Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.
Mice were fed either a high fat diet or a high fat diet containing 1% mannooligosaccharides (MOS) for twelve weeks. The effects of MOS on fat accumulation and excretion were examined. After twelve weeks, the percentage weight of the fat and hepatic triglyceride level were significantly lower in the MOS group than that of the control group (p < 0.05 and p < 0.01 respectively). Furthermore, the serum triglyceride level had a decreasing tendency in the MOS group (p = 0.058). On the other hand, the fecal triglyceride level as well as the amount of fat excreted significantly increased in the MOS group (p < 0.05). This study showed that the administration of MOS lessened the fat accumulation in the parametrial adipose tissue and the liver while at the same time increased the amount of fat being excreted. These results indicate that MOS may prevent the fat storage through inhibiting the intestinal absorption of dietary fat in a high fat diet.
Asbestos exposure is considered a social burden by causing mesothelioma. Despite the use of synthetic materials, multi-walled carbon nanotubes (MWCNTs) are similar in dimension to asbestos and produce mesothelioma in animals. The role of inflammatory cells in mesothelial carcinogenesis remains unclear. Here, we evaluated the differences in inflammatory cell responses following exposure to these fibrous materials using a luminometer and L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione) to detect reactive oxygen species (ROS). Rat peripheral blood or RAW264.7 cells were used to assess the effects on neutrophils and macrophages, respectively. Crocidolite and amosite induced significant ROS generation by neutrophils with a peak at 10 min, whereas that of chrysotile was ~25% of the crocidolite/amosite response. MWCNTs with different diameters (~15, 50, 115 and 145 nm) and different carcinogenicity did not induce significant ROS in peripheral blood. However, the MWCNTs induced a comparable amount of ROS in RAW264.7 cells to that following asbestos treatment. The peaks for MWCNTs (0.5–1.5 h) were observed earlier than those for asbestos (1–5 h). Apocynin and superoxide dismutase significantly inhibited ROS generation for each fiber, suggesting an involvement of NADPH oxidase and superoxide. Thus, asbestos and MWCNTs induce different oxidative responses in inflammatory cells, indicating the importance of mesothelial cell evaluation for carcinogenesis.
A liquid coffee drink containing mannooligosaccharides from coffee mannan (MOS) was administered to a group of healthy adults. Subsequently, the amount of fat excreted from the body was examined. The subjects were divided into two groups: One was administered a liquid coffee drink containing MOS 3.0 g/day whereas the other was administered a placebo drink for seven days. Both groups were fed the standardized meals during the experiment. In the amount of average excreted fat, the drink containing MOS intake showed a significant increase in comparison with the before intake group and placebo intake group (p < 0.05 and p < 0.001, respectively). In addition, fat utilization in MOS intake group was significantly lower than the before intake group and the placebo group (p < 0.05 and p < 0.001, respectively). These results suggested that the intake of MOS 3.0 g/day increased in the amount of excreted fat and decreased in fat utilization.
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