[Efficacy of the Extract from Fermentation of Soybean and Rice Bran on Hyperglycemia].

In recent years, lifestyle-related diseases such as hypertension and diabetes have been on the rise. These conditions can cause serious conditions such as myocardial and cerebral infarctions. Therefore, proper control of blood pressure and blood glucose levels is important issues in preventive medicine. Traditional fermented foods have been shown to have various functions, and their effects on lifestyle-related diseases have attracted particular attention. In this study, we investigated the effects of fermented soybeans and rice bran (OE-1) and supplements containing OE-1 on blood glucose levels and weight changes. We identified an inhibitory effect on elevated blood glucose levels upon administration of OE-1, and this effect was thought to be due to digestive enzyme inhibition. These effects of foods containing OE-1 are expected to have a positive effect on the prevention and improvement of lifestyle-related diseases as health foods.

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.

Suppression of type-I allergic responses by oral administration of grape marc fermented with Lactobacillus plantarum.

We investigated the inhibitory effects of fermented grape marc (FGM), lyophilized fine powder of skin, and seeds of Vitis vinifera Koshu grape prepared by fermentation with Lactobacillus plantarum, on type-I allergic responses in mice. Repeated oral administration of FGM, but not non-fermented grape marc (GM), to BALB/c mice primed with ovalbumin (OVA) resulted in a significant reduction of serum IgE levels, compared with those of immunized controls. After OVA challenge, increased numbers of eosinophils in bronchial alveolar lavage fluids (BALF) significantly decreased by treatment with FGM but not with GM. For passive cutaneous anaphylaxis (PCA) reaction, BALB/c mice received intradermal sensitization with anti-OVA IgE serum and were challenged intravenously with OVA containing Evans blue at 24 h after IgE sensitization. Oral administration of FGM at 30 min before OVA challenge significantly suppressed the PCA reaction. On the other hand, Lactobacillus alone and non-fermented GM did not show any suppressive effects. Interestingly, FGM samples prepared from grapes for red wine, such as Negroamaro (rich in resveratrol) or Tannat (rich in oligomeric procyanidin), did not suppress the reaction. These results indicate that oral administration of FGM, prepared from Koshu grape for white wine but not from grapes for red wine, could suppress both phases of type-I allergic responses. A fraction extractable with acetone was responsible for the suppressive effects of FGM.

Inhibitory effects of fermented grape marc from Vitis vinifera Negroamaro on antigen-induced degranulation.

To investigate the antiallergic effects of fermented grape marc from Negroamaro (N-FGM), we examined antigen (Ag)-induced degranulation of rat basophilic leukemia (RBL-2H3) cells. Among supernatants of N-FGM suspensions in water, ethanol, and dimethyl sulfoxide (DMSO), supernatants of DMSO-suspended N-FGM but not of nonfermented Negroamaro grape marc (N-GM) markedly suppressed the Ag-induced degranulation and phosphorylation of Syk in RBL-2H3 cells. Supernatants of DMSO-suspended N-FGM did not reduce the expression of FcepsilonRI on RBL-2H3 cells. Analyses of supernatants of N-FGM suspensions in water, ethanol, and DMSO by high-performance liquid chromatography revealed higher amounts of quercetin in supernatants of DMSO-suspended N-FGM than those in the other supernatants. Quercetin also suppressed the Ag-induced degranulation and phosphorylation of Syk but did not reduce the expression of FcepsilonRI on RBL-2H3 cells. These results suggest that inhibition of the Ag-induced degranulation and Syk phosphorylation by N-FGM might be due to the action of quercetin, as an active component in N-FGM.

Cytolysin-dependent escape of the bacterium from the phagosome is required but not sufficient for induction of the Th1 immune response against Listeria monocytogenes infection: distinct role of Listeriolysin O determined by cytolysin gene replacement.

Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-gamma), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-gamma production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-gamma production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-gamma induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-gamma-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-gamma that is essentially required to generate a Th1-dependent immune response.

Involvement of caspase-9 in the inhibition of necrosis of RAW 264 cells infected with Mycobacterium tuberculosis.

In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.

Dependence of the lethal effect of pore-forming haemolysins of Gram-positive bacteria on cytolytic activity.

Among bacterial haemolysins, cholesterol-dependent cytolysins (CDCs) produced by various Gram-positive bacteria are known to exhibit a lethal activity in mice. In this study, recombinant CDCs of streptolysin O, pneumolysin, ivanolysin O, listeriolysin O and several listeriolysin O mutants were constructed and the relationship between cytolytic activity and the lethal activity of each recombinant protein in mice was examined. Specific activity for cytolysis was determined by a quantitative haemolytic assay. Each protein was injected intravenously into mice and the lethal activity was evaluated by measuring the time until death of the mice. The four full-length CDC proteins exhibited lethal activity and their activities were highly proportional to their cytolytic activities. Inhibition of haemolytic activity resulted in the loss of lethal activity and non-haemolytic mutants of listeriolysin O did not exhibit any lethal activity. These data clearly indicate that the lethal effect of CDC proteins is dependent on the cytolytic activity.

Streptomycin-dependent exhibition of cytokine-inducing activity in streptomycin-dependent Mycobacterium tuberculosis strain 18b.

Peritoneal exudate cells of mice were stimulated with a streptomycin-dependent Mycobacterium tuberculosis strain, 18b. Gamma interferon production by natural killer cells depending on interleukin-12 and interleukin-18 was induced only in the presence of a high dose of streptomycin. This study suggested the requirement of active bacterial metabolism for this host response.