Biochemical characterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall.

The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3-14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth.

MHY884, a newly synthesized tyrosinase inhibitor, suppresses UVB-induced activation of NF-κB signaling pathway through the downregulation of oxidative stress.

The skin is the primary target of prolonged and repeated ultraviolet (UVB) irradiation which induces cutaneous inflammation and pigmentation. Nuclear factor κB (NF-κB) is the major factor mediating UVB-induced inflammatory responses through the expression of various proinflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We have previously reported that the synthetic novel compound 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) strongly suppressed tyrosinase activity and melanin synthesis in B16F10 melanoma cells. In the present study, we investigated the effect of MHY884 on the inhibition of UVB-induced NF-κB activation and its proinflammatory downstream proteins through the suppression of oxidative stress in an in vivo model of photoaging. Generation of reactive oxygen species (ROS) and peroxynitrite was measured in vitro and in B16F10 melanoma cells to verify the scavenging activity of MHY884. MHY884 suppressed oxidative stress both in vitro and in the melanoma cells in a dose-dependent manner. Next, melanin-possessing hairless mice were pre-treated with MHY884 and then irradiated with UVB repeatedly. Topical application of MHY884 attenuated UVB-induced oxidative stress, resulting in reduced NF-κB activity. Pre-treatment with MHY884 inhibited Akt and IκB kinase α/ß signaling pathways, leading to decreased translocation and phosphorylation of p65, a subunit of NF-κB. This result correlated with the expression levels of iNOS and COX-2 in the skin of MHY884-treated mice. Thus, the novel tyrosinase inhibitor MHY884 suppressed NF-κB activation signaling pathway by scavenging UVB-induced oxidative stress. The discovery of MHY884, a novel tyrosinase inhibitor that targets NF-κB signaling, is significant, because this compound is a promising protective agent against UVB-induced skin damage.

Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP).

BACKGROUND: Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). METHODS: The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo. RESULTS: The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50=0.53 µM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50=8.2 µM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP. GENERAL SIGNIFICANCE: The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.

Synthesis of novel azo-resveratrol, azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors.

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 µM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC(50) value of IC(50)=36.28 ± 0.72 µM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.

Mechanism of attenuation of pro-inflammatory Ang II-induced NF-κB activation by genistein in the kidneys of male rats during aging.

Angiotensin II (Ang II), a main effector of the renin-angiotensin system, is recognized as a pro-inflammatory mediator on age-related vascular inflammation. Ang II is one of the most important oxidative stress inducer, activates the redox-sensitive transcription factor, nuclear factor-κB (NF-κB) during aging. Genistein, a major component found in isoflavone, has anti-inflammatory activities that are often associated with its anti-oxidative activity. The purpose of this study is to document molecular mechanism of altered Ang II-related NF-κB activation during aging and inhibitory molecular events by genistein regarding to age-related Ang II-induced NF-κB activation. At present, we utilized young (6 months old), old (24 months old), and genistein-treated (2 and 4 mg/kg/day for 10 days) old rats. For our current study, we choose to use the kidney and rat endothelial cell line, YPEN-1 because of its vulnerability to age-related oxidative stress and inflammatory responsiveness. The results of the analysis showed that Ang II and AT1 expression increased during aging and that these increases were blunted by treatment with genistein. Furthermore, we investigated the inhibitory effects of genistein on the Ang II-induced redox imbalance in aged rat kidneys. Genistein reduced age-related increases in NF-κB activity and NF-κB-dependent pro-inflammatory genes expression. We also determined genistein attenuated Ang II-induced NF-κB activation through its anti-oxidant activity in YPEN-1 cells. Taken together, our present results show that genistein has potent anti-inflammatory effect resulting in the attenuation of the Ang II-induced NF-κB activation during aging. The most significant new finding from this study is that genistein exerts its anti-Ang II action during aging by suppressive effect of NF-κB activation. Based on these data, genistein may be an anti-Ang II agent that may be used in anti-inflammatory therapies.

Suppression of melanogenesis by a newly synthesized compound, MHY966 via the nitric oxide/protein kinase G signaling pathway in murine skin.

BACKGROUND: Ultraviolet B (UVB) radiation is the main physiological stimulus for skin pigmentation. Nitric oxide (NO) and the NO/PKG signaling pathway play an important role in UVB-induced melanogenesis, which is related to the induction of expression of tyrosinase. In an attempt to find a novel anti-melanogenic agent, we synthesized a new compound, 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl) phenol (MHY966). OBJECTIVE: The purpose of this study was to investigate the action of MHY966 on NO and the NO-mediated signaling pathway using in vitro and in vivo models of melanogenesis. METHODS: NO generation, melanin synthesis, and the expression of tyrosinase and PKG were measured in B16F10 melanoma cells to verify the anti-melanogenic effect of MHY966 in vitro. Next, melanin-possessing hairless mice were pre-treated with MHY966 and then irradiated with UVB repeatedly. Morphological, histological, and biochemical analyses including the expressions of PKG, tryosinase and nuclear MITF, and productions of nitric oxide, peroxynitrite and ROS were conducted. RESULTS: MHY966 effectively inhibited NO generation and subsequent melanin synthesis induced by sodium nitroprusside, an NO donor, and suppressed the expression of tyrosinase and PKG. Topical application of MHY966 dose-dependently attenuated UVB-induced pigmentation in a mouse model. This hypopigmentation effect induced by MHY966 treatment was mediated by the down-regulation of tyrosinase, PKG, and nuclear MITF, which was accompanied by decreased NO and NO-related oxidative stress. CONCLUSION: The novel compound, MHY966 had an inhibitory effect on NO generation and the NO-mediated signaling pathway leading to the down-regulation of tyrosinase. The significance of the present study is the finding of a promising anti-melanogenic agent targeting the NO/PKG signaling pathway.

Synthesis and preliminary in vitro biological evaluation of 5-chloro-2-(substituted phenyl)benzo[d]thiazole derivatives designed as novel antimelanogenesis agents.

We describe the design, synthesis, and biological activities of 5-chloro-2-(substituted phenyl)benzo[d]thiazole derivatives as novel tyrosinase inhibitors. Among them, 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) and 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl)phenol (MHY966) showed inhibitory activity higher than or similar to kojic acid, against mushroom tyrosinase. Therefore, we carried out kinetic studies on the two compounds with potent tyrosinase inhibitory effects. Kinetic analysis of tyrosinase inhibition revealed that all of these compounds are competitive inhibitors. MHY884 and MHY966 effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-melanocyte stimulating hormone (α-MSH). These data strongly suggest that the newly synthesized compounds MHY884 and MHY966 could suppress production of melanin via inhibition of tyrosinase activity.