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.     

Detection of a Fine Lamellar Gridwork After Degradation of Ocular Melanin Granules by Cultured Peritoneal Macrophages

In the present study we investigated by electron microscopy whether melanin granules derived from choroidal melanocytes and retinal pigment epithelium of cattle could be degraded in the phagolysosomes of cultured murine macrophages. It was found that degradation of ocular melanin is possible by the lysosomes of these macrophages. During degradation of the melanin granules an internal gridwork of fine concentric, highly ordered membranes, 3-4 nm thick, became visible. These membranes may represent remnants of the melanin polymer in the original melanosome or may result from self-assembly of degradation products. Early-stage melanosome-like structures also appeared during digestion of these melanin granules. Melanin granules that seemed to break down into smaller fragments without any visible internal structure were also observed.     

Does Melanin Turnover Occur in the Eyes of Adult Vertebrates?

This paper is a review of what is known about the turnover of melanin in iris, choroid, and retinal pigment epithelium (RPE) of the adult vertebrate eye. Differences in size and structure of choroideal and retinal pigment epithelial melanin granules are shown by electron micrographs. The classical stages of melanin synthesis, including the premelanosome, are shown in the RPE of adult hamsters that had been exposed to intense light. Degradation or synthesis of melanin also seem to occur in the melanocytes of the choroid in these animals. It is postulated that all three pigmented eye tissues (iris, RPE, and choroid) of adult vertebrates form melanin granules in vivo. However, nothing is known about the amount of this turnover.     

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.     

Microtubule motors transport phagosomes in the RPE,and lack of KLC1 leads to AMD-like pathogenesis

The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD.     

Characterization of brown film formed by Lentinula edodes

Lentinula edodes is a widely-produced mushroom in China that forms a brown film via pigment accumulation on mature mycelial surfaces to ensure high-quantity and high-quality fruiting body formation. Here, ultraviolet–visible, infrared spectra, and elemental analyses predicted that the pigment in the brown film was melanin. Electron microscopy revealed the size, morphological characteristics, accumulation, and morphogenesis of electron-dense material, which were similar to those of melanin, as well as subcellular structural changes during brown film formation. The electron-dense material appeared as granules, vesicles, and polymers. The accumulation of electron-dense materials on the cell wall was followed plasmolysis, plasma membrane disruption, electron-dense material accumulation in the interstitial space, and gradual accumulation on the outer cell wall. Dolipore septa degradation and morphogenetic cell death occurred during browning. In the final stage of browning, the dolipore septum disappeared and the cell was nearly empty. This study provides a cytological foundation for evaluating the regulation of brown film formation in L. edodes.     

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.     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

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.     

Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae

Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.     

Biogenesis of melanosomes in Kupffer cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

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.     

Ultimate Fate of Rod Outer Segments in the Retinal Pigment Epithelium

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Erythrophagocytosis and pigment cells of the amphibian liver]

The ultrastructure of Kupffer liver cells of adult frogs collected in winter and Kupffer cells during amphibian metamorphosis when larval red cells are replaced by adult red cells were investigated. It was revealed that Kupffer cells of the animals investigated had very large size and consisted of either the whole senescent erythrocyte or many phagosomes with small electron dense granules resembling ferritin. Phagosomes are oriented among numerous vesicular and vermiform profiles of agranular endoplasmic reticulum and big Golgi complex. Comparing our morphologic evidence with data of the literature that granules of melanin are synthesized and localized in frog liver Kupffer cells, we came to conclusion that pigment cells were formed as a result of erythrophagocytosis and therefore they were the depo of catabolism products. The comparison of functions of melano-macrophage centers of fish liver and of pigment cells of amphibia liver were discussed.     

The Thymic Microenvironment of the Common Sole,Solea solea

Abstract The thymus of the sole Solea solea contained lymphoblasts and thymocytes within a network of pale and dark epithelial cells. The pale cells were characterized by tonofilaments and desmosomes and some embraced rodlet cells within their cytoplasmic processes. The dark epithelial cells had numerous electron-dense inclusions and electron-lucent vacuoles. Lymphocytes were closely associated with the plasma membrane of both types of epithelial cells and with macrophages. Breakdown of effete lymphocytes appeared to be the main function of the macrophages. Some macrophages were multinucleated. Those containing melanin granules associated with phagosomes were classified as melanomacrophages. Pigment cells including melanophores and guanophores were present along the connective tissue trabeculae and surrounding the blood vessels. A few plasma cells and mucous cells were present.     

Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages

Summary In order to investigate the mechanism of synthesis and secretion of lysozyme (LZ) by human mononuclear phagocytes, the ultrastructural localization of LZ was studied by a pre-embedding direct immunoperoxidase method. Blood monocytes showed a reaction product for LZ in cytoplasmic granules, whereas cultured monocytes showed the reaction product in phagosomes as well as granules at 5 h of culture and in numerous large granules at 3 days of culture. In Kupffer cells, LZ was present in cytoplasmic granules, vacuoles and phagosomes. Some Kupffer cells showed a positive reaction for LZ in the rough endoplasmic reticulum, perinuclear cisterna and Golgi apparatus. Macrophages in the lymph nodes contained LZ in cytoplasmic granules. Bone marrow macrophages contained numerous phagosomes with electron-dense degradation products of erythrocytes, but the reaction product for LZ could not be clearly identified. The present study demonstrated that LZ is present in the granules of human mononuclear phagocytes and released into phagosomes. An in-vitro culture study, furthermore, demonstrated that macrophages produce LZ-containing large granules distinct from those of monocytes. However, findings that indicate the synthesis and secretion of LZ by cultured monocytes, as suggested previously by other investigators, were not observed in this study.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long-lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.     

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron‐induced peroxidation of lipids

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long‐lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration‐dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.