Number Of Melanosomes Research Articles

Melatonin protects the retina from experimental nonexudative age-related macular degeneration in mice.

Nonexudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. Currently, there are no available treatments for NE-AMD. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks. Several lines of evidence strongly support the involvement of oxidative stress in NE-AMD-induced retinal pigment epithelium (RPE) and outer retina damage. Melatonin is a proven and safe antioxidant. Our aim was analysing the effect of melatonin in the RPE/outer retina damage within experimental NE-AMD. The treatment with melatonin starting 48h after SCGx, which had no effect on the ubiquitous choriocapillaris widening, protected visual functions and avoided Bruch´s membrane thickening, RPE melanin content, melanosome number loss, retinoid isomerohydrolase (RPE65)-immunoreactivity decrease, and RPE and hotoreceptor ultrastructural damage induced within experimental NE-AMD exclusively located at the central temporal (but not nasal) region. Melatonin also prevented the increase in outer retina/RPE oxidative stress markers and a decrease in mitochondrial mass at 6weeks post-SCGx. Moreover, when the treatment with melatonin started at 4weeks post-SCGx, it restored visual functions and reversed the decrease in RPE melanin content and RPE65-immunoreactivity. These findings suggest that melatonin could become a promising safe therapeutic strategy for NE-AMD.

Organogold drug Auranofin exhibits anti-melanogenic activity in B16F10 and MNT-1 melanoma cells.

Auranofin (AF) is an organogold FDA-approved drug for treating rheumatism and has been repurposed for several pharmacological applications based on its anti-bacterial, anti-fungal and anti-inflammatory activities. To the best of our knowledge, there has been no study on effects of AF on melanogenesis yet. Hence, in this work, we studied the effect of AF on melanogenesis using B16F10 mouse melanoma cells and validated results in MNT-1 human melanoma cells. Melanogenesis assay was conducted with concentrations of AF determined to be nontoxic in B16F10 cells as well as HaCaT human epidermal cell line for a duration of 48h, followed by various assays to delineate mechanisms of melanogenesis inhibition. Ultrastructural analysis was conducted to study further if AF affected melanosome maturation and protein levels of a key melanogenic protein, tyrosinase, and the maturation signaling molecule, cyclic adenosine monophosphate (cAMP), was estimated. Our results demonstrate that AF at nontoxic concentrations of 0.25-1µM significantly inhibited melanin synthesis in a dose-dependent manner with significant inhibition of 32.85% at 1µM. The study ofmechanisms ofmelanogenesis inhibition revealed that AF inhibited tyrosinase activity in lysates of B16F10 cells but did not show a direct effect on purified mushroom tyrosinase activity or on copper chelation in a cell-free system, nor did it affect levels of B16F10 tyrosinase protein levels. However, AF significantly down-regulated cAMP levels, inhibited cellular ROS and increased number ofmelanosomes in immature stages, and also exhibited anti-melanogenic activity in B16F10-HaCaT cocultures. Furthermore, AF showed anti-melanogenic efficacy in MNT-1 cell monocultures and cocultures with an inhibition of intracellular tyrosinase activity. In summary, our results demonstrate a proof-of-principle for AF as a depigmenting agent for hyperpigmentation disorders and adjuvant for melanoma therapeutics.

Calebin-A, a Curcuminoid Analog Inhibits α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells

Hyperpigmentation skin disorders comprise melasma, age spots, and post-inflammatory hyperpigmentation. They are characterized by an aberrant upregulation of melanin pigment and pose a significant burden aesthetically. Calebin-A (CBA) is a natural curcuminoid analog derived from turmeric root (Curcuma longa) but, unlike curcumin, it has not been explored yet for anti-melanogenic activity. Hence, in the current study, we studied CBA for its effects on α-melanocyte stimulating hormone (αMSH)-stimulated melanogenesis in B16F10 mouse melanoma cells. Our results showed that CBA (20 μM) significantly suppressed αMSH-stimulated melanogenesis after 48 h treatment. The underlying mechanisms of CBA’s anti-melanogenic activity were studied, and it was shown that CBA did not affect either intracellular tyrosinase activity or the direct activity of tyrosinase enzyme. Additionally, CBA did not affect intracellular α-glucosidase activity but significantly inhibited direct α-glucosidase activity. CBA also directly scavenged 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals, consistent with potent antioxidant activity but did not inhibit intracellular reactive oxygen species (ROS). CBA increased acidification of cellular organelles and inhibited maturation of melanosomes by significantly reducing the number of mature melanosomes. Our results indicate that CBA may hold promise as a pigmentation inhibitor for hyperpigmentation disorders for cosmetic use by targeting pathways other than tyrosinase inhibition. Further studies to delineate the molecular signaling mechanism of melanogenesis inhibition and test anti-melanogenesis efficacy of CBA in human skin melanocytes and skin equivalents are warranted.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

PurposeOral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene.MethodsMice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy.ResultsC57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris.ConclusionsTreatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

Effect of narrow-band ultraviolet B radiation on the autophagy of cultured human melanocytes in vitro

Objective To evaluate the effect of narrow-band ultraviolet (NB-UVB) radiation on the autophagy of cultured human melanocytes in vitro, and to explore possible mechanisms underlying the treatment of vitiligo by NB-UVB. Methods In vitro cultured human melanocytes were divided into 4 groups to be irradiated with NB-UVB at different irradiation doses of 0 (control group) , 50, 100 and 200 mJ/cm2 (50-, 100- and 200-mJ/cm2 NB-UVB groups) respectively. After 24-hour treatment, the cells were collected, and monodansylcadaverin (MDC) staining was conducted to detect changes of autophagosomes in melanocytes. Western blot analysis was performed to determine the protein expression of autophagy signals including phosphorylated AMP-activated protein kinase (p-AMPK) , phosphorylated mammalian target of rapamycin (p-mTOR) , microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ (LC3 Ⅱ/Ⅰ) and P62, and transmission electron microscopy to observe ultrastructural changes of autophagosomes and melanosomes in the melanocytes. Statistical analysis was done by using one-way analysis of variance (ANOVA) for the comparison of Western blot results, and by Kruskal-Wallis H test for the comparison of the number of melanosomes, autophagosomes and autolysosomes. Results MDC staining showed that the percentages of autophagosome-positive melanocytes were significantly higher in the 100-, 200-mJ/cm2 NB-UVB groups (38.08% ± 4.10%, 40.23% ± 1.45%, respectively) than in the control group (21.83% ± 3.50%, both P < 0.05) and 50 mJ/cm2 NB-UVB group (23.66% ± 4.12%, both P < 0.05) . As Western blot analysis revealed, the 100-, 200-mJ/cm2 NB-UVB groups showed significantly increased expression of p-AMPK and LC3Ⅱ/Ⅰ, but significantly decreased expression of p-mTOR and P62 compared with the control group (all P < 0.05) . Transmission electron microscopy showed that the number of autophagosomes and autolysosomes was significantly higher in the 100-, 200-mJ/cm2 NB-UVB groups (5.12 ± 1.13, 5.25 ± 1.04) than in the control group (1.88 ± 1.18, both P < 0.05) . Meanwhile, the number of melanosomes was significantly higher in the 50-, 100- and 200-mJ/cm2 NB-UVB groups (39.12 ± 9.42, 57.38 ± 7.11, 59.75 ± 15.15, all P < 0.05) than in the control group (18.50 ± 4.18, all P < 0.05) . Conclusion NB-UVB radiation can not only promote the formation of melanosomes, but also activate the autophagy signal pathways in the melanocytes and promote the formation of autophagosomes and autolysosomes, which may be one of the mechanisms underlying the treatment of vitiligo by NB-UVB. Key words: Melanocytes; Vitiligo; Autophagy; Ultraviolet therapy; Melanosomes

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

Effect of epigallocatechin gallate on ultraviolet B-induced transfer and degradation of melanosomes in mice.

Objective To evaluate the effect of tea polyphenol epigallocatechin gallate (EGCG) on ultraviolet B (UVB) -induced skin pigmentation, transfer and degradation of melanosomes in mice, and to explore the role of autophagy in the mechanism of melanosome degradation. Methods A total of 32 ears from 16 female C57/BL6 mice were randomly and equally divided into 4 groups: acetone control group topically treated with acetone solution daily, EGCG group topically treated with 10 g/L EGCG acetone solution daily, UVB irradiation group irradiated with 500 mJ/cm2 UVB once a day and 2 hours later topically treated with acetone solution, UVB + EGCG group irradiated with 500 mJ/cm2 UVB once a day and 2 hours later topically treated with EGCG acetone solution. Ten days later, all the mice were sacrificed, and skin tissue samples were collected from the ears. Transmission electron microscopy was performed to observe ultrastructural changes of melanosomes and autophagosomes, immunohistochemical study to measure expression of protease-activated receptor 2 (PAR2) and microtubule-associated protein light chain 3 (LC3) in the epidermis, and Western blot analysis to determine the protein expression of PAR2, Ras-related protein Rab27a and LC3 in the epidermis. Results There was a significant difference in the number of melanosomes and autophagosomes among the acetone control group, EGCG group, UVB irradiation group and UVB + EGCG group (H= 12.249, 13.888, respectively, both P 0.05) . Western blot analysis revealed significant differences in the protein expression of PAR2 and Rab27a, as well as in the LC3-Ⅱ/LC3-Ⅰratio, among the 4 groups (F = 18.739, 25.967, 24.022, respectively, all P < 0.05) . Compared with the UVB irradiation group, the UVB + EGCG group showed significantly decreased expression of PAR2 (0.91 ± 0.54 vs. 3.12 ± 0.61, P < 0.05) and Rab27a (0.99 ± 0.16 vs. 1.42 ± 0.07, P < 0.05) , but significantly increased LC3-Ⅱ/LC3-Ⅰratio (1.67 ± 0.08 vs. 1.24 ± 0.07, P < 0.05) . Conclusion Topical EGCG treatment can effectively suppress UVB-induced skin pigmentation, which may be related to the inhibition of melanosome transfer and promotion of melanosome autophagy. Key words: Polyphenols; Ultraviolet rays; Autophagy; Melanins; Epigallocatechin gallate

Interplay between FGFR2b-induced autophagy and phagocytosis: role of PLCγ-mediated signalling.

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b‐induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor‐triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation.

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 520-525nm. 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 (450nm), 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.

Vitiligo – The story from within: A transmission electron microscopic study before and after narrow-band ultraviolet B

ABSTRACTMelanocyte loss is the main feature of vitiligo, but evidence refers to pathological multiplayers. Transmission electron microscopy was utilized to further explore vitiligo before and after narrow-band ultraviolet B (NB-UVB) therapy. Skin biopsies were retrieved from lesional and perilesional skin and compared to normal control skin. Sections were examined for melanocytes and keratinocytes and the number of melanosomes and thickness of basal lamina were measured. In lesional skin, keratinocytes revealed two types of degeneration with a significant increase in the mean thickness of basal lamina and decrease in the number of melanosomes. After treatment, lesional and perilesional skin showed variable ultrastructural features.

Ultra-structural effects of different low-level lasers on normal cultured human melanocytes: an in vitro comparative study.

The aim of this study was to investigate the effects of the different types of low-level laser therapy (LLLT) on the ultra-structure and number of melanosomes in normal cultured human melanocytes. Specific effects of various types of LLLT on the ultra-structure of melanosomes have not yet been reported. Melanocytes were exposed to LLLT at an energy level of 2.0J/cm2, using a blue (457nm), red (635nm), or ultraviolet (UV) (355nm) laser. After 72h of irradiation, the melanocytes were fixed in 2.5% glutaraldehyde (pH 7.2) phosphate buffer for 8h and analyzed by transmission electron microscopy. Four developmental stages (I to IV) of melanosomes were observed, and their numbers were counted manually. The percentage of stages I, II, III, and IV melanosomes was 12.8, 14.2, 22.6, and 50.3%, respectively, in the control (sham light). However, the melanosome percentages were 41.2, 5.4, 8.2, and 24.2% in stages I, II, III, and IV, respectively, in the blue laser-treated group; 58.4, 6.1, 9.3, and 26.2% for stages I, II, III, and IV, respectively, in the red laser-treated group; and 31.3, 11.1, 16.5, and 41.1% for stages I, II, III, and IV, respectively, in the UV laser-treated group. The present data show that the amount of stage I is significantly higher (P < 0.0001) in the LLLT-treated cells compared to the control, which indicates significant stimulation of melanogenesis. The red laser was more effective than the other lasers. Moreover, the effects of LLLT on the ultra-structure of melanosomes need to be studied in a larger number of subject groups.

Clinical and Histologic Analysis of the Efficacy of Topical Rapamycin Therapy Against Hypomelanotic Macules in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder leading to the aberrant activation of the mammalian target of rapamycin complex 1. Although the efficacy of mammalian target of rapamycin complex 1 inhibitors against tumors in patients with TSC, including facial angiofibroma, has been well investigated, their efficacy against hypomelanotic macules in patients with TSC is unknown. To evaluate objectively the efficacy of topical rapamycin treatment of hypomelanotic macules in patients with TSC and to elucidate the mechanisms of how rapamycin improves the macules. We performed a prospective, baseline-controlled trial of 6 patients with TSC and hypomelanotic macules in non-sun-exposed and sun-exposed skin at the Department of Dermatology, Osaka University, from August 4, 2011, through September 27, 2012. Rapamycin gel, 0.2%, was applied to the lesions twice a day for 12 weeks. Histologic examinations and blood tests were conducted at the start and completion of treatment. Blood rapamycin levels were analyzed at completion. Topical rapamycin treatment for hypomelanotic macules. Objective evaluation of rapamycin treatment of hypomelanotic macules in TSC with δ-L (L indicates the brightness of the color) levels on spectrophotometry at the start and completion (12 weeks) of treatment and at 4 and 12 weeks after discontinuation of treatment (16 and 24 weeks, respectively). Improvement of hypomelanotic macules (in δ-L values) was significant at 12 weeks (mean [SD], 2.501 [1.694]; P < .05), 16 weeks (1.956 [1.567]; P < .01), and 24 weeks (1.836 [1.638]; P < .001). Although efficacy tended to be prominent in sun-exposed skin, we did not observe significant differences (in δ-L values) between sun-exposed and non-sun-exposed skin at 12 weeks (mean [SD], 1.859 [0.629] and 3.142 [2.221], respectively), 16 weeks ( 1.372 [0.660] and 2.539 [2.037], respectively), and 24 weeks (1.201 [0.821] and 2.471 [2.064], respectively). No adverse events were observed, and rapamycin was not detected in the blood of any patient. Electron microscopic analysis of hypomelanotic macules revealed that topical rapamycin treatment significantly improved the uniformity of the melanosome numbers in the TSC melanocytes (pretreatment macules: mean [SD], 25.71 [21.90] [range, 5-63]; posttreatment macules: 42.43 [3.60] [range, 38-49]; P < .001). Moreover, rapamycin treatment induced the recovery of melanosomes in TSC-knocked-down melanocytes from depleted amounts (mean [SD], 16.43 [11.84]) to normal levels (42.83 [14.39]; P < .001). Topical rapamycin treatment was effective and safe against hypomelanotic macules arising from TSC. This efficacy of rapamycin was corroborated as stemming from the improvement of impaired melanogenesis in TSC melanocytes.

ADAM protease inhibitors reduce melanogenesis by regulating PMEL17 processing in human melanocytes

BackgroundADAMs (a disintegrin and metalloprotease) are a family of proteases involved in ectodomain shedding that play a role in various biological processes such as cell adhesion and migration. ADAM10 and ADAM17 are suggested to be involved in pigmentary disorders. ObjectiveWe examined the effect of ADAM protease inhibitors on the modulation of melanogenesis in normal human epidermal melanocytes (NHEM). MethodsNHEMs and B16F10 treated with ADAM protease inhibitors were analyzed. AlamarBlue cell proliferation assay, melanin content assay, tyrosinase activity assay, Western blotting analysis, electron microscopic analysis, and RNA interference were employed. ResultsIn NHEMs, melanin content was reduced by treatment with ADAM protease inhibitors. The inhibitors did not change the protein expression of tyrosinase, TRP-1, and MITF. In B16F10 cells, treatment of the cells with ADAM protease inhibitor diminished the α-MSH-induced increase in melanin content. Electron microscopy showed that the number of fibrillar and mature melanosomes was significantly reduced and that the vacuolar compartments were filled with dense unstructured aggregates after treatment with ADAM protease inhibitors. We therefore focused on the processing of PMEL17, a melanosomal glycoprotein that forms a fibrillar matrix on which melanin gets deposited. Proteolytic processing of PMEL17 is required to form functional fibrils during melanogenesis. Recently, γ-secretase and β-site amyloid precursor protein-cleaving enzyme 2 (BACE2) were found to cleave PMEL17. We found that ADAM protease inhibitors exerted effects on the processing of C-terminal and N-terminal fragments of PMEL17. Using BACE2 siRNA and γ-secretase inhibitor, we showed that ADAM protease inhibitor affected PMEL17 processing in a γ-secretase and BACE2-independent mechanism. ConclusionSeveral proteases, including ADAM proteases, can contribute to the formation of fibrils and their assembly into sheets in melanosomes.

Regulation of melanosome number, shape and movement in the zebrafish retinal pigment epithelium by OA1 and PMEL

ABSTRACTAnalysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for the generation of cylindrical melanosomes in zebrafish, which in turn is required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together, our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localisation in the apical processes.

Scaling and Root Planning decreases the Number of Melanosomes within Keratinocytes in Human Gingiva: Ultrastructural Analysis of Three Cases.

The aim of the present report was to evaluate the number of melanosomes within keratinocytes on pigmented gingiva, after and before scaling and root planning. Inflamed gingiva biopsies were taken from three patients (group 1). Forty days after scaling and root planning, biopsies were collected from the homologous contralateral areas (group 2). Samples were fixed in 2% glutaraldehyde-2.5% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M sodium cacodylate buffer, pH 7.4 for 4 hours, and then processed for transmission electron microscopy. Eighty electron micrographs were evaluated for recording the number of granules by a cross-section grid. The granules that were on intersections were recorded as well as the points that appeared on the cytoplasm for calculating the volumetric density (Vd), i.e the volume that the melanosomes occupied into the cytoplasm of keratinocytes. The presence of melanosomes in different stages of maturation and distribution into the cells were recorded with the aid of a magnifying glass. For the statistical analysis, a student t-test was applied. Results of the present report showed that melanosomes within keratinocytes were present in a higher number in inflamed gingiva A (11.08 ± 1.47), B (3.16 ± 0.38) and C (4.92 ± 0.89) and decreased after resolving of gingival inflammation A (9.46 ± 0.88), B (1.73 ± 0.25) and C (0.76 ± 0.18). There is a possibility that inflammation influences the intensity of gingival melanin pigmentation. The periodontal treatment appears to have an effect on gingival melanin pigmentation.

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

Iron upregulates melanogenesis in cultured retinal pigment epithelial cells

The purpose of our studies was to examine the relationship between iron and melanogenesis in retinal pigment epithelial cells, as prior observations had suggested that iron may promote melanogenesis. This relationship has potential clinical importance, as both iron overload and hyperpigmentation are associated with age-related macular degeneration (AMD). Human fetal retinal pigment epithelial cells and ARPE-19 cells were treated with iron in the form of ferric ammonium citrate, after which quantitative RT-PCR and electron microscopy were performed. Melanogenesis genes tyrosinase, tyrosinase-related protein 1, Hermansky–Pudlak Syndrome 3, premelanosome protein and dopachrome tautomerase were upregulated, as was the melanogenesis-controlling transcription factor, microphthalmia-associated transcription factor (MITF). Iron-treated cells had increased pigmentation and melanosome number. Multiple transcription factors upstream of MITF were upregulated by iron.

Melasma treatment using an erbium:YAG laser: a clinical, immunohistochemical, and ultrastructural study.

Melasma is a common pigmentary disorder that remains resistant to available therapies. The aim of the present study was to evaluate the efficacy of erbium:YAG lasers in the treatment of refractory melasma and investigate the histopathological and ultrastructural changes between melasma skin and adjacent control skin before and after surgery. Fifteen Egyptian female patients with melasma unresponsive to previous therapy of bleaching creams and chemical peels were included in this study. Full-face skin resurfacing using an erbium:YAG laser was performed. Clinical parameters included physician and patient assessment, and melasma area and severity index score were done. Adverse effects after laser resurfacing were recorded. Biopsies of lesions and adjacent healthy skin were stained using hematoxylin-eosin, immunohistochemically marked for Melan-A, and evaluated by electron microscopy. The amount of melanin, staining intensity, and number of epidermal melanocytes are increased in melasma lesions as compared to normal skin. Electron microscopic analysis revealed an increased number of mature melanosomes in keratinocytes and melanocytes, with more marked cytoplasmic organelles in melasma skin than in biopsy specimens from normal skin, suggesting increased cell activity. After surgery, the number of melanocytes and concentration of melanin decreased in melasma skin, and the mean melasma area and severity index score decreased dramatically. Erbium:YAG laser resurfacing effectively improves melasma; however, the almost universal appearance of transient postinflammatory hyperpigmentation necessitates prompt and persistent intervention.

Ultrastructural features of skin pigmentation in the lizardHeloderma suspectumwith emphasis on xanto-melanophores

The morphological origin of the dark and pink-orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark-grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta-layer contributes to increase the black intensity. In the orange-pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto-melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid-rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto-melanophores localized in the pink-orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.

Snow white, a Zebrafish Model of Hermansky-Pudlak Syndrome Type 5

Hermansky-Pudlak Syndrome (HPS) is a set of genetically heterogeneous diseases caused by mutations in one of nine known HPS genes. HPS patients display oculocutaneous hypopigmentation and bleeding diathesis and, depending on the disease subtype, pulmonary fibrosis, congenital nystagmus, reduced visual acuity, and platelet aggregation deficiency. Mouse models for all known HPS subtypes have contributed greatly to our understanding of the disease, but many of the molecular and cellular mechanisms underlying HPS remain unknown. Here, we characterize ocular defects in the zebrafish (Danio rerio) mutant snow white (snw), which possesses a recessive, missense mutation in hps5 (hps5I76N). Melanosome biogenesis is disrupted in snw/hps5 mutants, resulting in hypopigmentation, a significant decrease in the number, size, and maturity of melanosomes, and the presence of ectopic multi-melanosome clusters throughout the mutant retina and choroid. snw/hps5I76N is the first Hps5 mutation identified within the N-terminal WD40 repeat protein-protein binding domain. Through in vitro coexpression assays, we demonstrate that Hps5I76N retains the ability to bind its protein complex partners, Hps3 and Hps6. Furthermore, while Hps5 and Hps6 stabilize each other's expression, this stabilization is disrupted by Hps5I76N. The snw/hps5I76N mutant provides a valuable resource for structure-function analyses of Hps5 and enables further elucidation of the molecular and cellular mechanisms underlying HPS.