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Research Themes

  1. Phagocyte functions mediated by small GTP-binding proteins
  2. Activation mechanisms of superoxide generating NADPH oxidase
  3. Differentiation and maturation mechanisms of myeloid cells
  4. Functional and transcriptional analyses of antibacterial peptides
  5. Anti-inflammatory action of glucosamine

Research Projects

The ADP-ribosylation factors (ARFs), members of small GTP-binding proteins are involved in the intracelluar trafficking and cell movements in various cell types. Recently, ARFs are recognized as regulating molecules for phagocyte (neutrophil and macrophage) functions. We are now investigating the modulating mechanisms of ARFs and guanine-nucleotide-exchange proteins (GEFs), regulators for the activation of ARFs, in the expression of phagocyte functions [Figure], [Reference].

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NADPH oxidase is a superoxide generating enzyme that is present in phagocytes and composed of the membrane-bound cytochrome b and the cytosolic p47phox, p67phox, p40phox and p21rac. It is now established in other cell types that sphingoglycolipids form microdomain in the cell membrane and mediate the signal transduction.

To clarify the activation mechanism of NADPH oxidase, we are focusing on the function of lactosylceramide, a glycolipid exclusively expressed in phagocytes, in the signaling mechanism leading to the activation of NADPH oxidase [Figure], [Reference].

We are also analyzing the stability of oxidase components in neutrophils of patients with chronic granulomatous disease (CGD), an inherited disorder caused by defect in the NADPH oxidase[Figures 1, 2], [References 1, 2].

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Neutrophils are differentiated from a common myeloid progenitor cell in the bone marrow, and express specific proteins such as α-defensins. To elucidate the molecular mechanisms involved in the myeloid cell differentiation, we analyzed the promoters for the α-defensins, and their transcription factors specifically expressed during differentiation [Reference].

Furthermore, we have revealed that antibacterial peptides (CAP18/LL-37 and CAP11) are specifically expressed at the stages of myelocyte and metamyelocyte during neutrophil maturation in the bone marrow [References 1, 2].

In addition, we investigated the stage-specific expression of the NADPH oxidase components (membrane-bound cytochrome b, and the cytosolic p47phox, p67phox, p40phox and p21rac) during myeloid cell maturation (neutrophil and eosinophil), using bone marrow cells and promyelocytic leukemia cell lines (HL-60 and HL-60 clone 15) [Figure], [References 1, 2].

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Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (α-/β-defensins and cathelicidins), which contribute to the innate host defense by killing invaded microorganisms. It is now accepted that antibacterial peptides such as β-defensins and cathelicidin are expressed not only myeloid cells but also epithelial cells [Figure].

We investigated the actions of these epithelial cell-derived antibacterial peptides on tissue mast cells that modulate the inflammatory reactions [Figure], [References 1, 2, 3].

Moreover, we analyzed the transcriptional regulation of β-defensin gene in macrophages and pulmonary epithelial cells by LPS and LPS-stimulated mononuclear cell-derived cytokines, respectively [References 1, 2].

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Antibacterial cathelicidin peptides (human CAP18/LL-37 and guinea pig CAP11) possess lipopolysaccharide (LPS)-neutralizing activity as well as bactericidal activity. We evaluated the lipopolysaccharide LPS-neutralizing activities of LL-37 and guinea pig CAP11, using CD14-positive murine macrophage cell line RAW264.7 and endotoxin shock model. LL-37 and CAP11 exhibited the potent LPS-binding activities, and strongly suppressed the interaction of LPS with LPS-binding protein (LBP) that mediates the transport of LPS to CD14. Furthermore, LL-37 and CAP11 exerted the protective actions in murine endotoxin shock by blocking the binding of LPS to CD14-positive cells and suppressing the production of cytokines by these cells [Figure], [Reference].

Moreover, we are developing the peptide derivatives with enhanced bactericidal and LPS-neutralizing activities [Reference]

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Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility and elasticity of these tissues. In recent years, glucosamine has been widely used to treat osteoarthritis in humans and animal models. We evaluated the effects of glucosamine on neutrophil functions using human peripheral blood neutrophils. Glucosamine suppressed the superoxide anion generation and phagocytosis. Furthermore, glucosamine inhibited the release of granule enzyme lysozyme from phagocytosing neutrophils, and suppressed neutrophils chemotaxis.

In addition, glucosamine inhibited fMLP-induced upregulation of CD11b, polymerization of actin, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). Thus, it is likely that glucosamine suppresses the neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis [Figure1, 2], [References 1, 2, 3]

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