Novel method for isolating human melanoblasts from keratinocyte culture.

The characterization of melanoblasts is important for understanding their in vivo development, melanoma formation, and pigment-related disorders. However, no methods have been reported for the isolation of melanoblasts from human skin. Using a 'calcium-pulse' technique, involving the differentiation of human keratinocytes with high calcium and the subsequent spontaneous separation of the epidermal sheets, we effectively isolated human melanoblasts (keratinocyte-adapted melanoblasts, KaMBs) from keratinocyte culture. The KaMBs expressed early melanogenesis-related genes, including BRN2, which is a known melanoblast marker. Moreover, the KaMBs displayed much higher proliferative and growth capacities than the primary melanocytes. Considering that keratinocytes might provide an in vivo-like environment for KaMBs during isolation and in vitro culture, the 'calcium-pulse' technique offers an unprecedented, easy, and efficient method for the isolation of human melanoblasts, retaining the original characteristics of these cells.

Novel inhibitory function of miR-125b in melanogenesis.

MicroRNAs are known to be the important regulators of skin physiology and considered as new therapeutic targets to treat skin diseases. In this study, miR-125b was identified as a potent regulator of steady-state melanogenesis. We found that the expression of miR-125b was inversely related to pigment levels. A miR-125b mimic decreased the expression of pigmentation-related gene and melanin content, implying that miR-125b functions to decrease pigmentation. Moreover, we observed that the reduction in miR-125b expression in pigmented cells was at least partially due to the hypermethylation of the MIR125B-1 promoter, and miR-125b expression was regulated by intracellular cAMP levels.

Human dermal stem/progenitor cell-derived conditioned medium ameliorates ultraviolet a-induced damage of normal human dermal fibroblasts.

Adult skin stem cells are considered an attractive cell resource for therapeutic potential in aged skin. We previously reported that multipotent human dermal stem/progenitor cells (hDSPCs) can be enriched from (normal human dermal fibroblasts (NHDFs) using collagen type IV. However, the beneficial effects of hDSPCs on aged skin remain to be elucidated. In the present study, we analyzed the growth factors secreted from hDSPCs in conditioned medium (CM) derived from hDSPCs (hDSPC-CM) and found that hDSPCs secreted higher levels of bFGF, IGFBP-1, IGFBP-2, HGF, VEGF and IGF-1 compared with non-hDSPCs. We then investigated whether hDSPC-CM has an effect on ultraviolet A (UVA)-irradiated NHDFs. Real-time RT-PCR analysis revealed that the treatment of UVA-irradiated NHDFs with hDSPC-CM significantly antagonized the UVA-induced up-regulation of the MMP1 and the UVA-induced down-regulation of the collagen types I, IV and V and TIMP1 mRNA expressions. Furthermore, a scratch wound healing assay showed that hDSPC-CM enhanced the migratory properties of UVA-irradiated NHDFs. hDSPC-CM also significantly reduced the number of the early and late apoptotic cell population in UVA-irradiated NHDFs. Taken together, these data suggest that hDSPC-CM can exert some beneficial effects on aged skin and may be used as a therapeutic agent to improve skin regeneration and wound healing.

The retinoic acid-induced up-regulation of insulin-like growth factor 1 and 2 is associated with prolidase-dependent collagen synthesis in UVA-irradiated human dermal equivalents.

BACKGROUND: Ultraviolet (UV) A irradiation causes the degeneration of extracellular matrix in the skin dermis, mainly due to disrupted collagen homeostasis, resulting in the photo-aging of human skin. All-trans retinoic acid (ATRA) improves photo-aged human skin in vivo. OBJECTIVES: Although the effects of ATRA on collagen synthesis and MMP regulation are well known, the effects of ATRA on other collagen homeostasis-associated genes have not been elucidated. This study was aimed to study the factors that are pharmacologically associated with the effect of ATRA on collagen homeostasis. METHODS: The gene transcription profile of collagen homeostasis-associated genes was systematically evaluated in three-dimensional human dermal equivalents (HDEs) following UVA-irradiation and/or ATRA treatment. RESULTS: In addition to the expected changes in MMPs and collagen synthesis in HDEs in response to ATRA, prolidase, an important enzyme in the recycling of proline and hydroxyproline from degraded collagen molecules, was significantly decreased by UVA irradiation, and its down-regulation was antagonized by ATRA. Transfection with a prolidase-specific siRNA led to a significant decrease in procollagen synthesis in human fibroblasts. ATRA inhibited the UVA irradiation-induced decrease in prolidase activity through an insulin-like growth factor (IGF) receptor signaling pathway in HDEs. ARTA increased IGF1 and IGF2 production in HDEs, and neutralizing IGFs with anti-IGF antibodies abolished the effect of ATRA on proliase activity. CONCLUSIONS: These data demonstrate that ATRA regulates prolidase activity in HDEs via IGF receptor signaling, suggesting one of the pharmacological mechanisms by which improves photo-aged human skin.

Antioxidant effects of the ethanol extract from flower of Camellia japonica via scavenging of reactive oxygen species and induction of antioxidant enzymes.

The aim of this study was to investigate the antioxidant properties of the ethanol extract of the flower of Camellia japonica (Camellia extract). Camellia extract exhibited 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species (ROS) scavenging activity in human HaCaT keratinocytes. In addition, Camellia extract scavenged superoxide anion generated by xanthine/xanthine oxidase and hydroxyl radical generated by the Fenton reaction (FeSO(4) + H(2)O(2)) in a cell-free system, which was detected by electron spin resonance spectrometry. Furthermore, Camellia extract increased the protein expressions and activity of cellular antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. These results suggest that Camellia extract exhibits antioxidant properties by scavenging ROS and enhancing antioxidant enzymes. Camellia extract contained quercetin, quercetin-3-O-glucoside, quercitrin and kaempferol, which are antioxidant compounds.

New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS).

The immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the water-in-oil (W/O) emulsion system were studied. The terminal amine group was used as the important functionality for GOD immobilization on the silica substrate. When only TEOS is used as a silica source, the disordered mesoporous silica microspheres are obtained. As the molar ratio of APTMS to TEOS (R(AT)) increases, the surface area and pore volume of the silica particles measured by nitrogen adsorption and desorption method and SEM decrease rapidly. Particularly, the largest change of the surface morphology is observed between R(AT)=0.20 and R(AT)=0.25. The amount and the adsorption time of immobilized enzyme were measured by UV spectroscopy. About 20wt% of GOD was immobilized into the silica substrates above R(AT)=0.60 and was completely adsorbed into the substrate of R(AT)=0.80 with lapse of 4h after addition. In the measurement of the thermal stability, GOD dissolved in buffer solution loses nearly all of its activity after 30 min at 65 degrees C. In contrast, GOD immobilized on the surface-modified silica particles still retains about 90% of its activity after the same treatment. At this temperature, the immobilized glucose oxidase retained half of its initial activity after 4h. It is shown that the suitable usage of functionalizing agent like APTMS as well as the control of surface morphology is very important on the immobilization of enzyme.

The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-water double emulsion by controlling pH and electrolyte concentration.

This study presents a new approach that can stabilize effectively L-ascorbic acid in water-in-oil-in-water (w/o/w) double emulsions. Basically, the behavior of L-ascorbic acid in the aqueous phase was observed, considering its molecular deformation. Then, it was found that the stability determined in the aqueous phase by high-performance liquid chromatography (HPLC) showed that the collapse of ionization of L-ascorbic acid played a crucial role in protecting the molecular deformation. Then, the stable aqueous system was incorporated into the internal aqueous phase of the double emulsions. From the HPLC analysis, it was observed that the L-ascorbic acid in an appropriate system showed high molecular stability for a long time. Moreover, in the measurement of in vitro skin permeation, the L-ascorbic acid stabilized in this study showed considerable skin permeation ability, indicating its potential applicability in pharmaceutics and cosmetics.

Depigmenting activity and low cytotoxicity of alkoxy benzoates or alkoxy cinnamte in cultured melanocytes.

To obtain effective and safe topical depigmenting agents, we synthesized hydroxybenzoates, alkoxybenzoates, and 3,4,5-trimethoxycinnamate containing a thymol moiety and screened then for high-level inhibitory activity against melanin synthesis in cultured melanocytes. Eight compounds were tested for their depigmenting effect and cytotoxicity using a murine melanocyte cell line. We found that 3,4,5-trialkoxybenzoates and 3,4,5-trimethoxycinnamate, synthesized by conjugating 3,4,5-trialkoxybenzoic acids and 3,4,5-trimethoxycinnmic acid with thymol, showed a potent depigmenting effect and low cytotoxicity. Compound 4h, 5-methyl-2-(methylethyl)phenyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate, showed the most potent depigmenting effect (IC50=10 microM) with low cytotoxicity (IC50=200 microM).

Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent.

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.

Nano-structured biphasic polymer film on the hair surface from PEGylated polymer latexes.

In this study, biphasic polymer latexes were synthesized by surfactant-free-emulsion polymerization of butyl methacrylate, poly(ethylene glycol) methyl ether methacrylate, and 2-(methacryloyloxy) ethyl trimethyl ammonium chloride. The latexes synthesized were composed of hydrophobic core phase and hydrophilic shell phase. Nano-structured film morphology could be obtained by annealing the biphasic polymer latexes between the two transition temperatures. It was found that the unique film morphology gave a viscoelastic property to the film. Scanning electron microscope and atomic force microscope images revealed that the biphasic polymer latexes deposited effectively onto the entire hair surface upon conditioning with 1 wt% polymer concentration in water. Consequently, they formed a smooth polymer membrane thereon, showing a high potential for a new hair cosmetic ingredient.