Although many hypo-pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole-animal model for phenotype-based screening of melanogenic inhibitors or stimulators. We used both the well-known melanogenic inhibitors (1-phenyl-2-thiourea, arbutin, kojic acid, 2-mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost-effectiveness, and physiological relevance.

ABSTRACT. A seven-year-old male elk (Cervus elaphus nelsoni) was euthanized and necropsied after having a 3-week history of body weight loss, emaciation, excessive salivation, teeth grinding, fever, anorexia, and respiratory distress. The elk was imported into Korea from Canada on March 9, 1997. Gross pathologic findings were restricted to a diffuse fibrinous pneumonia. Microscopic lesions included mild neuronal vacuolation and spongiform change in the neuropil of selected brain stem nuclei and generalized astrocytosis. Immunohistochemistry for protease-resistant prion protein (PrP res ) was positive in all brain sections but more pronounced in the section of the obex of the medulla. And the PrP res was also detected by western immunoblotting in the brain and spinal cord. All the remaining elk and deer that had been in contact with this elk were destroyed and negative for chronic wasting disease (CWD). To our knowl edge, this is the first case of CWD occurring outside of the U.S.A. and Canada.

Toll-like receptors (TLRs) play a critical role in sensing microbial components and inducing innate immune and inflammatory responses by recognizing invading microbial pathogens. Lipopolysaccharide-induced dimerization of TLR4 is required for the activation of downstream signaling pathways including nuclear factor-kappa B (NF-kappaB). Therefore, TLR4 dimerization may be an early regulatory event in activating ligand-induced signaling pathways and induction of subsequent immune responses. Here, we report biochemical evidence that 6-shogaol, the most bioactive component of ginger, inhibits lipopolysaccharide-induced dimerization of TLR4 resulting in the inhibition of NF-kappaB activation and the expression of cyclooxygenase-2. Furthermore, we demonstrate that 6-shogaol can directly inhibit TLR-mediated signaling pathways at the receptor level. These results suggest that 6-shogaol can modulate TLR-mediated inflammatory responses, which may influence the risk of chronic inflammatory diseases.

The development of effective skin-lightening agents is an increasingly important area of research aimed at the treatment of hyperpigmentation induced by UV irradiation or by medical conditions such as melasma, postinflammatory melanoderma and solar lentigo. Although some inhibit tyrosinase, identifying and understanding the mechanisms of action of other agents is an important goal if more effective pigmentation inhibitors are to be developed. We present here that an extract of Lepidium apetalum (ELA) decreased UV-induced skin pigmentation in brown guinea pigs and melanogenesis of HM3KO human melanoma cells. Interestingly, ELA did not reduce melanogenesis in HM3KO cells unless they were co-cultivated in keratinocyte-conditioned medium prepared by culturing keratinocytes with ELA. Under these conditions, ELA decreased tyrosinase mRNA and protein expression as well as melanin content via an ELA-mediated increase in keratinocyte IL-6 production which in turn was shown to decrease in the expression Mitf, a transcription factor implicated in tyrosinase gene expression and melanocyte differentiation. The results reveal that ELA may be an effective inhibitor of hyperpigmentation caused by UV irradiation or by pigmented skin disorders through a mechanism involving IL-6-mediated downregulation of Mitf rather than a direct inhibition of tyrosinase activity.

Current therapies are still unsatisfied for skin pigmentation disease conditions. Furthermore, Asian women prefer lighter skin color and there is a great demand to develop more safe and effective skin whitening agents. Recently, many efforts have been paid to develop new therapeutic agents against pigmentation abnormalities, especially using novel biologically active compounds from natural plants. Many tyrosinase inhibitors that suppress melanogenesis have been actively studied with the aim of developing new whitening agents. 1-3)Medicinal plants are most suitable for pharmacological research and drug development, because their constituents can be used not only as therapeutic agents but also as starting materials or models for the synthesis of drugs or pharmacologically active compounds. We are interested in re-evaluation of traditional Chinese herbs on melanogenesis from huge resources of famous whitening formulations and recipes in ancient literature. Traditional Chinese medicine has been used for the treatment of chloasma for a long history. Many famous ancient formulations and recipes are still in use to treat pigmentation disorders. Though these drugs were reported to be clinically effective in China, the mechanism and the active components have not been studied yet. Evaluation of Chinese herbal medicine in the treatment of skin pigmentation abnormalities may be beneficial for the development of new and more efficient remedies. In China, search for depigmentation medicine from traditional Chinese herbs is currently focused on those having inhibitory activity to tyrosinase. In a previous screening study performed by Japanese researchers with mushroom tyrosinase, several extracts of crude traditional Chinese drugs showed highly inhibitory activity. 4) Results from these studies provided important information about traditional Chinese herbs on melanogenesis. However, melanin biosyntheis is a complicated process involving many factors including the key enzymes tyrosinase, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), cytokines from autocrine and paracrine and those related to melanin transportation and decomposition. 5,6) Mushroom tyrosinase test is a simple method but with some disadvantages. Plant tyrosinase is different from mammalian tyrosinase because of its unique requirements for substrate and cofactor as well as its different sensitivity to inhibitors. 7,8) Several papers published in Chinese indicated that many plant extracts showing inhibitory activity to mushroom tyrosinase in vitro did not reduce pigmentation activity in cells. Also, some compounds tested on mammalian tyrosinase did not give comparable results with mushroom tyrosinase.9) Thus, this study was undertaken to evaluate the depigmentation effect of traditional Chinese herbs based on melanocyte cell culture assays. MATERIALS AND METHODS Preparation of Herb ExtractsIdentification of the herbs used in this study was provided by the Union Bioengineering Institute (Beijing, China). The raw herbs were extracted wit...

EPR spenra of Fe3+ ions in single crystals of KliOPOr (KTP), synthesized by the Eux method, have been investigated at mom temperature by employing a Bruker Q-band spectrometer. From the angular dependence of the EPR spectm two Fe3+ centres denoted as C1 and C2 have been identified, in agreement with the previous analysis by other investigators. In this study, for the first time we have fully identified two groups of four magnetically inequivalent Fe3+ sites each belonging to the centres C1 and C2. Two sets of triclinic spin-Hamiltonian parameters, which simultaneously fiited EPR data for the four sites belonging to the centres C1 and Q, were determined. The direction cosines of the principal axes of the g-tensor as well as the second-order zero-field splitting (ZFS) tensor are found to be given by the relations: Imn, imn, lrirn, and i%n for each four F$+ sites of C1 as well as C2, respectively, consistent with the crystallographic point group mm2 of KTP.

The SWI/SNF chromatin-remodeling complex functions as a transcriptional regulator and plays a significant role in cell proliferation, differentiation and embryonic development. SRG3, a homologue of human BAF155, is a core component of the mouse SWI/SNF chromatin remodeling complex. Mutant mice deficient in Srg3 expression are peri-implantation lethal. To investigate the role of SRG3 in the post-implantation stage, we generated SRG3 transgene-rescued (Srg3-/-Tg+) embryos by inducing exogenous gene expression. These Srg3-/-Tg+ embryos overcame early embryonic lethality and extended the life span until mid-gestation. However, the embryos displayed significant defects in blood vessel formation and fetal circulation within the yolk sac around embryonic day 10.5, which led to developmental retardation and death. We found that SRG3 expression was absent in the visceral endoderm of Srg3-/-Tg+ yolk sacs, while SRG3 was normally expressed in wild-type embryos. In addition, expression of angiogenesis-related genes, including Angiopoietin1, Tie2, EphrinB2, Ihh and Notch1, was markedly reduced in Srg3-/-Tg+ yolk sacs. During normal angiogenesis, maturation of the visceral endoderm development is observed in the yolk sac. However, in Srg3-/-Tg+ yolk sacs, the visceral endoderm did not develop normally. Our results indicate that SRG3 is required for angiogenesis and visceral endoderm development in the yolk sac.

Tyrosinase, a type I membrane glycoprotein, is synthesized and glycosylated in the endoplasmic reticulum (ER) and Golgi. The enzyme is subsequently transported to melanosomes where it participates in melanogenesis. Previous studies showed that the disruption of early ER N-glycan processing by deoxynojirimycin (DNJ), an inhibitor of alpha-glucosidase, suppresses tyrosinase enzymatic activity and melanogenesis. However, the disruption of late glycan processing, mainly performed by ER and Golgi alpha-1,2-mannosidases, on tyrosinase enzymatic activity and melanogenesis remains to be investigated. Following treatment of HM3KO human melanoma cells with deoxymannojirimycin (DMJ), an inhibitor of alpha-1,2-mannosidase, transport of tyrosinase to the melanosome, enzymatic activity, and melanogenesis were reduced in a dose-dependent manner. However, DMJ did not directly inhibit tyrosinase enzymatic activity and expression. Interestingly, an extract of Streptomyces subrutilus culture medium (ESSCM) containing DMJ and DNJ as the main components inhibited glycosylation and transport of tyrosinase to the melanosome as well as melanin synthesis, but with no negative effects on cell viability. These inhibitory effects of ESSCM were stronger than those of DMJ or DNJ alone. Tyrosinase glycosylation and melanogenesis in HM3KO melanoma cells were more effectively inhibited by DMJ and DNJ combined than DMJ or DNJ alone. Accordingly, we propose that ESSCM is a potential candidate for treating undesirable hyperpigmentation conditions, such as melasma, postinflammatory melanoderma, and solar lentigo.