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Secreted frizzled-related protein 5 (SFRP5) plays a pivotal role in regulating the development of many tissues and organs, however, as an inhibitor of Wnt signaling, the role of SFRP5 in vitiligo remains unknown. Hence, we speculated that SFRP5 might be associated with melanogenesis in melanocytes by regulating Wnt signaling in vitiligo. In this study, we found that SFRP5 was overexpressed in the skin lesions of patients with vitiligo. Compared with that in normal epidermal melanocytes (PIG1), the expression of SFRP5 was increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5). The results showed that SFRP5 overexpression inhibited melanin synthesis in PIG1 cells through downregulation of microphthalmia-associated transcription factor (MITF) and its target proteins via suppression of the Wnt/β-catenin signaling pathway. Accordingly, SFRP5 silencing increased melanin synthesis and activated the Wnt signaling pathway in PIG3V cells. Moreover, SFRP5 overexpression also downregulated the transcriptional activity of T cell factor/lymphoid enhancer factor (TCF/LEF) in PIG1 cells. Furthermore, this inhibitory effect of SFRP5 on melanin synthesis was reversed by treatment with the β-catenin agonist, SKL2001. The inhibitory action of SFRP5 in pigmentation was further confirmed in vivo using a nude mouse model. Hence, our results indicate that SFRP5 can inhibit melanogenesis in melanocytes. Additionally, our findings showed that SFRP5 plays a vital role in the development of vitiligo, and thus may serve as a potential therapeutic target for vitiligo.

Background Tyrosinase plays a key role in the formation of skin melanin. The excessive accumulation of skin melanin will cause the serious aesthetic problems for human beings. Hypothesis/purpose To find the potent tyrosinase inhibitors using computational simulation from TCM Database@Taiwan. Study design Inhibitors of tyrosinase have been thought as potential drugs for the decrease of melanin synthesis in the process of pigmentation. To develop new tyrosinase inhibitors, we performed a virtual screening from Traditional Chinese medicine (TCM) and Druglike Databases using the best 3D QSAR pharmacophore model as a 3D search query. Methods A total of 109 compounds were obtained after filtering by Lipinski's rule of five. Finally, 148 compounds (22 from training set, 17 from test set, 109 from TCM and Druglike databases) were selected for further docking studies. De Novo Evolution designed the top 10 candidates from the docking results. Results Hypo1 was selected as the best quantitative pharmacophore model, because Hypo1 has characters of the highest cost difference (353.773), the lowest RMS (1.985), the lowest Error (121.440), and the best correlation coefficient (0.933). By the analysis of interaction amino acids in the top 10 hits including two controls, HIS42, HIS60, HIS204, HIS208, ARG209 and VAL218 are identified as the key binding site residues, ARG209 and VAL218 are the critical residues for the inhibitory activity of tyrosinase. This finding is consistent with the results from literatures. Conclusion De Novo Evolution study suggested Tyrosinase_1*_Evo_4, Tyrosinase_23*_Evo_7, magnolone.cdx_15_Evo_4, compound_2.cdx_2_Evo_2, Compound_B_Evo_5, Compound_C_Evo_9, Compound_D_Evo_6 and malabaricone_C.cdx_3_Evo_10 as the potential tyrosinase inhibitor candidates. De Novo Evolution study also suggested compound_2.cdx_2_Evo_2 as the most potential tyrosinase inhibitor candidate. A total of ten novel leading compounds were identified to have the favorable interaction with tyrosinase by the docking analyses.

Melanocyte homoeostasis and their response to ultraviolet radiation (UVR) are mediated to a large extent by keratinocyte-derived factors, many of which have been well-characterized. Lee et al. describe novel effects of adenosine 5'-triphosphate (ATP), which is secreted by keratinocytes and can stimulate melanogenesis by melanocytes following UVA exposure. The investigators attribute the melanogenic effect of ATP to binding purinergic receptors type 2 X7 (P2X7), which are expressed on human melanocytes, leading to activation of the protein kinase C pathway. This report is the first to identify expression of specific purinergic receptors on human melanocytes, and it suggests ATP as a signaling molecule that stimulates pigmentation. Follow up on these results should clarify the physiological role of ATP in mediating the tanning response to solar UVR.

Introduction The study investigated a natural solution for the hypopigmentary disorder, progressive macular hypomelanosis (PMH). The aim was to determine whether gold nanoparticles synthesized from Aspalathus linearis (Burm.f.) R.Dahlgren could be effective for PMH. Methods Gold nanoparticles (ALAuNPs) were synthesized using the ethanolic extract of A. linearis (ALEtOH) and tested, along with the extract for its regulatory effect of melanogenesis in human melanocytes and its antibacterial activity against Cutibacterium acnes (ATCC 6919 strain). Melanosome transfer was determined in a co-culture of normal human melanocytes and keratinocytes and was quantified using flow cytometry. Results The ALAuNPstab (AuNPs stabilized with gum arabic) and ALAuNPnon-stab (non-stabilized) exhibited significantly (P Conclusion This study confirmed that A. linearis and its biosynthesised gold nanoparticles stimulated melanin synthesis.

A new series of chalcone derivatives 1-18, bearing isoxazole moieties were designed and synthesized, and biologically evaluated for their activity on mushroom tyrosinase and melanin synthesis in murine B16 cells. The result indicated that most of prepared compounds 1-18 showed potent activating effect on tyrosinase, especially for 1-2, 4, 6-7, 9 and 15. Among them, compounds 2, 4 and 9 demonstrated the best activity with EC50=1.3, 2.5 and 3.0μmol·L-1 respectively, much better than the positive control 8-methoxypsoralan (8-MOP, EC50=14.8μmol·L-1); In B16 cells, all the tested compounds exhibited a stronger activity on melanogenesis than 8-MOP (with the value of 115%). It was interesting that derivatives substituted with halogen (1, 2, 4, 5, 7, 9) were generally more potent. Compounds 2 (463%) and 18 (438%) with 3 and 4-fold potency compared with 8-MOP respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.

Furocoumarins, isolated from Psoralen corylifolia L., were found to be the most effective drug in the treatment of vitiligo nowadays. Twenty-five furocoumarin derivatives were thus designed and synthesized in order to improve the melanogenesis in B16 cells for the first time. Among them, twenty-three compounds were more potent than the positive control (8-MOP), the commonly used drug for vitiligo in clinic. Noticeably, compounds 6m (350.5%) and 6p (313.1%) based on the scaffold of 6k (2H-benzofuro[2,3-h]chromen-2-one) were nearly 3-fold stronger than 8-MOP (114.50%). The in vitro melanin synthesis evaluation of these structurally diverse analogues had also led to an outline of structure-activity relationship.

Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I1, a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.

Kojic acid (KOJ) is a melanin synthesis inhibitor widely used as skin lightening agent in topical preparations. Unfortunately it is easily susceptible to photo-oxidation, phenomenon responsible for chemical and organoleptic modifications. The aim of this work was the intercalation of KOJ in hydrotalcite-like compounds (HTlc) in order to stabilize KOJ and to reduce its photolability. Hydrotalcite containing Zn and Al (ZnAl-HTlc) was used as host to obtain the final compound ZnAl-HTlc-KOJ. The intercalation was carried out, after many attempts, by ionic exchange mechanism by means of the strong base EtO

Zinc alpha-2-glycoprotein is secreted by a variety of normal and malignant epithelial cells and overexpression by tumors has been implicated in cancer cachexia. To investigate biologic properties of zinc alpha-2-glycoprotein further, stable transfectants of recombinant human zinc alpha-2-glycoprotein were created in the B16F10 murine melanoma cell line. Both B16-recombinant human zinc alpha-2-glycoprotein clones with strong expression of zinc alpha-2-glycoprotein and vector-transfected B16 cells treated with exogenous zinc alpha-2-glycoprotein had decreased melanin production in vitro. Furthermore, B16-recombinant human zinc alpha-2-glycoprotein clones formed amelanotic tumors in vivo, despite their melanin production in vitro. Although no qualitative differences in tyrosinase mRNA expression could be detected by reverse transcription-polymerase chain reaction, B16-recombinant human zinc alpha-2-glycoprotein tumors had decreased levels of tyrosinase protein and minimal tyrosinase activity. Purified zinc alpha-2-glycoprotein also decreased tyrosinase activity in vector-transfected B16 tumor sections in vitro. Taken together, these studies demonstrate that zinc alpha-2-glycoprotein inhibits melanin production by B16 melanoma cells via post-transcriptional effects on tyrosinase protein. As zinc alpha-2-glycoprotein decreases melanin synthesis more strongly in vivo than in vitro, however, it is likely that zinc alpha-2-glycoprotein affects melanin synthesis through indirect mechanisms as well. Zinc alpha-2-glycoprotein also inhibits melanin production by melan-A primary melanocytes in vitro. As zinc alpha-2-glycoprotein is normally produced by epidermal keratinocytes, these studies raise the possibility that epidermal-derived zinc alpha-2-glycoprotein may play a part in normal regulation of melanin production in vivo, in addition to its previously described role in cancer cachexia.

Purpose: To evaluate the clinical significance of 5-S-cysteinyldopa (5-S-CD), a major intermediate in melanin synthesis, as a potential diagnostic tumor marker for uveal malignant melanoma. Methods: The levels of 5-S-CD in the serum were measured by high-performance liquid chromatography in 16 patients with primary uveal melanoma. The levels of 5-S-CD were also measured in both aqueous and vitreous humor in 10 patients with uveal melanoma. The serum of healthy volunteers and patients with skin diseases other than melanoma, and the intraocular fluids of patients with cataract and vitreoretinal diseases were used as controls. Results: Serum concentrations of 5-S-CD in patients with uveal melanoma in the absence of extraocular metastases were close to those of controls; however, serum concentrations of 5-S-CD were significantly elevated in patients with extraocular metastases of melanoma. Concentrations of 5-S-CD in the intraocular fluids, especially vitreous humor, were higher in patients with uveal melanoma than in controls. Conclusions: 5-S-CD in intraocular fluids may serve as a useful biochemical marker for the diagnosis of uveal melanoma. Serum 5-S-CD may contribute to the assessment of the presence and progression of extraocular metastases in patients with uveal melanoma.

Most hypopigmenting reagents target the inhibition of tyrosinase, the key enzyme involved in melanin synthesis. In this issue, Choi et al. report that NADPH:quinone oxidoreductase-1 (NQO1) increases melanin synthesis, probably via the suppression of tyrosinase degradation. Because NQO1 was identified by comparing normally pigmented melanocytes with hypopigmented acral lentiginous melanoma cells, these results suggest various hypotheses regarding the carcinogenic origin of the latter.

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with 13C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.

Money, N. P., and Howard, R. J. 1996. Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement.Fungal Genetics and Biology20,217–227. A novel approach to turgor measurement has confirmed a link between melanin synthesis and appressorial turgor pressure in the rice blast fungusMagnaporthe grisea.The new technique employs a cold stage to measure the melting point of ice within individual cells. Turgor is then calculated from the difference between the melting points of intracellular ice and ice in the fluid surrounding the cell. Conidia ofM. griseawere incubated on various surfaces, and appressoria developed within 6 h. Measurements from conidia showed that wild-type and melanin-deficient strains generated the same pressures, which decreased before germ tube emergence. By contrast, wild-type appressoria generated much higher pressures than appressoria of a melanin-deficient buf−mutant. These experiments strengthen the hypothesis that melanin confers pathogenicity by facilitating the development of high pressures within appressoria.

Purpose To report a case of bilateral bull's-eye maculopathy in a patient who ingested uva ursi, a known inhibitor of melanin synthesis, for 3 years before the onset of symptoms. Design Observational case report. Methods Both eyes of a female patient were examined in the clinical practice setting. Results A 56-year-old woman who ingested uva ursi for 3 years noted a decrease in visual acuity within the past year. Ocular examination including fluorescein angiography revealed a typical bull's-eye maculopathy bilaterally. Conclusions Uva ursi is a known inhibitor of melanin synthesis. It is necessary to broaden the list of potential retinal toxic drugs to include herbal adjuvants such as Uva Ursi and to elicit a history of their use in patients with unexplained ocular findings.

Melanin synthesis in the mouse involves the interaction of many pigmentation loci. Tyrosinase, the product of the albino (c) locus, catalyses the first step of the pathway. The brown (b) locus protein has significant homology to tyrosinase and controls black/brown coat coloration, but its function is controversial. To investigate the function of the b-protein and its interaction with tyrosinase, we established cell lines expressing both tyrosinase and the b-protein by transfecting tyrosinase-expressing fibroblasts with a b-protein expression vector. The tyrosinase-expressing parent line does not have l-dopachrome tautomerase activity, but this enzyme is detectable in double transfectants as well as in fibroblasts expressing the b-protein alone. Cells expressing both proteins have a higher steady-state level of tyrosinase than fibroblasts expressing tyrosinase alone, and contain elevated levels of melanin intermediates. This is thought to result from interaction of tyrosinase with the b-protein. Only phaeomelanin is detectable in fibroblasts expressing tyrosinase alone, whereas double transfectants synthesise significantly more phaeomelanin and detectable eumelanin.

Black pigment extracted from wings of Precis coenia was identified as melanin by solubility characteristics and incorporation of radiolabeled melanin precursors. Different colours in wing scales appeared successively, starting with white followed by red, black and grey pigments. At corresponding times [14C]-tyrosine and [14C]-3,4-dihydroxyphenylalanine injected into pupae were incorporated most intensively into black and grey scales. By contrast, red scales were labeled by 3,4-dihydroxyphenylalanine (DOPA) and, to a lesser extent, by tyrosine when injected at the time of red pigment synthesis. Labeled β-alanine, a component of the sclerotizing agent N-β-alanyldopamine, was incorporated into all scales with the exception of intensely black scales. It is discussed whether the mechanical stability of black scales may be due to melanin itself whereas the stability of all other scales may be due to N-β-alanyldopamine. Amounts of extractable melanin increased during visible pigment formation in black and grey scales. When [14C]-tyrosine was offered to isolated wings in Grace's medium before melanization, 93% of incorporated activity was found in the hydrolysable fraction and only 7% in the melanin fraction. However, when supplied during intensive melanin synthesis up to 70% was incorporated into melanin. The same incorporation pattern resulted after injection into intact developing pupae. Incorporation of [14C]-tyrosine into melanin began earlier, and was higher in presumptive black scales than in grey scales. Incorporation into white scales was always low. DOPA decarboxylase (DDC) activity in whole wings was low during the pupal stage but increased at the time when scale melanization started. DDC activity in different coloured wing pieces increased first in presumptive black pattern elements, the eyespots. DDC activity in the eyespots was 3.5 times higher than in presumptive white parts of the wing; it was intermediate in grey areas. This demonstrates that wing colour pattern as well as colouration intensity are related to selective activity of DDC in time and different locations resulting in different amounts of melanin in grey and black scales.

SIR—A rise in newly diagnosed cases of melanoma and lack of effective treatment have stimulated the search for new methods to detect and treat the disease early. Melanoma is a pigmented tumour. A non-pigmented form that lacks tyrosinase (the key catalyst in melanin synthesis) is very uncommon. By virtue of its high affinity for melanin, methylene blue (MTB) accumulates selectively in melanoma cells, and is a useful carrier for radioisotopes. 1,2 One of the radioderivatives, 211 At-MTB ( 211 Astatine, t1/2=7·214 h) prevents metastatic spread and controls the growth of melanoma when given to human melanoma-bearing animals. 3,4 Evaluation of 211 At-MTB in human beings showed that uptake in melanoma cells is sufficiently high to enable also detection of early metastases. 5

Colony differentiation in Microdochium bolleyi, i.e. the transformation of hyaline cells into melanin-pigmented chlamydospores, was found to be markedly affected by medium composition and light, potato dextrose agar producing the most intense pigmentation of the media tested. Spectra of alkaline extracts of M. bolleyi melanin absorbed in the ultraviolet region; logarithmic plots of such spectra between 380 and 540 nm gave a series of straight lines. Under alternating light/dark regimes, zones of dark pigmentation were produced that coincided with the light phases. This rhythm was found to be exogenous in nature and ceased when uniform external conditions were re-established. The implications of these findings are discussed.

Structural differences in melanins were studied by Curie-point pyrolysis-gas chromatography-mass spectrometry, electron spin resonance spectroscopy and chloroquine binding. Pyrolysis-gas chromatographic data on the polymer allow the calculation of a UU index that expresses the relative amount of unindolized DOPA-derived units in the melanins. It is shown that the content of unindolized units in the polymer depends strongly on the oxidation time and DOPA concentration used during the melanin synthesis. Comparison of the chloroquine binding to melanins and the pyrolysis data showed that there is an inverse relationship between the affinity of chloroquine and the UU index.

The presence of copper during growth of Aureobasidium pullulans in both liquid and solid media had a profound effect on morphology of the organism. In addition to stimulating melanin synthesis copper also promoted formation of filaments or hyphae during later stages of growth. The taxonomic implications of these findings are discussed.

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L -3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-products(s) inhibiton or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.

Recent advances in the biology of mammalian pigmentation are reviewed. The multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone forms the focal point for melanin metabolism within mammalian epidermis. Within an epidermal melanin unit, melanosomes are synthesized by melanocytes and transferred to keratinocytes where they are degraded as they ascend to the epidermal surface. During the past 25 years, technical advances in biology and biochemistry have fostered a multi- disciplinary approach to research on mammalian pigmentation. Emphasizing this perspective, we have examined the current state of knowledge of the form and function of epidermal melanin units from the levels of biologic organization ranging from the molecules relevant to melanin synthesis through the skin as a totally integrated system. To an unusual degree, advances in melanin pigmentation have resulted from the integration of clinical medicine and basic science.

The concentration of dopa, dopamine and cysteinyldopa were measured in various parts of the mature bovine eye, using a fluorescent assay. Special attention was paid to cysteinyldopa levels, since this compound is thought to be a specific marker of melanin turnover . Levels of cysteinyldopa in the iris and choroid/retinal pigment epithelium were indicative of the presence of active melanin synthesis in the adult eye. This data is presented as confirmatory evidence for the recent documentation of active tyrosinase activity in adult mammalian uveal tracts.

The purpose of this study was to investigate with high resolution autoradiography, the biosynthesis and intracellular transport of melanin within the mammalian pigment cell. The site of melanin synthesis within the pigment cell has been a topic of controversy for many years (1), and many diverse cell areas and organelles have been proposed as likely candidates (2-9). Despite much work the problem remains unsettled.

The concept that mast cells are capable of melanin synthesis was proposed at the turn of the century (1-4), and was based on the observation of melanin granules in some mast cells. Critics of this theory (5, 6) explained the presence of melanin in mast cells on the basis of phagocytosis rather than synthesis.