Three-Dimensional Analysis of Melanosomes Isolated from B16 Melanoma Cells by Using Ultra High Voltage Electron Microscopy

Melanosomes, isolated by centrifugal separation from culture broth of B16 melanoma cells derived from mouse, were observed by scanning electron microscopy (SEM), and by transmission electron microscopy (TEM). Some interesting structural features were found inside and outside of the melanosomes. By SEM observation, the melanosomes were ellipsoid shape, their surface was not smooth and was covered with rough substructure, 10 to 20 nm particles. By TEM, uneven structure and micro particles were observed in the melanosomes. Furthermore, three-dimensional analysis was tried by using the ultra-high voltage electron microscopy(UHVEM). Micrographs of the melanosomes were taken at various tilted angles by UHVEM, after preparing 500 nm thickness specimens stained with lead citrate. From the micrographs collected, the three-dimensional structures were reconstructed by using i-mode software. Melanin stained by lead and non stained parts was clearly observed in the reconstructed structure. Non stained parts were round, regular size, and distributed widely in the melanosomes.

Significance of aberrant melanosomes in the diagnosis of malignant melanoma

In order to clarify the significance of the aberrant melanosomes in thediagnosis of malignant melanoma,17 cases of malignant melanoma were studied with elec-tron microscopy,and 30 cases of nevus were studied likewise for comparison.Aberrantmelanosomes were present in 12 cases of pigmented and 2 cases of amelanotic malignantmelanoma.They were also found in 4 cases of congenital nevus.The findings suggest thatthe presence of aberrant melanosomes is of significance for the diagnosis of malignantmelanoma but this has been overemphasized.It is believed that the diagnosis of malignantmelanoma depends upon a comprehensive judgement of all the ultrastructural findings un-der electron microscopy.

Epimedin B exhibits pigmentation by increasing tyrosinase family proteins expression,activity,and stability

Epimedin B(EB)is one of the main flavonoid ingredients present in Epimedium brevicornum Maxim.,a traditional herb widely used in China.Our previous study showed that EB was a stronger inducer of melanogenesis and an activator of tyrosinase(TYR).However,the role of EB in melanogenesis and the mechanism underlying the regulation remain unclear.Herein,as an extension to our previous investigation,we provide comprehensive evidence of EB-induced pigmentation in vivo and in vitro and elucidate the melanogenesis mechanism by assessing its effects on the TYR family of proteins(TYRs)in terms of expression,activity,and stability.The results showed that EB increased TYRs expression through microphthalmia-associated transcription factor-mediated p-Akt(referred to as protein kinase B(PKB))/glycogen synthase kinase 3β(GSK3β)/β-catenin,p-p70 S6 kinase cascades,and protein 38(p38)/mitogen-activated protein(MAP)kinase(MAPK)and extracellular regulated protein kinases(ERK)/MAPK pathways,after which EB increased the number of melanosomes and promoted their maturation for melanogenesis in melanoma cells and human primary melanocytes/skin tissues.Furthermore,EB exerted repigmentation by stimulating TYR activity in hydroquinone-and N-phenylthiourea-induced TYR inhibitive models,including melanoma cells,zebrafish,and mice.Finally,EB ameliorated monobenzone-induced depigmentation in vitro and in vivo through the enhancement of TYRs stability by inhibiting TYR misfolding,TYR-related protein 1 formation,and retention in the endoplasmic reticulum and then by downregulating the ubiquitination and proteolysis processes.These data conclude that EB can target TYRs and alter their expression,activity,and stability,thus stimulating their pigmentation function,which might provide a novel rational strategy for hypopigmentation treatment in the pharmaceutical and cosmetic industries.

The mechanisms of color production in black skin versus red skin on the heads of New World vultures

A determination of how the color of animal integument is produced is a starting point for investigations into the function and evolution of coloration.The mechanisms that give rise to the color of bare skin of New World vultures are largely unexplored.Here,we investigate the source of color production in the bare skin of Turkey Vultures(Cathartes aura)and Black Vultures(Coragyps atratus).Using UV-vis reflectance spectroscopy,we found evidence that hemoglobin is the primary pigment responsible for the red coloration of the bare skin on the heads of Turkey Vultures,and that eumelanin is responsible for the black coloration of the bare skin on the heads of Black Vultures.Light microscopy of incisional skin samples further supported these mechanisms of color production by revealing the presence of numerous blood vessels near the surface of the Turkey Vulture skin,and a high concentration of melanosomes in the skin of Black Vultures.Using high performance liquid chromatography(HPLC),we detected carotenoids within the skin of both species with significantly higher total concentrations of carotenoids in the skin of Turkey Vultures compared to the skin of Black Vultures.The carotenoids detected were dietary carotenoids that typically produce yellow coloration when accumulated in integument and were present in low concentrations.We hypothesize that the dietary carotenoids present do not contribute to the color of the skin,but rather help to compensate for the lack of melanosomes found in Turkey Vulture skin.The presence of additional carotenoids may act as an antioxidant to minimize UV damage when the bare Turkey Vulture head skin is exposed to direct sunlight for prolonged periods of time when soaring and scavenging for food.

Pigment Distribution and Secretion in the Mantle of the Pacific Oyster(Crassostrea gigas)

The color of Mollusca shells is one of the most important attributes to consumers.At the cellular level,black color is mainly from the melanin produced by melanocytes.The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis,storage,and transportation.How the complex pigmentation process in the Crassostrea gigas is established remains an open question.The objectives of this studies are to examine the morphological characteristics of melanosomes or melanin of mantle pigmentation in the Pacific oyster,thereby investigating its contribution to shell color.The results show that pigmented granules of the mantles vary among the three lobes,and the melanosomes at different stages are enriched in distinct cargo molecules,which indicate the remarkable difference between the marginal mantle and central mantle.Examination of mantle histology reveals that the mantle margin of the oyster is characterized by three different folds,including the outer secretory,middle sensory,and inner muscular fold.Ferrous ion chelating assays against the tyrosine hydroxylase indicate that a large amount of melanin is localized in the inner surface of the middle fold.Transmission electron microscopy analyses show that the mantle edge is composed of tall columnar and cuboidal epidermal cells and some pigmented melanocytes intersperse among these cells.The numbers of melanosomes among the three lobes are different.In the inner fold and the middle fold of the mantle,some single dispersion,or aggregation of melanosomes with different degrees of melanization are found in the outer surface.Numerous melanosomes are distributed in the epithelium of the outer fold of the mantle,and mainly are at the apical microvillar surface near the lumen.However,melanosomes are occasionally observed in the central mantle,and they are relatively less.This work provides new insights into the process of melanin deposit in the mantle and shell pigmentation in C.gigas.

Characteristics of normal human retinal pigment epithelium cells with extremes of autofluorescence or intracellular granule count

Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation.High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration(AMD);however,this has not been confirmed in histology so far.Here,based on our previous dataset of RPE granule characteristics,we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence(AF)intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy(HR-SIM)and laser scanning microscopy(LSM).Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules.In addition,total AF intensity per cell and granule density(number of granules per cell area)were determined.For the final analysis,RPE cells with total granule number below 5th or above the 95th percentile,or a total AF intensity±1.5 standard deviations above or below the mean were included,and compared to the average RPE cell at the same location.Data are presented as mean±standard deviation.Results:Within 420 RPE cells examined,42 cells were further analyzed due to extremes regarding total granule numbers.In addition,20 RPE cells had AF 1.5 standard deviations below,28 RPE cells above the mean local AF intensity.Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity.RPE cells with high granule content have nearly twice(1.8 times)as many granules as an average RPE cell.Conclusions:In normal eyes,outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells,suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level.The AF of a cell is related to the composition of intracellular granule types.Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscin´s role in aging and AMD further into perspective.

Differences in MITF gene expression and histology between albino and normal sea cucumbers (Apostichopus japonicus Selenka)

Albino Apostichopus japonicus occur both in the wild and in captivity. The offspring of albino A. japonicus also suffer from albinism. The formation of melanin in the melanocytes is dependant on microphthalmia-associated transcription factor (MITF). To investigate the role of MITF in controlling albinism, we cloned the full-length MITF cDNA from A. japonicus and compared MITF mRNA expression in albino and normal A. japonicus. In addition, we used light and electron microscopy to compare histological samples of normal and albino A. japonicus. The body wall of albino adults was characterized by significantly lower levels of MITF expression and lower numbers of epidermal melanocytes, which also contained less melanin. In albino juvenile offspring, MITF expression levels were significantly lower 32 d after fertilization and there were fewer, and less developed, epidermal melanocytes. Thus, we conclude that albino A. japonicus have fewer melanocytes and a reduced ability to synthesize melanin, likely because of lower expression of MITF.

Ultrastructure of Amelanotic Melanocytes from Human Hair Follicles

To investigate the ultra structure of amelanotic melanocytes （AMMC）. Methods： The hair follicles obtained from normal human scalp by 0.50% collagenase type V treatment were washed with 0.1 mol/L phosphate buffer salt （PBS）. Hair-follicle cell suspensions were prepared by trypsin treatment and cultured in melanocyte medium. Remaining keratinocytes were removed by differential trypsinization. 100μg/ml geneticin was used to eliminate the contaminating fibroblasts. At third passage, the cells were trypsinized, and then washed in phosphate-buffered saline and processed for transmission electron microscopy. Results： Under transmission electron microscope, the cultured cells showed round or oval shape, with single large nuclear and the karyotheca were double deck. There were obvious euchromosome within the nucleus, and sparse heterochromosome. There were various organelles in the cytoplasm, including plentiful melanosomes with nearly similar size, mitochondria, rough endoplasmic reticule （RER） and ribosome. The electron density granules in most of the melanosomes disposed along concentric circularities. Golgi apparatus in the cells was inconspicuous. Conclusion： The ultra structure of AMMC from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional immature of AMMC.

Effect of Hoechunyangkyeok-San Extract on Melanogenesis

Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.

Examination of wnt signaling mediated melanin transport and shell color formation in Pacific oyster(Crassostrea gigas)

Mollusca exhibit remarkable diversity in shell coloration,attributed to the presence of melanin,a widely distributed pigment with various essential roles,such as mechanical strengthening,antioxidation and thermoregulation.However,the regulatory network governing melanogenesis and melanin transport in molluscs remains poorly understood.In this study,we conducted a systematic analysis of melanin distribution and transport in the Pacific oyster,utilizing light microscopy and high-resolution transmission electron microscopy.In addition,we characterized CgWnt1 and CgWnt2b-a in Crassostrea gigas,and analyzed Wnt signaling in melanocyte formation.Expression analysis revealed that these genes were predominantly expressed in the mantle of black-shelled individuals,particularly in the outer fold of the mantle.Furthermore,we employed RNA interference and inhibitors to specifically inhibit Wnt signaling in both in vivo and in vitro.The results revealed impaired melanogenesis and diminished tyrosinase activity upon Wnt signaling inhibition.These findings suggest the crucial role of Wnt ligands and downstream factors in melanogenesis.In summary,our study provides valuable insights into the regulatory mechanism of shell pigmentation in C.gigas.By demonstrating the promotion of melanogenesis through Wnt signaling modulation,we contribute to a better understanding of the complex processes underlying molluscan melanin production and shell coloration.