough investigation should be made to exclude the preexistence or coexistence of a primary lesion elsewhere. Herein we report the case of a 60-year-old male patient with primary malignant melanoma of the duodenum, which was misdiagnosed as lymphoma or undifferentiated carcinoma in frozen consultation. The patient had achieved disease-free survival for more than 46 months without any evidence of recurrence after surgery. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1221457317710561.     

银耳菌糠黑色素的理化性质及抗氧化和抑菌活性

银耳菌糠中存在一种银耳的伴生菌——炭团菌(俗称香灰菌),其菌丝生长到一定阶段后会产生大量的黑色素,具有广泛的应用价值。本研究从银耳菌糠中提取黑色素,研究其理化性质、抗氧化活性及抑菌作用。通过紫外-可见光谱、傅里叶红外光谱对提取的黑色素进行鉴定,表明银耳菌糠黑色素具有黑色素的典型特征。通过对银耳菌糠黑色素理化性质的研究,表明银耳菌糠黑色素是一种趋于黑色并略带红色和黄色的粉末;该黑色素易溶于碱性溶液;具有较好的热稳定性和光稳定性,其稳定性受氧化剂和还原剂的影响较小,受Ca²⁺、Cu²⁺的影响较明显。通过总抗氧化能力(FRAP法)、自由基清除能力检测银耳菌糠黑色素的抗氧化活性,研究表明黑色素具有较高的抗氧化活性,羟自由基、ABTS自由基清除的EC₅₀值分别为0.429 mg/mL和0.016 mg/mL。本研究还检测了黑色素对革兰氏阳性、革兰氏阴性细菌的抑菌能力,结果表明该黑色素在浓度为3.2 mg/mL时对供试菌株的抑菌率超过90%,且对革兰氏阳性菌会较敏感。本研究为银耳菌糠的有效利用及其黑色素产品的开发提供了理论基础,具有较高的经济价值。     

纤维素酶-超声波协同提取黑木耳黑色素工艺及其抗氧化活性分析

黑色素是黑木耳的主要活性成分之一,在黑木耳的药理活性上发挥着重要作用。为了提高黑木耳黑色素的提取得率,实验采用正交法和响应面法对纤维素酶-超声波协同提取黑木耳黑色素的提取工艺进行了优化,并对最优条件下提取的黑木耳黑色素体外抗氧化活性进行了分析。实验结果表明,纤维素酶-超声波协同提取黑木耳黑色素的最优条件为:酶添加量12mg,酶解温度40℃,酶解pH 5.0,酶解时间120min,NaOH浓度1.27mol/L,料液比1:40,超声功率300W,超声时间52min,超声温度60℃。在最优条件下,黑木耳黑色素提取得率可达到10.48%,相比于实验设置的未添加纤维素酶的超声波组黑色素提取得率提高了12.93%。抗氧化结果表明,采用纤维素酶-超声波协同提取的黑色素相比于未加纤维素酶提取的黑色素清除ABTS、DPPH和羟基自由基的能力更强。研究结果为黑木耳黑色素的高效提取及其产品的应用开发奠定了基础。     

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.     

Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.     

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.     

Convergent, modular expression of ebony and tan in the mimetic wing patterns of Heliconius butterflies

The evolution of pigmentation in vertebrates and flies has involved repeated divergence at a small number of genes related to melanin synthesis. Here, we study insect melanin synthesis genes in Heliconius butterflies, a group characterised by its diversity of wing patterns consisting of black (melanin), and yellow and red (ommochrome) pigmented scales. Consistent with their respective biochemical roles in Drosophila melanogaster, ebony is upregulated in non-melanic wing regions destined to be pigmented red whilst tan is upregulated in melanic regions. Wing regions destined to be pigmented yellow, however, are downregulated for both genes. This pattern is conserved across multiple divergent and convergent phenotypes within the Heliconii, suggesting a conserved mechanism for the development of black, red and yellow pattern elements across the genus. Linkage mapping of five melanin biosynthesis genes showed that, in contrast to other organisms, these genes do not control pattern polymorphism. Thus, the pigmentation genes themselves are not the locus of evolutionary change but lie downstream of a wing pattern regulatory factor. The results suggest a modular system in which particular combinations of genes are switched on whenever red, yellow or black pattern elements are favoured by natural selection for diverse and mimetic wing patterns.     

Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response

Melanin’s influence on the chromosome aberration frequency induced by radiation in human lymphocytes and mouse bone marrow cells has been studied. We revealed earlier that melanin significantly decreases the frequencies of different radiation-induced mutations in animal germ cells. Melanin protection in somatic cells has been found to be less effective. The melanin effect in somatic cells depends on radiation dose: the lower the damage level, the better the melanin protection. In order to determine the influence of melanin at low radiation doses, the adaptive response was investigated in mouse bone marrow cells in vivo. The level of chromosome aberrations in these cells after fractionated irradiation of 0.2 Gy+1.5 Gy with a 4-h interval was about half that after a single dose of 1.7 Gy. If melanin was injected prior to irradiation, the aberration level decreased by a factor of about two in both cases. This observed result may be due to the potential radioprotective effect of melanin and to the absence of any adaptive response, whereas in the case of melanin application between the priming and challenge doses, the combined effect of the adaptive response as well as melanin protection resulted in a 4-fold decrease of chromosome aberrations. These results allow us to draw the following conclusions: adaptive response can be prevented by a radioprotector such as melanin, and melanin is capable of completely removing low-dose radiation effects. Received: 2 December 1998 / Accepted in revised form: 15 September 1999     

Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns

Body coloration affects how animals interact with the environment. In insects, the rapid evolution of black and brown melanin patterns suggests that these are adaptive traits. The developmental and molecular mechanisms that generate these pigment patterns are largely unknown. We demonstrate that the regulation and function of the yellow and ebony genes in Drosophila melanogaster play crucial roles in this process. The Yellow protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. Conversely, Ebony is required to suppress some melanin formation, and is expressed in cells that will produce both melanized and non-melanized cuticle. Ectopic expression of Ebony inhibits melanin formation, but increasing Yellow expression can overcome this effect. In addition, ectopic expression of Yellow is sufficient to induce melanin formation, but only in the absence of Ebony. These results suggest that the patterns and levels of Yellow and Ebony expression together determine the pattern and intensity of melanization. Based on their functions in Drosophila melanogaster, we propose that changes in the expression of Yellow and/or Ebony may have evolved with melanin patterns. Consistent with our hypothesis, we find that Yellow and Ebony are expressed in complementary spatial patterns that correlate with the formation of an evolutionary novel, male-specific pigment pattern in Drosophila biarmipes wings. These findings provide a developmental and genetic framework for understanding the evolution of melanin patterns.     

异齿裂腹鱼仔鱼的黑色素分布

本文运用组织学方法对异齿裂腹鱼仔鱼发育过程中除眼睛以外的黑色素分布进行研究。结果表明: 异齿裂腹鱼孵化出膜后,黑色素在各个组织器官的出现顺序为: 脑颅外膜→围心腔、背部皮肤→腹腔内膜、脊髓腔。其中,出膜5 d时脑颅外膜处出现黑色素;7 d时围心腔和背部皮肤出现黑色素;10 d时腹腔内膜、脊髓腔出现黑色素。异齿裂腹鱼出膜10 d后,其皮肤和内脏器官(脑颅、围心腔、腹腔内膜、脊髓腔)均具有黑色素,且主要分布在背侧。黑色素在异齿裂腹鱼仔鱼发育过程中的出现时序和分布规律可能与其所处的强紫外辐射环境有关。本研究可为研究鱼类黑色素的紫外防护机制提供参考,并为今后高原鱼类仔鱼的培育条件优化提供理论支撑。     

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.