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Prolonged stress results in elevation of glucocorticoid (GC) hormones, which can have deleterious effects in the brain. The hippocampus, which has a high concentration of glucocorticoid receptors, is especially vulnerable to increasing levels of GCs. GCs have been suggested to endanger hippocampal neurons by exacerbating the excitotoxic glutamate-calcium-reactive oxygen species (ROS) cascade. In an effort to reveal the mechanisms underlying GC-mediated hippocampal neurotoxicity, we aimed to clarify the molecular pathway of GC-induced ROS increase by using organotypic hippocampal slice cultures. Assays for ROS, using 2',7'-dichlorodihydrofluorescein diacetate fluorescence, showed that treatment of synthetic GC, dexamethasone (DEX) significantly enhanced ROS levels. Time course and dose response analyses indicated that peak amount of ROS was generated at 4 h after treatment with 50 micromol/L DEX. By contrast, other steroid hormones, progesterone and estradiol did not influence ROS production. N-acetyl-L-cysteine completely suppressed ROS produced by DEX. Propidium iodide staining exhibited prominent cell death in the hippocampal layer after 96 h of DEX treatment. RU486, a GC receptor antagonist, almost completely blocked the effect of DEX on ROS production and cell death, indicating that DEX-induced ROS overproduction and hippocampal death are mediated via GC receptors. Real-time reverse transcriptase PCR analysis demonstrated that after DEX treatment the level of glutathione peroxidase mRNA was decreased whereas that of NADPH oxidase mRNA was significantly enhanced. These findings suggest that excess GCs cause hippocampal damage by regulating genes involved in ROS generation.

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Arbutin has been used as a whitening agent in cosmetic products. Melanin, the major pigment that gives color to skin, may be over-produced with sun exposure or in conditions such as melasma or hyperpigmentary diseases. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes; therefore, inhibitors of the tyrosinase enzyme could be used for cosmetic skin whitening. A recent study has reported that arbutin decreases melanin biosynthesis through the inhibition of tyrosinase activity. However, this inhibitory mechanism of arbutin was not sufficiently demonstrated in skin tissue models. We found that arbutin both inhibits melanin production in B16 cells induced with alpha-MSH and decreases tyrosinase activity in a cell-free system. Furthermore, the hyperpigmentation effects of alpha-MSH were abrogated by the addition of arbutin to brownish guinea pig and human skin tissues. These results suggest that arbutin may be a useful agent for skin whitening.

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Ehrlichia chaffeensis is an obligatory intracellular bacterium which infects macrophages and monocytes. Double immunofluorescence labeling was used to characterize the nature of E. chaffeensis inclusion in the human promyelocytic leukemia cell line THP-1. E. chaffeensis was labeled with dog anti-E. chaffeensis serum and fluorescein isothiocyanate-conjugated anti-dog immunoglobulin G (IgG). Lissamine rhodamine-conjugated anti-mouse IgG was used to label various mouse monoclonal antibodies. Ehrlichial inclusions did not fuse with lysosomes, since they were not labeled with anti-CD63 or anti-LAMP-1. The ehrlichial inclusions were slightly acidic, since they weakly accumulated 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine and stained weakly positive for vacuolar type H ؉ ATPase. Some ehrlichial inclusions were labeled positive with antibodies against HLA-DR, HLA-ABC, and ␤ 2 microglobulin, while other inclusions in the same cell were labeled negative. The inclusions were labeled strongly positive for transferrin receptors (TfRs) and negative for the clathrin heavy chain. Time course labeling for TfRs showed that up to 3 h postinfection, most of the ehrlichial inclusions were negative for TfRs. After 6 h postinfection, 100% of the ehrlichial inclusions became TfR positive and the intensity of labeling was increased during the subsequent 3 days. Reverse transcription-PCR showed a gradual increase in the level of TfR mRNA postinfection, which reached a peak at 24 h postinfection. These results suggest that ehrlichial inclusions are early endosomes which selectively accumulate TfRs and that the ehrlichiae up-regulate TfR mRNA expression.

Polyphenolic compounds were examined for their effects on suppressing adipocyte differentiation in 3T3-L1 cells. Most polyphenolic compounds inhibited adipocyte development from 3T3-L1 cells to some extent. Among them, rutin was the most effective in suppressing adipocyte differentiation in a dosage dependant manner. Activity of glycerol-3-phosphate dehydrogenase (GPDH), which has a central position in lipogenesis in adipose cells, was also decreased by rutin addition at the induction stage. RT-PCR results demonstrated that mRNA expression of adipogenic transcription factors such as peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha) in 3T3-L1 cells were remarkably down regulated by rutin treatment. For further investigation on anti-adipogenic activities of rutin, it was orally administered (25 and 50 mg/kg b.w/daily) with high-fat diet (64.4% of total calories as fat) to C57BL/6 mice. Body weight gains were less in high-fat diet + rutin fed groups (HFR) than high-fat diet alone fed group (HF) after 4 weeks. Total cholesterol contents in blood were significantly lower in HFR groups. When mRNA expressions of PPARgamma and C/EBPalpha in hepatocytes were compared between the control HF and HFR groups, their expressions in hepatocytes of HFR groups were significantly suppressed. These results indicate that rutin inhibits adipogenic development in pre-adipocytes and hepatocytes by down regulating expressions of key adipogenic transcription factors.

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