Stimulation Of Tyrosinase Activity Research Articles

Evaluation of a panel of furochromenones as the activator and inhibitor of tyrosinase.

Tyrosinase is a copper-containing enzyme involved in the biosynthesis of melanin pigment. While the excess production of melanin causes hyperpigmentation of human skin, hypopigmentation results in medical conditions like vitiligo. Tyrosinase inhibitors could be used as efficient skin whitening agents and tyrosinase agonists could be used for enhanced melanin synthesis and skin protection from UV exposure. Among a wide range of tyrosinase-regulating compounds, natural and synthetic derivatives of furochromenones, such as 8-methoxypsoralen (8-MOP), are known to both activate and inhibit tyrosinase. We recently reported a synthetic approach to generate a variety of dihydrofuro[3,2-c]chromenones and furo[3,2-c]chromenones in a metal-free condition. In the present study, we investigated these compounds for their potential as antagonists or agonists of tyrosinase. Using fungal tyrosinase-based invitro biochemical assay, we obtained one compound (3k) which could inhibit tyrosinase activity, and the other compound (4f) that stimulated tyrosinase activity. The kinetic studies revealed that compound 3k caused 'mixed' type tyrosinase inhibition and 4f stimulated the catalytic efficiency. Studying the mechanisms of these compounds may provide a basis for the development of new effective tyrosinase inhibitors or activators.

Microwave-assisted extraction of Lawsonia inermis L. leaves: Method validation, optimization, and tyrosinase-stimulating activity

Vitiligo is a long-term skin condition characterized by the development of pale white patches due to a lack of melanin. Lawsonia inermis L. leaves, which contain lawsone, have been explored as a potential alternative treatment for vitiligo. This study aimed to optimize the microwave-assisted extraction of L. inermis leaves and evaluate its ability to stimulate tyrosinase activity, which is relevant to vitiligo treatment. The Box-Behnken design was employed, with three factors being varied: microwave power, time, and cycle. Two responses were monitored: extraction yield and lawsone content. To determine the lawsone content, validated high-performance liquid chromatography was utilized, which demonstrated good linearity, specificity, precision, and accuracy. The optimal conditions that provided the highest extraction yield and lawsone content were then used to evaluate the extract’s ability to stimulate tyrosinase activity. The results revealed that a microwave power of 300 W and a microwave time of 20 s for one cycle yielded an extraction yield of 12.99% ± 0.10% and a lawsone content of 94.85 ± 3.12 mg/g extract. The accuracy of the prediction, with a percent error of less than 10%, confirmed the reliability of the computer program used for data prediction. Furthermore, the extract exhibited significant stimulation of tyrosinase activity, indicating its potential usefulness in vitiligo treatment. The optimal microwave-assisted extraction method identified in this study holds promise for maximizing extraction yield and lawsone content in L. inermis leaves extract. This extract could serve as a basis for developing topical products for the treatment of vitiligo.

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.

A 7-Hydroxy 4-Methylcoumarin Enhances Melanogenesis in B16-F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potentials of the naturally occurring 7-hydroxy coumarin derivatives 7-hydroxy 5,6-dimethoxycoumarin (7H-5,6DM), 7-hydroxy 6,8-dimethoxycoumarin (7H-6,8DM), 7-hydroxy 6-methoxycoumarin (7H-6M), and 7-hydroxy 4-methylcoumarin (7H-4M) in the melanogenic cells model for murine B16F10 melanoma cells. The initial results indicated that melanin production and intracellular tyrosinase activity were significantly stimulated by 7H-4M but not by 7H-5,6DM, 7H-6,8DM, or 7H-6M. Therefore, our present study further investigated the melanogenic effects of 7H-4M in B16-F10 cells, as well as its mechanisms of action. In a concentration-dependent manner, 7H-4M increased intracellular tyrosinase activity, leading to the accumulation of melanin without affecting the viability of B16-F10 cells. Our study further investigated the effects of 7H-4M on melanogenesis, including its ability to promote tyrosinase activity, increase melanin content, and activate molecular signaling pathways. The results indicate that 7H-4M effectively stimulated tyrosinase activity and significantly increased the expression of melanin synthesis-associated proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and TRP2. Based on our findings, we can conclude that 7H-4M has the ability to activate the melanogenesis process through the upregulation of cAMP-dependent protein kinase (PKA) and the cAMP response element-binding protein (CREB). Additionally, our study showed that 7H-4M induced melanogenic effects by downregulating the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3β (GSK-3β) cascades, while upregulating the JNK and p38 signaling pathways. Finally, the potential of using 7H-4M in topical applications was tested through primary human skin irritation tests. During these tests, no adverse reactions were induced by 7H-4M. In summary, our results indicate that 7H-4M regulates melanogenesis through various signaling pathways such as GSK3β/β-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

Potential medicinal plants for progressive macular hypomelanosis

Progressive macular hypomelanosis (PMH) is a hypopigmentation disorder caused by the bacterium identified as Propionibacterium acnes. The current treatments for PMH are antibiotics together with ultra violet radiation; however, UV radiation is not a recommended method to increase melanin production. Currently, there are no known plants used traditionally or medicinally for PMH. The objective of this study was to find plants that could stimulate tyrosinase activity induce melanin production and inhibit P. acnes' growth.Seventeen ethanol plant extracts, used traditionally in Africa for skin diseases, were screened for their antibacterial activity against P. acnes, their effect on monophenolase activity of tyrosinase and their cytotoxicity and stimulation of melanin production on mouse melanocytes (B16-F10).Hypericum revolutum Vahl subsp. revolutum (Hypericaceae) and Withania somnifera L. Dunal (Solanaceae) (twigs and leaves), combined with the known drug tetracycline, exhibited significant antibacterial activity against P. acnes, with the minimum inhibitory concentration ranging from 5.47μg/ml to 14.06μg/ml. The combination of a known drug with other antibacterial compounds not only decreases the concentration needed to inhibit bacterial growth, but also decreases the chances of bacterial resistance. W. somnifera was the only plant extracts that resulted in an increase in the monophenolase activity of tyrosinase. Four compounds typically present in plant extracts, namely coumarin, quercetin, withaferin and winthanone, were docked into the active site of tyrosinase enzyme to determine the interaction with active site residues. Mouse melanocytes (B16F10) treated with H. revolutum, W. somnifera (leaves) and Terminalia prunoides showed an increase in total melanin content as compared to untreated cells at 12μg/ml, 12μg/ml and 150μg/ml respectively.Considering both the antibacterial activity and the stimulatory effect of the treatment on melanin production, H. revolutum and W. somnifera (leaves) could be considered as potential plants for further studies for PMH.

Biological interactions of fluorinated chalcones: Stimulation of tyrosinase activity and binding to bovine serum albumin

The evaluation of tyrosinase activity of CH, CH3F, CH4F, CH23F, CH25F, CH35F and CH2356F as well as of the interactions of CH and fluorinated chalcones CH3F, CH4F and CH2356F with bovine serum albumin (BSA) in a PBS buffer solution (pH=7.4), at 288K, 293K and 298K, were investigated by using spectroscopic techniques, zeta potential and computational methods. Tyrosinase enzymatic activity was measured by UV–vis spectrometry of the oxidation of l-DOPA in the presence of the enzyme and chalcones: the unsubstituted chalcone inhibits tyrosinase activity, whereas its fluorinated derivatives showed the opposite effect, whose sample CH2356F showed the highest percentage activation. Studies of quenching of the BSA fluorescence by CH, CH3F, CH4F and CH2356F show a combination of static and dynamic quenching mechanisms. In addition FRET mechanism can occurs with high probability. For CH the binding constant (Ka) is in the range of 105M−1 indicating a moderate association between chalcone and BSA. On the other hand, for the fluorinated derivatives CH3F, CH4F and CH2356F Ka=104M−1, which indicates that the presence of fluorine atoms on the chalcone structure decreases the binding ability. The thermodynamic parameters ΔH° and ΔS° show that the BSA:CH association is enthalpically and entropically driven, whereas for fluorinated derivatives it is only entropically driven. The negative ΔG° values found in all cases are consistent with a spontaneous albumin:chalcone association. Circular dichroism and zeta potential data show that the binding does not affect significantly the albumin structure. The binding site study clearly indicates that the main binding pocket for the samples is the Trp-212-containing binding site. The largest docking score values also suggest the same binding site. Molecular docking results suggested that the presence of fluorine atoms decrease the number of amino acid residues that can stabilize the interaction of the fluorinated chalcones with BSA. For CH3F and CH2356F the type of interaction between the amino acid residues and ligand structure did not change significantly.

Platelet-derived growth factor regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner

BackgroundAlthough many kinds of keratinocyte-derived factors are known to regulate the proliferation and differentiation of human melanocytes, it is not well defined whether dermis-derived factors work in a similar way. ObjectiveThe aim of this study is to clarify whether dermal factors are involved in regulating the proliferation and differentiation of human melanocytes. MethodsHuman epidermal melanoblasts were cultured serially in a serum-free growth medium. Platelet-derived growth factor-BB (PDGF-BB) was supplemented to the medium, and the effects on the proliferation of melanoblasts/melanocytes and the differentiation of melanocytes were studied. ResultsPDGF-BB stimulated the proliferation of melanoblasts cultured in melanoblast-proliferation medium, but inhibited the proliferation of melanocytes cultured in melanocyte-proliferation medium. By contrast, PDGF-BB stimulated the differentiation, dendritogenesis, and melanogenesis of melanocytes through the stimulation of tyrosinase activity and the expressions of tyrosinase and tyrosinase-related protein-1. ConclusionThese results suggest that PDGF-BB regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner. PDGF-BB seems to be one of the dermal factors that regulate the proliferation and differentiation of human melanocytes.

B16F10 멜라노마세포에서 과기음가미방의 멜라닌 생성 촉진 효과

ABSTRACT Objectives : Since hypopigmentation is known to increase the risk of skin cancer, melanogenesis in the skin needs to be regulated. Here, we evaluated the melanogenesis sti mulatory effects of a modified Goagium herbal remedy (HR) and HR+ox bile (Bos taurus domesticus) extract (OBE) to address hypopigmentation disorders. Methods : B16F10 melanoma cells were treated with different dosages of HR and HR+OBE for 24 to 48 h after 1 h of 10 nM α-melanocyte stimulating hormone (α-MSH). After th e treatment, cell viability, tyrosinase activity, melanin synthesis and the expression of genes related to melanin synthesis were measured and the regulation of the α-MSH signalling through cAMP responding element binding protein (CREB) was determined. Results : HR and HR+OBE with the ranges of 15~100 µg/mL did not affect cell viability in melanoma cells. The 1 h treatment of HR+OBE (50 and 100 µg/mL) potentiated the phosphorylation of CREB by enhancing α-MSH signaling and its 24 h treatment increased CREB expression. Consistent with CREB potentiation, their treatment for 24 h, the expression of microphthalmia-associated transcription factor (MIFT), tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 were increased in realtime PCR. Ultimately, the 48 h treatment of HR+OBE (50 and 100 µg/mL) increased tyrosniase activity and melanin contents in the melanoma cells in comparison to the control. Conclusions : HR+OBE (50 and 100 µg/mL) increases melanin synthesis in B16F10 melanoma cells via the stimulation of tyrosinase activity and expression of MIFT, tyro sinase, TRP-1 and TRP-2. HR+OBE can be used as the a possible treatment for hypopigmentation of the skin.Key words : tyrosinase, MIFT, melanin, CREB, hypopigmentation

Abstract 5242: Effect of total saponins of Gynostemma pentaphyllum on melanogenesis and anti-melanoma effect in B16 cells.

Abstract Melanogenesis is a physiological process of melanin production in response to UV exposure, which is modulated through multi-signaling pathways including cAMP/PKA, Wnt/β-catenin and MAPK signaling cascades. In this study, we investigated both the melanogenesis and anti-melanoma effects of total saponins of Gynostemma pentaphyllum (GpS). Our results showed that non-toxic dosages of GpS stimulated tyrosinase activity and increased melanin content in a dose-dependent manner. Western blot analysis showed that GpS treatment significantly up-regulated the expression levels of the melanogenic proteins, tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) in a dose-dependent manner. The p-CREB, which is the down-stream target of PKA is also elevated upon GpS treatment. We further observed that H89, a PKA inhibitor, attenuated the GpS induced tyrosinase activity, melanin content, and the protein expression of p-CREB and the nuclear β-catenin. To identify the active saponins, GpS was fractionated in the MCI-CHP 20P column and eluted with methanol in a gradient of 50 to 100% . The resulting ten fractions were tested for their melanogensis activities. We found that Fractions 5 to 9 showed the strongest effect in melanin synthesis in B16 cultures, while up-regulated the expression levels of TYR and MITF, while Fractions 7 to 9 increased nucleus β-catenin protein expression. Further purification of GpS is under way. Our early works showed that GpS exhibits strong anti-cancer effects in both in cellular and animal model. We were wonder whether GpS would exert anti-cancer effect against melanoma. To address this question, we tested and showed that GpS inhibited B16 cells migration in a dose-dependent manner. Meanwhile, GpS also increased G1/S ratio of the GpS-treated B16 cells, suggesting that GpS may act as G1 inhibitor. Our study seems to be in line with the recent reports that stimulation of melanogenesis might be associated with the anti-melanoma effect by decreasing proliferation and invasiveness of melanoma (Chien, et al.,2009). Based on the above results, we proposed that GpS might exert the anti-melanoma effect by stimulating the melanogenesis through the activation of cAMP/PKA and Wnt/β-catenin signaling pathways. Citation Format: Ting Fung Tsang, William Chi Shing Tai, Wendy Wen Luan Hsiao. Effect of total saponins of Gynostemma pentaphyllum on melanogenesis and anti-melanoma effect in B16 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2013-5242

Abrogating effect of a xanthophyll carotenoid astaxanthin on the stem cell factor-induced stimulation of human epidermal pigmentation

We established a model for the stem cell factor (SCF)-associated stimulation of human epidermal equivalent (HEE) pigmentation. The addition of SCF (at 5nM) gradually stimulated the visible pigmentation of HEEs over 14days of treatment. A time course study using real-time RT-PCR and western blotting analysis demonstrated that the expression of all melanocyte-specific genes and proteins examined was gradually up-regulated over 7-10days of treatment with SCF. The addition of astaxanthin (Ax) at concentrations of 1, 4, or 8μM markedly abolished the SCF- but not the endothelin (EDN)1-elicited increase in visible pigmentation over 14days in a dose-dependent manner, with almost complete inhibition at 8μM. While no degeneration of the epidermal tissue was visible at day 14 by HE staining, melanin deposition throughout the epidermis was markedly reduced in the Ax-treated HEEs at day 14 compared to untreated controls. Ax significantly reduced the eumelanin content of HEEs to the non-SCF-stimulated level at concentrations of 4 or 8μM compared with untreated controls. Real-time RT-PCR and western blotting of Ax-treated HEEs revealed that the SCF-stimulated expression of tyrosinase (TYR), TYR-related protein-1 (TYRP1), and Pmel17, as well as microphthalmia-associated transcription factor (MITF), is significantly suppressed by Ax at the transcriptional and translational levels. Studies using cultured normal human melanocytes revealed that pre-treatment with Ax interrupts the SCF- but not the EDN1-induced stimulation of TYR activity, and there was no direct inhibitory effect of Ax on TYR activity in vitro. These findings indicate that Ax attenuates SCF-stimulated pigmentation by directly interrupting SCF-associated intracellular signaling linkages through increased expression of MITF, which leads to the stimulated expression of melanogenic genes and proteins in a reactive oxygen species depletion-independent mechanism.

Mangosteen leaf extract increases melanogenesis in B16F1 melanoma cells by stimulating tyrosinase activity in vitro and by up-regulating tyrosinase gene expression

Melanin synthesis is stimulated by various effectors, including α-melanocyte stimulating hormone (α-MSH), cyclic AMP (cAMP)-elevating agents (forskolin, isobutylmethylxantine, glycyrrhizin) and ultraviolet light. Our investigation focused on the identification of the melanogenic efficacy of mangosteen (Garcinia mangostana) leaf extract with regard to its effects on melanogenesis in B16F1 melanoma cells, since it has been known to possess strong anti-oxidant activities. The mangosteen leaf extract was found to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner without any significant effects on cell proliferation. Cytotoxicity of the extract was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the highest concentration of the extract that did not affect cell viability was 32 µg/ml. Formation of melanin from cultured B16F1 melanoma induced by extract treatment was estimated using spectrophotometry. In order to clarify the subsequent mechanism of tyrosinase activation by the extract, the levels of tyrosinase expression in B16F1 melanoma were examined using an intracellular tyrosinase assay and tyrosinase zymography. Up-regulation of intracellular tyrosinase expression seemed to correlate with an increase in microphtalmia-associated transcription factor (MITF) protein levels since MITF is the key factor for genes involved in melanogenesis. Both of the results showed that tyrosinase activity was markedly enhanced from extract-treated cells. The overall results suggest that mangosteen leaf extract may be a promising candidate for the treatment of hypopigmentation disorder and useful for self-tanning cosmetic products.

SOX9 and the tanning response: something new under the sun

SOX9 and the tanning response: something new under the sun

Interleukin-1α Stimulates the Differentiation of Melanocytes but Inhibits the Proliferation of Melanoblasts from Neonatal Mouse Epidermis

Interleukin (IL)-1alpha is one of the important cytokines involved in regulating immunological reactions in the mouse skin. However, it is not known whether IL-1alpha regulates the proliferation and differentiation of mouse epidermal melanocytes. In this study, to investigate the role of IL-1alpha in the regulation of the proliferation and differentiation of mouse epidermal melanocytes, IL-1alpha was supplemented to serum-free primary cultures of epidermal cell suspensions from the initiation of the primary culture (keratinocytes and melanoblasts-melanocytes) as well as to pure cultures of melanoblasts-melanocytes (keratinocyte-depleted cultures, after 14 days), and its effect was tested. IL-1alpha inhibited the proliferation of undifferentiated melanoblasts irrespective of the presence or absence of keratinocytes, whereas the cytokine inhibited the proliferation of differentiated melanocytes only in the presence of keratinocytes. Moreover, IL-1alpha induced the differentiation of melanocytes and, in addition, stimulated tyrosinase activity, melanin synthesis, and dendritogenesis of melanocytes irrespective of the presence or absence of keratinocytes. These results suggest that IL-1alpha is involved in inhibiting the proliferation of neonatal murine epidermal melanoblasts and in stimulating the differentiation, melanogenesis, and dendritogenesis of melanocytes. The results also suggest that IL-1alpha inhibits the proliferation of differentiated melanocytes in cooperation with keratinocyte-derived factors.

Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis

The murine recessive yellow ( Mc1r e ) is a loss-of-function mutation in the receptor for α-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r e mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine ( l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r e mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.

Co-occurrence of the multicopper oxidases tyrosinase and laccase in lichens in sub-order peltigerineae.

Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.

Hormone influence on melanogenesis and spots formation

Hormone influence on melanogenesis and spots formation

Hot-Water Extracts from Adzuki Beans (Vigna angularis) Stimulate Not Only Melanogenesis in Cultured Mouse B16 Melanoma Cells but Also Pigmentation of Hair Color in C3H Mice

A hot-water extract of adzuki was obtained by boiling beans of adzuki (Vigna angularis). This hot-water extract was fractionated using HP-20 column chromatography. Its distilled water fraction (WEx) was found to stimulate tyrosinase activity in cultured mouse B16 melanoma cells and hair color pigmentation in C3H mice. At concentrations of 1-3 mg/ml, WEx stimulated melanogenesis without inhibiting cell growth. During this effect, WEx activated tyrosinase-inducing activity in the cells, but did not activate tyrosinase, which exists at an intracellular level. In this study, WEx increased cyclic adenosine-3',5'-monophospate (cAMP) content in the cells and protein kinase A (PKA) activity, and stimulated translocation of cytosolic protein kinase C (PKC) to the membrane-bound PKC. These results suggest that the addition of WEx activates the adenylcyclase and protein kinase pathways and, as a result, stimulates melanogenesis. WEx was found to have pigmentation activity on hair color in C3H mice. It might be useful in anti-graying, protecting human skin from irradiation.

N-Methyl-d-aspartate Receptor Stimulation Activates Tyrosinase and Promotes Melanin Synthesis in the Ink Gland of the Cuttlefish Sepia officinalis through the Nitric Oxide/cGMP Signal Transduction Pathway: A NOVEL POSSIBLE ROLE FOR GLUTAMATE AS PHYSIOLOGIC ACTIVATOR OF MELANOGENESIS

The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway.

The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.

Comparative Biological Activities of α-MSH Antagonists in Vertebrate Pigment Cells

We have previously reported that melatonin was an effective lightening agonist in the teleostSynbranchus marmoratus,the amphibiansRana pipiensandBufo ictericus,and in the lizardAnolis carolinensis.The hormone, previously applied to the preparations, effectively inhibited α-MSH darkening activity in a dose-independent manner, and was also able to reverse MSH-induced darkening. We presently describe the inhibitory effect of the indoleamine on the murine melanoma cell proliferation. Interestingly, the hormone also stimulated tyrosinase activity, with a correlated increase in melanin content. We also demonstrate that in a diverse lizard species,Urosaurus ornatus,the indoleamine was totally ineffective. The competitive MSH antagonistic activity of H-His-d-Arg-Ala-Trp-d-Phe-Lys-NH2has been demonstrated previously inR. pipiensandU. ornatus.Herein, its inhibitory activity is also reported in another lizard species,A. carolinensis.However, this MSH analogue was inactive inS. marmoratus,and in murine melanoma cells. On the other hand, the 7 thru 10 α-MSH fragment, Ac-Phe-Arg-Trp-Gly-NH2, although ineffective inS. marmoratusandR. pipiens,was an α-MSH antagonist inA. carolinensis.Surprisingly, in the melanoma cell line, the MSH fragment exhibited no agonist or antagonist activity, but dramatically potentiated the MSH-induced increase in tyrosinase activity. These data might suggest that the fragment is participating either in the process of facilitation or in positive cooperativity. The present results, taken together with our previously reported data, demonstrate a major interspecies diversity of the MC1 subtype of melanocortin receptor, and point out the relevance of the membrane microenvironment for the final receptor configuration.