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Cells are often exposed to exogenous and endogenous redox disturbances and exert their protective mechanisms in response to stimuli. The KEAP1-NRF2 system plays pivotal roles in counteracting oxidative damage. Due to the transient nature of NRF2 activation, the identification of cells in which NRF2 is activated in response to systemic stimuli is sometimes not easy. To examine the electrophilic stress response at a single-cell resolution, we aimed to develop a new reporter mouse in this study. A cell-tracing strategy exploiting Cre recombinase-mediated activation of a reporter gene was chosen for stable detection of reporter expression instead of real-time monitoring of the cellular response. We established a transgenic mouse line expressing the Neh2-Cre recombinase fusion protein. As Neh2 is an amino-terminal domain of NRF2 that serves as a degron and mediates KEAP1-dependent degradation and electrophile-inducible stabilization, Neh2-Cre was expected to be activated in response to electrophiles. The Neh2-Cre transgenic mouse was crossed with the ROSA26-loxP-stop-loxP-tdTomato reporter mouse (ROSA-LSL-tdTomato mouse). The compound mutant reporter mice exhibited accumulation of tdTomato-positive cells in various organs after repeated administration of CDDO-Im, one of the NRF2-inducing electrophiles. The mice were also successfully used for the detection of cells that experienced a cisplatin-induced electrophilic stress response.

Many studies have examined the protective effects of antioxidative agents against diabetic nephropathy, using various models. 6-Methylsulfinylhexyl isothiocyanate (6-MSITC) isolated from wasabi (Wasabia japonica MATUM) induces glutathione S-transferase in vitro, thus 6-MSITC may act as an antioxidant in vivo. The aim of this study was to examine whether wasabi powder (WP) and 6-MSITC suppress oxidative stress in vivo and inhibit the impairment of renal function and diabetic nephropathy, using type 2 diabetic mice. KK-Ay type 2 diabetic mice were assigned to three groups (n = 10 each); control mice were fed normal chow (CRF-1) and two experimental groups were fed CRF-1 containing 0.5% WP or 0.03% 6-MSITC for 4 wk. Urine volume, urinary albumin excretion, and creatinine clearance were significantly lower in the 6-MSITC group than in the control group. There was statistically no difference in TBARS or other biomarkers of oxidative stress among the three groups. However, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), one of the markers of oxidative stress tended to be lower in the 6-MSITC group than in the control group. In conclusion, the present results show that a sufficient supply of dietary 6-MSITC may prevent or delay renal dysfunction in diabetes by protecting against oxidative stress, and that dietary 6-MSITC may have beneficial effects on diabetic complications in type 2 diabetic mice.

機能性成分である6-MSITCの,各種わさび及び加工わさび製品における含量を分析した.本ワサビ5品種の6-MSITC含量を部位別に測定した結果,いずれの品種も根茎における含量が最も高く,次いで根,茎,葉の順であった.一方,西洋ワサビの6-MSITC含量は低かった.本ワサビ根茎の6-MSITC含量が最も高かった品種はみつきであり,最も含量の低かった品種との間には約1.6倍の差があった.みつきは収穫量も高く,優れた品種であることがわかった.加工わさび製品25種類の6-MSITC含量を分析した結果,メーカーや製品の種類によって,大きな差があった.6-MSITC含量には本ワサビ原料,特に根茎の配合率が大きく関与していることが明らかになった.以上のことより,食事の中で効率よく6-MSITCを摂取するためには,本ワサビ根茎を食べるのがもっとも望ましく,また,加工わさび製品でも,本ワサビ根茎が多く配合されている製品を選ぶことが重要であると考えられた.

We have established a reliable procedure for electroporation in the marine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replicon were found to be stably maintained in the Vibrio cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mg2+in order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm-1. Under optimal conditions, about 105transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

Wasabi has been used as an important spice in Japanese foods. The wasabi leaves were used as a cosmetic material, but its biological activities have not yet been examined. We investigated the effect of isosaponarin derived from wasabi leaf on collagen synthesis in human fibroblasts. The production of type I collagen in human fibroblasts was increased with treatment of wasabi leaf extract. Isosaponarin isolated from wasabi leaves belonged to the group of flavone glycoside, and was the key compound in collagen synthesis from the wasabi leaf ingredients. Isosaponarin increased the type I collagen production at the mRNA gene level. The treatment of isosaponarin did not influence the production of transforming growth factor-beta (TGF-beta) protein, but increased the production of TGF-beta type II receptor (TbetaR-II) protein and TbetaR-II mRNA. Prolyl 4-hydroxylase (P4H) protein and P4H mRNA were increased by treatment with isosaponarin. Heat shock protein 47 (HSP47) was not increased by treatment with isosaponarin. These results suggested that isosaponarin increased collagen synthesis in human fibroblasts, caused by up-regulated TbetaR-II and P4H production.

We investigated the effect of wasabi rhizome extract on atopic dermatitis (AD) model mice. The wasabi extract was fed to the HR-1 hairless mice, which develop AD-like symptoms with a special diet (HR-AD diet). The extract was expected to reduce the symptoms induced. Wasabi rhizome-containing HR-AD diet (5% and 10%) reduced the scratching behavior, and the 10% wasabi rhizome HR-AD diet significantly reduced scratching behavior on days 28, 35 and 42. Plasma components (histamine, eotaxin, IgE and thymus and activation-regulated chemokine (TARC)) were decreased in the 10% wasabi rhizome HR-AD diet. In histopathological examinations (toluidine blue (T.B.), major basic protein (MBP), CD4, IL-4, IL-5, eotaxin, TARC and IgE), the wasabi rhizome-containing HR-AD diet (5% and 10%) significantly reduced the number of positive stained cells. These results suggested that the wasabi rhizome extract improved the AD-like symptoms of HR-1 hairless mice.