The aim of the present study was to establish a progressive steatohepatitis mouse model because few reported animal models of non-alcoholic steatohepatitis (NASH) show the progression from fatty liver to steatohepatitis. C57BL/6N mice were fed a high-fat diet (HFD) to develop obesity and were either administered carbon tetrachloride (CCl4 ) eight times (0.05 mL/kg, s.c., once, followed by 0.1 mL/kg, s.c., seven times) or not. Serum parameters and hepatic histopathology were examined. In a separate experiment, CCl4 was administered subcutaneously from 0 to eight times to HFD-fed obese mice to investigate progressive changes. Markers of oxidative stress, inflammation and apoptosis, as well as histopathological changes in the liver, were analysed. The HFD-fed obese mice showed fatty liver but not steatohepatitis. In contrast, HFD-fed mice administered CCl4 eight times showed histopathological features of steatohepatitis (fatty liver, inflammation, hepatocellular ballooning and fibrosis) and increased serum alanine aminotransferase levels. However, the multiple administration of CCl4 to obese mice reduced the ratio of reduced glutathione to oxidized glutathione, superoxide dismutase activity and mitochondrial DNA copy number, leading to the development of chronic oxidative stress, increased numbers of apoptotic cells and increased levels of both tumour necrosis factor-α and transforming growth factor-β mRNA. The resulting inflammation led to increased hydroxyproline content in the liver and fibrosis. The present study demonstrates that multiple administration of CCl4 to HFD-fed obese mice induces chronic oxidative stress that triggers inflammation and apoptosis and leads to the development of fibrosis in the liver, resulting in progression from fatty liver to steatohepatitis. This murine model will be useful in the research of hepatic disorders.

Oligobrachia mashikoi, a frenulata, is a marine invertebrate living in an unusual habitat in Tsukumo Bay (20-25 m deep), Japan. It lacks a mouth and a gut, and instead possesses specialized cells called bacteriocytes, in which endosymbiotic bacterial cells are kept. The endosymbiosis involves specific interactions and obligatory metabolic exchanges between the host and endosymbiotic bacterial cells. Analyses of 16S rRNA genes from the endosymbiotic cells indicated that there are at least seven phylotypes of endosymbionts in O. mashikoi, and these phylotypes are closely related Gammaproteobacteria among which sequence homology was more than 97.6%. The analyses suggested that an adult worm predominantly accommodates one of the seven phylotypes. In situ hybridization targeting 16S rRNA demonstrated that the distribution patterns of the bacterial cells in adult worms were very similar among at least two phylotypes of the endosymbionts. The findings suggest that O. mashikoi has a strict selection mechanism for its endosymbiont.

it is important to determine how the bacteriocytes are organised in the trophosome. As ｔhe first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections.Secondly, visualisation of the actual distribution of the bacteriocytes was attempted using whole-mount in situ hybridisation with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin and eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient-deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole-mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three-dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. 2We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.

Beard worms (Siboglinidae, Polychaeta), which lack a mouth and a digestive tract, harbor thioautotrophic or methanotrophic bacteria in special cells called bacteriocytes. These endosymbionts have been considered to be trapped at a specific larval stage from the environment. Although many species of beard worms have been discovered in various abyssal seas, Oligobrachia mashikoi inhabits Tsukumo Bay which is only 25 m deep. At least seven types of endosymbionts (endosymbiont A-G) have been distinguished in O. mashikoi. In this study, we investigated the distribution pattern of free-living cells related to the major endosymbiont (endosymbiont A) in Tsukumo Bay by quantitative PCR targeting the 16S rRNA gene. The endosymbiont A-related phylotype was detected in almost all sediment samples collected from 23 points in Tsukumo Bay, ranging in copy number of the 16S rRNA gene from 2.22×10 4 to 1.42×10 6 copies per gram of dry-sediment. Furthermore, the free-living cells made up less than 9% of the total eubacterial population, suggesting that the O. mashikoi larvae precisely select candidates for their endosymbiont from bacterial flora in the environment. This is the first report on the ecological characterization of a free-living bacterium related to the endosymbiont of the siboglinid polychaete, O. mashikoi.

The protective effect of isoflavones on skin damage from ultraviolet (UV) radiation and their bioavailability were investigated in ovariectomized hairless mice fed diets composed of fermented soymilk containing aglycone forms of isoflavones or control soymilk containing glucose-conjugated forms of isoflavones. The erythema intensity of dorsal skin was significantly higher in ovariectomized mice than in sham-operated mice (p < 0.05). The erythema intensity and epidermal thickness of dorsal skin were significantly lower in the fermented soymilk diet group than in the control diet group (each p < 0.05). Levels of cyclobutane pyrimidine dimers in dorsal skin were significantly lower in the fermented soymilk diet group than in the control group (p < 0.05). Serum and dorsal skin isoflavone concentrations were significantly higher in the fermented soymilk diet group than in the soymilk diet group (p < 0.05). These results indicate that oral administration of a fermented soymilk diet increases isoflavone concentrations in the blood and skin, effectively scavenging the reactive oxygen species generated by UV irradiation and exerting an estrogen-like activity, with a consequent protective effect on skin photodamage in hairless mice.