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For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of beta-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with alpha-agglutinin. Three beta-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these beta-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit beta-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying beta-glucosidase.

The expression levels of various cytokines increase with the progression of HIV-1 infection. However, the effects of antiretroviral therapy (ART) on serum cytokine levels have not been fully determined. In this study we measured serum cytokine levels of 35 HIV-1-infected Japanese adults. We first performed a cross-sectional study and observed that TNF-α, IL-6, IL-10, IL-18, and IL-7 levels all showed significant increases in those with advanced disease, and that this had a significant negative correlation with the CD4 cell count. However, IFN-γ levels did not show this relationship. A longitudinal study in 18 HIV-1-infected patients with a CD4 cell count <350/μL revealed that the introduction of ART reduced cytokine levels. Significant reductions of IL-7, IL-10, IFN-γ, and IL-18 levels were observed on days 30, 60, 90, and 90 after the initiation of ART, respectively. These results indicate a discrepancy between cross-sectional and longitudinal studies of serum levels of IFN-γ. To clarify this, we investigated serum IFN-γ levels in each patient. In 5 of the 15 patients IFN-γ levels did not decrease, even after ART initiation, and remained at 5 pg/mL or higher on day 120 after ART initiation. Higher IFN-γ levels (>5 pg/mL) were also observed in 2 of 7 asymptomatic patients, and 2 of 11 patients who underwent ART for 1 year or longer. These data demonstrate that IFN-γ levels in some patients increased and remained high even after the initiation of ART, which was a specific observation different from those of the other cytokines.

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<b><i>Background:</i></b> Lysophosphatidic acid (LPA), a prototypic member of a large family of lysophospholipids, has been recently shown to play a role in immune responses to respiratory diseases. The involvement of LPA in allergic airway inflammation has been reported, but the mechanism remains unclear. <b><i>Object:</i></b> We analyzed the biological activity of LPA in vitro and in vivo and investigated its role in allergic inflammation in mice using an LPA receptor 2 (LPA2) antagonist. <b><i>Methods:</i></b> We used a murine model with acute allergic inflammation, in which mice are sensitized and challenged with house dust mite, and analyzed airway hyperresponsiveness (AHR), pathological findings, Th2 cytokines, and IL-33 in bronchoalveolar lavage fluid (BALF) and lung homogenates. The effect of LPA on Th2 differentiation and cytokine production was examined in vitro using naive CD4⁺ T cells isolated from splenocytes. We also investigated in vivo the effects of LPA on intranasal administration in mice. <b><i>Results:</i></b> The LPA2 antagonist suppressed the increase of AHR, the number of total cells, and eosinophils in BALF and lung tissue. It also decreased the production of IL-13 in BALF and IL-33 and CCL2 in the lung. LPA promoted Th2 cell differentiation and IL-13 production by Th2 cells in vitro. Nasal administration of LPA significantly increased the number of total cells and IL-13 in BALF via regulating the production of IL-33 and CCL-2-derived infiltrating macrophages. <b><i>Conclusion:</i></b> These findings suggest that LPA plays an important role in allergic airway inflammation and that the blockade of LPA2 might have therapeutic potential for bronchial asthma.

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A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.