Melanin synthesis and regulation in vivo and commonly used melanin inhibitors from natural products and traditional Chinese medicine / 中国中药杂志

Melanin is an important factor affecting human skin color. This study defines its synthetic pathways and regulatory pathways have the practical significance for the treatment of diseases caused by pigmentation problems, such as chloasma. Besides, it also provides a theoretical basis for screening out melanin inhibitors and developing whitening and freckle products. At present, the melanin inhibitors used in whitening and freckle products mainly come from natural products and traditional Chinese medicine. This article first introduces the melanin biosynthesis pathway with tyrosinase as the core, defines the synthesis, transport and catalytic activity of tyrosinase as the three key factors affecting melanin synthesis, and then reviews two common types of melanin inhibitors, namely tyrosinase synthesis inhibitors and tyrosinase inhibitors derived from natural products and traditional Chinese medicine. This article provides guidance for the development of new melanin inhibitors, and puts forward the idea for combining and synergizing inhibitors according to different mechanisms, in order to develop new whitening formulas.

Heat Shock Protein Hspa5 Interacts with and Protects Tyrosinase Activity

In this study, heat shock protein, Hspa5 was cloned, expressed and purified subsequently confirmed that it interacted with the tyrosinase (TYR) in vitro. Then, using the crystal structure of the homologous protein from the bacteria as a template, a homology model of human TYR was constructed. This model was further applied to investigate the molecular docking with Hspa5. The model showed that the interaction between the TYR and Hspa5 was mainly maintained by some hydrogen bonds in a quite low energy state. The results indicated that TYR was protected in different denaturation conditions by Hspa5. It was concluded that Hspa5 served as a molecular chaperone of TYR, which could help to better understand the molecule regulation mechanism of TRY in many kinds of diseases.

Hesperidin Suppresses Melanosome Transport by Blocking the Interaction of Rab27A-Melanophilin

We investigated the inhibitory effects of hesperidin on melanogenesis. To find melanosome transport inhibitor from natural products, we collected the structural information of natural products from Korea Food and Drug Administration (KFDA) and performed pharmacophore-based in silico screening for Rab27A and melanophilin (MLPH). Hesperidin did not inhibit melanin production in B16F10 murine melanoma cells stimulated with alpha-melanocyte stimulating hormone (alpha-MSH), and also did not affect the catalytic activity of tyrosinase. But, hesperidin inhibited melanosome transport in melanocyte and showed skin lightening effect in pigmented reconstructed epidermis model. Therefore, we suggest that hesperidin is a useful inhibitor of melanosome transport and it might be applied to whitening agent.

The effect of H 1 and H 2 receptor antagonists on melanogenesis.

Background: Histamine was found to stimulate melanogenesis in cultured human melanocytes specifically mediated by histamine H 2 receptors via protein kinase A activation. Based on this finding, the effect of topically applied H 2 antagonist on UVB-irradiated Guinea pigs' skin was examined and found to be suppressive on the post-irradiation melanogenesis. Aims: In this study, we tried to explore the role of topically applied H 1 and H 2 receptor antagonists, in inhibition of UVB-induced melanization. Methods: The effect of topically applied H 1 and H 2 receptor antagonists in inhibition of melanization was done clinically and histochemically using Fontana Masson and DOPA reactions compared with placebo. Results: The post-irradiation pigmentation was found to be brownish/black instead of the original light brown color. This color change occurred below the shaved orange-red fur suggesting a switch of melanogenesis from pheomelanin to eumelanin. The induced pigmentation was suppressed by topically applied H 2 antagonist while both H 1 antagonist and vehicle had no effect. The microscopic examination showed that the keratinocytes in the H 2 antagonist-treated areas contained few melanosomes while the nearby dendrites are full of them. Conclusion: H 2 antagonists' inhibition of UVB-induced pigmentation is not only due to suppression of melanization but also due to a specific action on melanosomes' transfer.

A Simple Assay Method for Melanosome Transfer

Pigmentation is induced by production of melanin in specialized organelles termed melanosomes and by transfer of these organelles from melanocytes to surrounding keratinocytes. The chemical basis of melanogenesis is relatively well known but the mechanism of melanosome transfer is not well studied. Various pigmentary disorders and cosmetic applications require the use of depigmenting agents. Currently available topical agents used for the reduction of pigmentation mainly include tyrosinase inhibitors and/or melanocyte-cytotoxic agents. Recently, several agents have been introduced to inhibit melanosome transfer from melanocytes to keratinocytes. However, an experimental model for melanosome transfer is not well established. In this study, a simple assay method using flow cytometry is described.

Effects of Nicotinamide on Melanin Transportation in Human Skin Melanocytes / 环境与健康杂志

Objective To study the effects of nicotinamide on the human skin melanocytes and try to explore the potential mechanism of nicotinamide on the calcium signal transduction,cytoskeleton.Methods The primary cultured human skin melanocytes from foreskin were selected as the target cells in the present study.0.00,0.05,0.25,1.25 and 6.25 mg/ml nicotinamide were applied respectively.Western blot,fluorescent probes(Flu-3AM),flow cytometry analysis and time-lampse microscope digital skill were used to evaluate the effects of nicotinamide on melanosome motility and the melanosome distribution in melanocytes.Results The results showed that nicotinamide had a potential effect on regulating free calcium concentration in a dose-dependent manner(y=76.461 2-5.435x,r=0.97);The activity of Na+-K+-Ca2+-ATPase was down regulated with the increasing concentration of nicotinamide.The expression of cellular dynein was also altered by nicotinamide;Na +-K+-Ca2+-ATPase activity was kept normal when given 0.05,0.25 mg/ml nicotinamide,while the dynein protein expression was inhibited(P

The Effects of Keratinocyte-derived Factors Involved in Transforming the Morphologies of Melanocyte and in Translocating Melanosomes to Keratinocyte / 대한해부학회지

Keratinocyte-derived factors are involved in regulating the proliferation, differentiation or melanogenesis of melanocytes. To investigate the effects of keratinocyte-derived factors on the skin pigmentation, human melanocytes and keratinocytes were cultivated in the forms of pure melanocyte or keratinocyte culture system, co-culture system (melanocytes and keratinocytes were cultivated together in a vessel but they were separated with semipermeable membrane) or mixed culture system (melanocytes and keratinocytes were cultivated together in a vessel in mixed form). The authors studied the cellular features (dendritogenesis and area) and the tyrosinase activities and observed the electron microscopic structures for melanosome transfer. Melanocyte in co-culture system increased in the area (p.0.05) but not in the dendritogenesis compared with the melanocyte in pure culture system. The tyrosinase activities of co-culture system on the 2nd and 4th day revealed higher compared with the ones of the pure and mixed culture system (p.0.05). In the co-culture system, the tyrosinase activities were gradually decreased with the lapse of time and vice versa in the pure and mixed culture system. On the 6th day culture, all of the three melanocyte culture systems showed the same tyrosinase activities. In spite of high tyrosinase activities in the medium, there were no tyrosinase activities in keratinocytes with the co-culture system. In transmission electron microscopic findings, there were scant of melanosomes in keratinocytes with the co-culture and mixed culture systems. In conclusion, keratinocyte-derived factors modulate the activities and melanogenesis of melanocytes but there were no effects on melanosome transfer to keratinocytes.

The Motile Properties of Vimentin Intermediate Filament in B16 Melanoma Cells / 대한피부과학회지

BACKGROUND: Intermediate filaments as well as microtubule and microfilament are major components of cytoskeleton of human cells. Melanocytes have vimentin intermediate filament, which have not been well investigated as other cytoskeletons, especially in their function. OBJECTIVE: The purpose of this study was to observe the motile characteristics of vimentin intermediate filament in living B16 melanoma cells. METHODS: The motile properties of vimentin intermediate filament have been studied in living B16 melanoma cells using green fluorescent protein(GFP). cDNA expressing GFP-vimentin fusion protein was cloned and transfected into living B16 melanoma cells. Living cells were observed under fluorescent microscope and confocal microscope. Time-lapse images were collected and analysed. RESULTS: GFP-vimentin is incorporated into the endogenous vimentin networks. Time-lapse observations of vimentin fibrils demonstrate that they are constantly changing their configurations. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Short filamentous structures('squiggle') are also seen actively translocating. Melanosomes also are changing their position back-and-pro constantly. They are co-localized very well with kinesin molecules in B16 melanoma cells. CONCLUSION: The vimentin intermediate filament and melanosomes in B16 melanoma cells have very active movement, which seem to have close relation with kinesin motor proteins.

A Case of Oculocutaneous Albinism

Oculocutaneous albinism resulting from genetic defect of melanin synthesizing system is characterized by pale skin, straw-colored hair, hypopigmentation of the iris, hypoplasia of fovea, photophobia, low visual acuity and strabismus. In general, oculocutaneous albinism can be distinguished by its clinical feature and hair follicle incubation test but should be diagnosed by electron microscopic findings of the skin which is exposed to sunlight. We experienced a case of 6-year-old female oculocutaneous albinism that showed clinical typical features and was diagnosed through electron microscopic finding of many immature melanosomes of the skin in the back of the hand. We report this unusual case with literature review.

The Investigation of the Melanocytes in a Cultured Skin Equivalent Model / 대한해부학회지

Melanocytes grown in the pure monolayer culture lack the three-dimentional organization and the cellular interactions that exist in vivo. These can be partially overcome by growing melanocytes together with other epidermal cells in cultured skin equivalent models. In this study, skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in ratio 10 : 1 onto artificially constructed dermis. They were cultured in DMEM/F12 (1 : 1) for 4 days and then lifted to the air-liquid interface and maintained in DMEM/F12 (3 : 1) for 10 days. Histological and electronmicroscopic examinations of the cultured skin equivalants revealed a structure that closely resembled human interfollicular epidermis; 1. Melanocytes, were identified by positive staining with melanoma-specific antibodies (NKI/C3 and S-100 protein) and prominent cytoplasm with rich cell organelles, were located in the basal layer. 2. Melanocytes contained predominently early stage melanosomes and prominent Golgi apparatus. Mature melanins, were usually abundant in normal skin, were hardly seen both in melanocytes and in neighbouring keratinocytes. 3. Melanocytes were surrounded by keratinocytes but did not form intercellular junctions with them. 4. Keratinocytes were charaterized by microfilament bundles and intercellular junctions such as desmosomes and hemidesmosomes with neighbouring keratinocytes and artificial dermis. The melanocyte in the above skin equivalents had a strong resemblance to the one of normal human skin and therefore this model can be used as artificial skin for the transplantation and in the investigation of melanocytes' role to the UV stimuli.

Clear cell sarcoma: 1 case report / 영남의대학술지

Clear cell sarcoma is a rare soft tissue sarcoma that occurs tendons and aponeuroses, usually of the lower extremities in young adult. The exact histogenesis is not definitely established, We experienced a case of 58 year-old female who presented with a 3.2x2.2cm sized mass located in the subcutaneous tissue on the left lower thigh. The mass was well circumscribed and grayish firm. Two small satellite nodules were also seen. Histologically the tumor was composed of round to fusiform cells with clear or pale eosinophilic cytoplasm and separated into compact nests or short fascicles by delicated fibrous septa. The melanin pigments and hemosiderin were seen. Tumor cells showed positive reaction for S-100 protein and HMB-45. The ultrastructural examination showed abundant mitochondria and melanosomes.

Clinical Features and Histopathological Characteristics of Nevus Depigmentosus / 대한피부과학회지

BACKGROUND: Nevus depigmentosus was first reported in 1884 by Lesser. It is defined as a congenital non-progressive hypopigmented macule or patch that is stable in its relative size and distribution throughout the life of the individual. The etiopathogenesis and histopathological characteristics of nevus depigmentosus are not fully established. OBJECT: The purpose of this study is to investigate the clinical and histopathological characteristics and pathogenesis of nevus depigmentosus. METHODS: Clinieal survey was carried out on forty-nine patients with nevus depigmentosus and two skin biopsies were taken from eighteen patients; from the central part of the depigmented lesion and the border of the lesion including the perilesional normal skin. The sections were stained with hematoxylin-eosin, Fontana-Masson and S-100 protein. The ultrastructural evaluation were also done to detect alternation of melanocytes. RESULTS: The results are as follows ; 1. The lesions were mostly (91.8%) present before the age of three, but some lesions appeared in childhood (8.2%). 2. The lesions were most frequently found on the trunk (42.9%), followed by the face and scalp (20.4%). 3. There were 33 patients (67.3%) with the isolated type, 15 patients (30.6%) with the dermatomal type and one patient with the whorled type. 4. Histopathological studies have shown that the stainability of Fontana-Masson in the lesions of nevus depigmentosus was decreased compared with perilesional nomal skin, but there were no changes in the number of melanocytes. 5. There was a great reduction in the number of melanosomes in melanocytes and keratinocytes of nevus depigmentosus. In keratinocytes, there was some aggregations of melanosomes and some of them showed membrane bound architecture. CONCLUSION: The results of this study support the fact that nevus depigmentosus is caused by functional defects of melanocytes and morphological abnonnalities of melanosomes.

Role of calcium in melanosome aggregation within Labeo melanophores.

In isolated scale melanophores of Labeo rohita the melanosome aggregating effect of K+ was inhibited in Ca2+ deprived medium. Moreover, the Ca2+-antagonists, verapamil and lanthanum inhibited the action of K+ in concentration dependent manner. The elevation of extracellular Ca2+ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca2+ appeared to be required only for K+ - induced aggregation and not melanosome aggregation per se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca2+ free medium.

Meningeal Melanocytoma Associated with Ota's Nevus: Report of a case

Primary meningeal melanocytoma of the central nervous system is extremely rare. We report a case of meningeal melanocytoma associated with Ota's nevus as a recurrent form in a 53-year old male. The meningeal melanocytoma was removed from right parietooccipital lobe 4 years ago and recurred in right parietal, occipital and left frontal lobes. Ultrastructurally, the tumor cells were characterized by the presence of numerous melanosomes and premelanosomes in their cytoplasm. Moreover, the tumor was lacking in histologic and ultrastructural features of pigmented meningioma, melanotic schwannoma and prolonged clinical course was different from primary meningeal melanoma or metastatic malignant melanoma.

Pigmented Squamous Cell Carcinoma

An 84-year-old woman had an ovoid shallow ulcer with an elevated, indurated, pigmented border on the left cheek. Histological examination revealed a moderately differentiated squamous cell carcinoma and a solar keratosis with abundant melanocytes and melanin pigment. Ultrastructurally, the keratinocytes contained numerous melanosomes in their cytoplasms and the melanocytes in the squamous cell carcinoma and the solar keratosis had mature melanosomes.

Melanosome in Retinal Pigment Epithelium: Distribution, Type and Activity of Acid Phosphatase

Distribution of melanosome, types of melanosome in retinal pigment epithelium(RPE) and activity of acid phosphatase in melanosome were studied in rabbits. The eyes were observed using electron microscopy and enzyme cytochemical electron microscopy. The majority of melanosomes were located near the apical region of RPE. So melanosomes can absorb light that passes through the neural retina rapidly and remove free radicals that develop in RPE effectively, thereby benefiting the optical system and protecting visual outer segments. Melanosomes in RPE were classified as two types; ellipsoid and spherical or oval. Ellipsoid types were loccated parallel to the apical process and spherical or oval types, vertically or obliquely to apical process. Mature and immature melanosome showed mostly positive responses for activity of acid phosphatase, indicating that melanosome commonly incorporates into the lysosomal system of the retinal pigment epithelium. But some melanosomes showed negative responses for activity of acid phosphatse, suggesting that melanosome has a stable and inert property. The observed premelanosome showed negative response. Two types of melanosomerelated complex granules were identified: melanosome with a cortex of enzymereactive material(melanolysosome) and melanosome with a cortex of lipofuscin(melanolipofuscin). They were located near or below the melanosomes in the cytoplasm. These findings indicate that a relationship between melanosome and the lysosomal system of the retinal pigment epithelium exists, and suggest that melanosome undergoes modification or degradation there. Also, the findings of premelanosome and positive responses for the activity of acid pnosphatase indicate that melanosome continues to be synthesized at a low rate in adult eye.

Melanosome in Retinal Pigment Epithelium: Distribution, Type and Activity of Acid Phosphatase

Distribution of melanosome, types of melanosome in retinal pigment epithelium(RPE) and activity of acid phosphatase in melanosome were studied in rabbits. The eyes were observed using electron microscopy and enzyme cytochemical electron microscopy. The majority of melanosomes were located near the apical region of RPE. So melanosomes can absorb light that passes through the neural retina rapidly and remove free radicals that develop in RPE effectively, thereby benefiting the optical system and protecting visual outer segments. Melanosomes in RPE were classified as two types; ellipsoid and spherical or oval. Ellipsoid types were loccated parallel to the apical process and spherical or oval types, vertically or obliquely to apical process. Mature and immature melanosome showed mostly positive responses for activity of acid phosphatase, indicating that melanosome commonly incorporates into the lysosomal system of the retinal pigment epithelium. But some melanosomes showed negative responses for activity of acid phosphatse, suggesting that melanosome has a stable and inert property. The observed premelanosome showed negative response. Two types of melanosomerelated complex granules were identified: melanosome with a cortex of enzymereactive material(melanolysosome) and melanosome with a cortex of lipofuscin(melanolipofuscin). They were located near or below the melanosomes in the cytoplasm. These findings indicate that a relationship between melanosome and the lysosomal system of the retinal pigment epithelium exists, and suggest that melanosome undergoes modification or degradation there. Also, the findings of premelanosome and positive responses for the activity of acid pnosphatase indicate that melanosome continues to be synthesized at a low rate in adult eye.

Effect of Sulfhydryl Compounds on Melanosomal Morphology of Epidermal Melanocytes in UV - Irradiated Black Mice / 대한피부과학회지

The present study was performed to evaluate the effect of sulfhydryl compounds, cysteine and glutathione, on the size of melanosomes and the ratio of melanosormai stages of epidermal melanocytes in UV-irradiated black mice. The results were as follows; 1. Both of cysteine and glutathione showed significant diminution in the short axis of melanosomes and the percentage of stage 4 melanosomes of epidermal melanocytes in C57BL black mice skin. 2. The length of short axis of melanosomes in glutathione-treated group is smaller than those in cysteine-treated group at the end of 3rd week of intraperitoneal injection. The percentage of stage IV melanosomes significantly decreased in glutathione-treated group and cysteine-treated group at the end of 3rd week and 5th week respectively. 3. In glutathione-treated group, the short axis of melanosomes and the percentage of stage 4 melanosomes both decreased in proportional to the period of intraperitoneal in]ection.