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.

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Under physiological circumstances, cell membrane damage is not evident in biliary systems, despite the fact that hydrophobic bile salts are known to induce such damage by their detergent effects. The aim of this study was to determine the cytoprotective effects of liposomes and hydrophilic bile salts against hydrophobic bile salt-induced cell membrane damage, with the use of hemolysis of erythrocytes as a model of cytotoxicity. Washed human erythrocytes were incubated for 10, 30, 60, 90, and 120 min in buffered media (pH 7.45) containing increasing concentrations of different bile salts (1, 2.5, 5, 25, 50 mM). The cytotoxicity of the bile salts was found to be dose and time dependent and was correlated to the degree of the hydrophobicity of the bile salts as determined by the retention factor in reversed-phase high-performance liquid chromatography. Hydrophobic bile salt-induced hemolysis was reduced by liposomes and hydrophilic bile salts. Cytoprotection by liposomes was related to the degree of saturation of the fatty acyl chains, and cytoprotection by hydrophilic bile salts was related to their hydrophilicity. These in vitro findings indicate that vesicles may play a role in protection against cell membrane damage by hydrophobic bile salts in biliary systems and that such damage may be caused by an imbalance between hydrophobic and hydrophilic bile salts.

A deletion mutant of influenza virus haemagglutinin (HA; headless HA) lacking the globular region was expressed in CV-I cells and detected with a monoclonal antibody, C179, which recognizes a conformational epitope in the middle of the stem region of HA and neutralizes all H 1 and H2 subtypes. The cDNA coding for the headless HA was constructed from influenza virus A/Okuda/57 (H2N2), which was also used to select C179. The conformational epitope recognized by C 179 was highly stable even after removal of the globular region. The survival rate of mice immunized with the headless HA and challenged with lethal influenza virus A/FM/1/47 (HIN1) was significantly higher than that of the control mice. The headless HA has the potential to induce cross-protection against influenza virus infection.

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E 2 (PGE 2 ), 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 PGE 2 is one of key players in carcinogenesis. Thus, we confirmed that PGE 2 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 PGE 2 production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE 2 elevation in chronic inflammation site.Key words agaro-oligosaccharide; anti-inflammation; cancer chemoprevention Agarose, the main component of polysaccharide in agar, is composed of the alternating residues of 3-O-linked β-Dgalactopyranose (Gal) and 4-O-linked 3,6-anhydro-α-Lgalactopyranose (Ah-Gal).1) When agarose is treated by mild acidic conditions, the α1 3 linkage is hydrolyzed, and then yielding agaro-oligosaccharides (AGOs) that have repeating agarobiose (AB) units, with Gal at the non-reducing end and Ah-Gal at the reducing end (Fig. 1). We previously reported that AGOs suppress the elevated levels of nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β and interleukin-6 in lipopolysaccharide (LPS)-stimulated monocytes and macrophages.2) These pro-inflammatory mediators have been implicated in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA) and carcinogenesis.3-5) Elevated levels of NO and PGE 2 have been observed in the synovial fluid of RA patients, and excessive NO or PGE 2 contribute to inflammation-related carcinogenesis. [6][7][8][9][10] The activated macrophages at the sites of inflammation persistently express inducible NO synthase (iNOS), resulting in the production of a large amount of NO which interacts with other free radicals (superoxide anions) to form the highly toxic peroxynitrite. 11)Also, overproduced PGE 2 increases the blood flow and potentiates edema and hyperalgesia. 12) It has been shown that AGOs can suppress the excess NO and PGE 2 production via heme oxygenase-1 induction.2) Further, with respect to NO suppression, we confirmed that iNOS expression is inhibited by AGOs treatment, and showed the inhibition of pro-inflammatory mediators release in activated macrophages in vitro. Thus, AGOs were expected to elicit their beneficial effects on subjects having chronic inflammation state such as carcinogenesis.The aim of this study is to demonstrate whether AGOs exert the anti-tumor-promoting activity on well-established mouse...

Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 10 2 , maintaining linearity with r 2 > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for largescale metabolome analysis of plasma for biological studies and clinical biomarker screening.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m 2 , n = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γglutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves longterm migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γglutamyltransferease (GGT) enzyme activity (r = −0.46; p = 2.5 × 10 −7 ), which was indicative of a low-risk (n = 64) as compared to a high-risk (n = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.

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