Anti-tumor-promoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis.

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E2(PGE2), and pro-inflammatory cytokines in activated monocytes/macrophages, via heme oxygenase-1 induction. In this report, we initially demonstrated that AGOs intake inhibited NO production in activated peritoneal macrophages. Then, we tested for the ability of AGOs to prevent tumor promotion on the two-stage mouse skin carcinogenesis model. As a result, AGOs feeding led to delayed tumor appearance and decreased tumor number. It is known that PGE2 is one of key players in carcinogenesis. Thus, we confirmed that PGE2 production was suppressed by AGOs intake in TPA-induced ear edema model. We also demonstrated that cyclooxygenase-2 and microsomal PGE synthase-1, rate-limiting enzymes in PGE2 production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE2 elevation in chronic inflammation site.

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes.

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.

Oligosaccharides from agar inhibit pro-inflammatory mediator release by inducing heme oxygenase 1.

We investigated whether agaro-oligosaccharides have any immunological effects on RAW264.7 mouse macrophages and human monocytes in vitro. We demonstrate that agaro-oligosaccharides suppressed the elevated levels of nitric oxide, prostaglandin E(2), and such pro-inflammatory cytokines as tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 in lipopolysaccharide-stimulated monocytes and macrophages. We also demonstrate that those effects of agaro-oligosaccharides on activated monocytes and macrophages may have been caused by heme oxygenase-1 induction. It is therefore proposed that agaro-oligosaccharides might be a good candidate for a functional food to prevent inflammatory diseases.

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe.

Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei.

Diabetes mellitus is a chronic disease that is characterized by hyperglycemia caused by insufficient insulin action. We have explored the edible ingredients from folk medicines in Japan that contain substances complementing insulin action, such as the induction of adipocyte differentiation and the enhancement of glucose uptake. We eventually found that the ethanol extract from a Japanese herb "Ashitaba", Angelica keiskei, contained two major chalcones of 4-hydroxyderricin (4-HD) and xanthoangelol that showed strong insulin-like activities via a pathway independent of the peroxisome proliferator-activated receptor-gamma activation. The 4-HD especially showed the preventive effects on the progression of diabetes in genetically diabetic KK-Ay mice.

[Development of the detection system for Mycobacterium tuberculosis DNA by using the isothermal DNA amplification method ICAN].

The isothermal and chimeric primer-initiated amplification of nucleic acids(ICAN) is a new isothermal DNA amplification method composed of exo- Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the detection system, combined the ICAN with luminescence detection by a probe hybridization, for Mycobacterium tuberculosis DNA targeting the IS6110 insertion element. We examined performance tests of the system. This system was able to detect one copy of Mycobacterium tuberculosis DNA for only 3.5 hours, and performance of the system was equivalent or better to the Roche PCR system. We also examined a detection system by using magnetic beads, which system could shorten detection time for 2.5 hours. It was shown that the ICAN system was an efficient and sensitive detection system for Mycobacterium tuberculosis DNA from mass samples.

Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein.