Evidence for melanogenesis in the retinal pigment epithelium of adult cattle and golden hamster.

1. The ultrastructure of the retinal pigment epithelium (RPE) of adult Syrian golden hamsters and cattle was examined with respect to pigment granules and phagosomes involved in degradation of disk membranes from rod outer segments. 2. In the RPE of cattle, phagosomes were found that contained an electron-dense melanin-like material that was not autofluorescent and therefore not lipofuscin. 3. Disk membranes of rods are about 4 nm thick and become enlarged (7-20 nm) and electron-dense during degradation in the RPE. 4. Additionally electron-dense vesiculo-globular bodies (10-100 nm) were found in phagosomes during disk membrane degradation and in mature melanin granules. 5. In the RPE of adult hamsters that had been exposed to intense light, premelanosomes containing unmelanised filaments with a striated periodicity were found in the cytoplasm or in association with mature melanin granules. Early and late stage melanosomes were also present. Phagosomes in the RPE contained degraded disk membranes, melanin-like material and melanofilaments. 6. Dopa oxidase was detected ultrastructurally within shed disk membranes that were in close contact with the microvilli of the RPE. 7. The possibility of melanogenesis within phagosomes during disk membrane degradation is discussed.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.     

Fine structure of melanocytes and macrophages in the Harderian gland of the mouse

The presence of dendritic cells containing melanin granules has been demonstrated employing silver impregnation and electron microscopy in the interstitial tissue of the Harderian gland of the mouse. Two types of melanocytes, either with or without the various developmental stages of melanin granules, were found in the gland. Cells with developing granules were more dendritic and contained a large number of cytoplasmic organelles. The other cells were ellipsoidal or slender in shape and contained few cytoplasmic organelles and a large number of fully melanized granules, but no developing granules. In general, the granules of the Harderian gland melanocytes resembled granules from other organs (particularly the skin of the eyelids). The general size range of the granules was 0.2-0.9 micron. Each granule was enclosed by a membrane. The Harderian gland macrophages contained fully pigmented melanin granules of various sizes. The granules were enclosed by a membrane either singly or in groups. Some of the melanin granules within the phagosomes showed signs of degradation, revealing the underlying matrix.     

Transport of Endocytosed Material Into Melanin Granules in Cultured Choroidal Melanocytes of Cattle—New Insights Into the Relationship of Melanosomes With Lysosomes

Cultured choroidal melanocytes from cattle were incubated with gold labeled albumin. After phagocytosis of the labeled protein, the label appeared inside the melanin granules, as was observed under the electron microscope. Melanin granules associated with gold particles were also exocytosed into the culture medium by the melanocytes. The results of this study show that endosomes or phagosomes are transported from the cell surface of a melanocyte to the melanin granules. Therefore, melanin granules are part of the lysosomal degradation pathway. The possibility that albumin is degraded by proteases present in lysosomes and melanosomes and that the tyrosine released during degradation is used as substrate by tyrosinase and thereby converted to melanin is discussed. The present study additionally shows that the choroidea of cattle can be used as a source for cell culture of melanocytes.     

Skeletal muscle–melanocyte association during tadpole tail resorption in a tropical frog,Clinotarsus curtipes Jerdon (Anura,Ranoidea)

We tested the hypothesis that melanin has a role as a molecule within the thyroid-mediated cascade. Light microscopic and ultrastructural changes in the skeletal muscle during tail resorption in tadpoles of the tropical frog Clinotarsus curtipes Jerdon (Anura: Ranoidea) were observed. Light microscopic analysis at metamorphic stage XVIII showed a melanized epidermis. A gradual migration of melanocytes from the epidermis to the dermis and filopodia of melanocytes pervading the skeletal muscle preceded tail resorption. The invasion of melanocytes into the muscle bundles coincided with the breakdown of the muscle bundles into sarcolytes and the arrival of macrophages at this site. This would suggest that the melanocyte–sarcolyte association signals the arrival of macrophages at these sites as metamorphosis progressed. Melanophages, macrophages with melanin granules, were observed at the climax stage of XXIII. The sarcolytes and the melanin granules were phagocytosed by macrophages so as to completely cleanse the exocytic muscle debris and the melanin granules. The presence of large melanomacrophage centers in the tadpole liver at metamorphic climax suggests that these phagocytic macrophages were further processed in the liver and, likely, in the spleen. It is proposed that melanin, a byzantine molecule, has a role in the cascade of events leading to tail resorption in anuran tadpoles.     

Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.     

Melanin granule metabolism in the iris and retinal pigment epithelium in adult tritions after completion of eye regeneration

It was concluded that the newly synthesized melanin granules were replaced in the pigmented tissues of the newt eye on the basis of redistribution of the cells of pigment epithelium of retina and iris labelled by 3H-DOPA 2.5 and 6.5 months after the isotope injection. The replacement of melanin granules and, correspondingly, melanin synthesis proceed more actively in the peripheral zones of the pigment epithelium of retina. The depigmentation of cells preceding the melanin synthesis appears to be realized with the participation of macrophages.     

Current Understanding on the Role of Retinal Pigment Epithelium and its Pigmentation

Retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that is strategically placed between the rod and cone photoreceptors and the vascular bed of the choriocapillaris. It has many important functions, such as phagocytic uptake and breakdown of the shedded photoreceptor membranes, uptake, processing, transport and release of vitamin A (retinol), setting up the ion gradients within the interphotoreceptor matrix, building up the blood-retina barrier, and providing all transport from blood to the retina and back. This short review focuses on the role of the pigment granules in RPE. Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of zinc and drugs, and, therefore, serve as a versatile partner of the RPE cell. Melanin plays a role in the development of the fovea and routing of optic nerves. New findings show that the melanin granules are connected to the lysosomal degradation pathway. Most of these functions are not yet understood. Deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness.     

Current understanding on the role of retinal pigment epithelium and its pigmentation.

Retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that is strategically placed between the rod and cone photoreceptors and the vascular bed of the choriocapillaris. It has many important functions, such as phagocytic uptake and breakdown of the shedded photoreceptor membranes, uptake, processing, transport and release of vitamin A (retinol), setting up the ion gradients within the interphotoreceptor matrix, building up the blood-retina barrier, and providing all transport from blood to the retina and back. This short review focuses on the role of the pigment granules in RPE. Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of zinc and drugs, and, therefore, serve as a versatile partner of the RPE cell. Melanin plays a role in the development of the fovea and routing of optic nerves. New findings show that the melanin granules are connected to the lysosomal degradation pathway. Most of these functions are not yet understood. Deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : II. Studies on the Mechanism of Formation of Autophagic Vacuoles Produced by Chloroquine

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1–2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.     

An electron microscopical study on the genesis of lipofuscin, melanin and haemosiderin in the haemopoietic tissues of fish

The mode of origin of the pigments within the macrophages of the haemopoietic tissues of some fish species was studied with the electron microscope. Lipofuscin appears to be derived from damaged cellular components, such as effete mitochondria, through the peroxidation of their unsaturated lipids. Haemosiderin is almost certainly derived from the breakdown of haemoglobin from effete erythrocytes. Melanin appears to be derived from phagocytosis of melanin granules or their precursor organelles from melanin-containing cells. Both lipid peroxidation and haemoglobin breakdown produce free radicals and cations which are potentially toxic. Melanin absorbs free radicals and has strong affinity for cations and it is probable that they are neutralized by the melanin in macrophages. The electron micrographs published here illustrate the association of the lysosomal apparatus with pigment formation in fish melano-macrophages. These findings appear valid for all the species examined and may apply to all fish. It has been suggested that fish melano-macrophage centres represent primitive analogues of the germinal centres of higher animals. This study reveals that melanocyte-like cells outnumber melano-macrophages in the kidney of rainbow trout, Salmo gairdneri. Moreover, like melano-macrophages, these cells increase markedly in number during starvation.     

A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. therefore, the melanosome is a specialized lysosome.     

Colourful parrot feathers resist bacterial degradation

The brilliant red, orange and yellow colours of parrot feathers are the product of psittacofulvins, which are synthetic pigments known only from parrots. Recent evidence suggests that some pigments in bird feathers function not just as colour generators, but also preserve plumage integrity by increasing the resistance of feather keratin to bacterial degradation. We exposed a variety of colourful parrot feathers to feather-degrading Bacillus licheniformis and found that feathers with red psittacofulvins degraded at about the same rate as those with melanin and more slowly than white feathers, which lack pigments. Blue feathers, in which colour is based on the microstructural arrangement of keratin, air and melanin granules, and green feathers, which combine structural blue with yellow psittacofulvins, degraded at a rate similar to that of red and black feathers. These differences in resistance to bacterial degradation of differently coloured feathers suggest that colour patterns within the Psittaciformes may have evolved to resist bacterial degradation, in addition to their role in communication and camouflage.     

Melanin granule in skin window macrophages.

Pigment granules have been studied in macrophages of skin window preparations. These granules usually appeared blue with Romanowsky stains, and stained positively for melanin but negatively for iron. There were significantly more pigment granules per macrophage in sun-tanned individuals and in coloured subjects and in the latter, the granules were usually larger and darker. Presumably, the source of the melanin is damaged or degenerating pigment cells of the skin and reflects a normal in vivo phenomenon.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Ultrastructural study on hepatic melanin in Xenopus laevis

Summary The livers of Xenopus laevis, grouped by chronological age (0.5,2 and 3 yrs), were studied electron microscopically. Ultrastructurally most of the melanin granules in the mature female liver showed an-internal structure similar to the melanin granules of the oocytes. The hepatic melanin granules of immature females and of all males were pleomorphic and failed to show the characteristic internal structure similar to those of the oocytes. The oocyte is the probable source of most of the hepatic melanin of the mature female.     

Melanogenesis in dermal melanocytes of Japanese Silky chicken embryos

The Japanese Silky chicken (SK) shows dermal and visceral hyperpigmentation. This study characterizes ultrastructurally the melanin granules developing in dermal melanocytes of the dorsal skin of SK, in an attempt to better understand the processes of melanogenesis in these permanently ectopic cells. The steps of melanogenesis are similar to those described for epidermal melanocytes, with melanosomes going from stage I to IV but, in SK, the maturation occurs in the cell body, as well as in the cytoplasmic processes. At stage III, the deposition of melanin is cumulative and can aggregate in rounded structures, which combine to turn into the mature granule. The final destiny of mature melanosomes is still unclear, although it was observed that dermal macrophages can accumulate melanin granules in their phagosomes. Even with the close proximity between melanocytes and other dermal cells, the transference of melanosomes was not observed. Our findings indicate that melanogenesis in dermal melanocytes in SK has the same morphological characteristics found in epidermal melanocytes, but the functional aspect still remains to be elucidated.     

Synthesis and assembly of fungal melanin

Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.     

Melanin endocytosis by cultured mammalian cells : A model for melanin in a cellular environment

The incorporation of natural eumelanin from bovine eyes and synthetic 3,4-dihydroxy-phenylalanine (dopa) melanin into Chinese hamster ovary (CHO) cells is reported. The process is linear for at least 8 h. Electron microscopy showed phagocytosis of melanin, either as a single granule or in groups of granules, into cell lysosomes with subsequent degradation of the granule. The general features of the ingestion and degradation processes mimic those of the incorporation of melanosomes into keratinocytes. CHO cells with ingested melanin in general revealed properties very similar to those of the pigment-free CHO cell: cell division, oxygen consumption and plating efficiency were not greatly altered by moderate concentrations of pigment. This suggests that the CHO cell system may be useful for the study of pigment in a cellular environment; pigment-free CHO cells are well characterized and can serve as a good control. Preliminary applications are reported: demonstrations of (1) incorporation of metal ions (Al3+) into CHO cells using melanin as a carrier; (2) the ability of melanin to enhance the rate of oxygen consumption during photo-irradiation of the cells.     

The role of melanin in the antagonistic interaction between the apple scab pathogen Venturia inaequalis and Microsphaeropsis ochracea

The objective of this study was to examine the role of melanin in the interaction between the mycoparasite Microsphaeropsis ochracea and the apple scab pathogen Venturia inaequalis. Melanin was extracted from the cell wall of the pathogen and its chemical and physical properties determined on the basis of biochemical tests and visible and infrared spectra. The physical and chemical characteristics of V inaequalis melanin were similar to the those of synthetic dihydroxyphenylalanine (DOPA) melanin. Precursors of the four known melanin biosynthetic pathways were tested for their ability to restore the pigmentation of an albino strain of V inaequalis. Scytalone, an intermediate of the 1,8-dihydroxynaphthalene (DHN) pathway, was the only precursor to restore the dark-brown pigmentation. Tricyclazole and pyroquilon, two antipenetrant fungicides, specific inhibitors of DHN melanin synthesis in Pyricularia oryzae, were used to confirm the melanin pathway in V. inaequalis wild type. A reddish-brown pigment was obtained due to the accumulation of shunt products of the DHN melanin pathway instead of a dark-brown pigment, suggesting that the melanin extracted from V inaequalis was a DHN melanin. Furthermore, growth of an albino mutant of V. inaequalis on scytalone-amended medium resulted in the formation of dark granules similar to those seen in wild-type isolates. Transmission electron microscopic observations of M. ochracea grown in the presence of melanin showed that the granules accumulated gradually along fungal cell walls to form a uniform dark coating.