We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.
The effect of 10-hydroxy-trans-2-decenoic acid (10H2DA), a major fatty acid component of royal jelly, was investigated on LPS-induced cytokine production in murine macrophage cell line, RAW264 cells. 10H2DA inhibited LPS-induced IL-6 production dose-dependently, but did not inhibit TNF-α production. 10H2DA inhibited LPS-induced NF-κB activation in a dose-dependent fashion. In addition, NF-κB activation induced by over-expression of either MyD88 or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was also inhibited by 10H2DA. Degradation of IκB-α and phosphorylation of IκB kinase-α were not inhibited by 10H2DA. On the other hand, reduction of LPS-induced IκB-ζ expression was discovered. Production of lipocalin-2 and granulocyte colony-stimulating factor (G-CSF), which is dependent on IκB-ζ, was also inhibited by 10H2DA, whereas that of IκB-ζ-independent cytokines/chemokines, such as IFN-β, murine monocyte chemotactic protein-1 (JE), macrophage inflammatory protein (MIP)-1α and MIP-2, was not. Together, 10H2DA specifically inhibited LPS-induced IκB-ζ expression, followed by inhibition of IκB-ζ-dependent gene production. These results suggest that 10H2DA is one of the components of royal jelly to show anti-inflammatory effects and could be a therapeutic drug candidate for inflammatory and autoimmune diseases associated with IκB-ζ and IL-6 production.
Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin‐receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto‐trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stx‐neutralizing activity. Galacto‐trehalose was designed as a Gb2 mimicking, unnatural Stx‐ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto‐trehalose copolymer showed neutralizing activity against Stx‐1 but not Stx‐2 in a HeLa cell cytotoxicity assay. It was thought that galacto‐trehalose copolymer could be a lead compound for the treatment of Stx‐mediated diseases, although it requires modification to show neutralizing activity to Stx‐2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx‐2 than those with lower density. However, the density‐dependency of the neutralizing activity was less obvious against Stx‐1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx‐1‐ and Stx‐2‐producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx‐1 and the more clinically relevant Stx‐2 in vitro and effectively inhibit Stx toxicity in vivo.
Royal jelly acid, 10-hydroxy-trans-2-decenoic acid (10H2DA), is a major lipid component of royal jelly, which is the exclusive diet of queen honeybees. Previously, we showed partial inhibition of lipopolysaccharide (LPS)-induced NF-κB activation by 10H2DA. In this study, the ability of 10H2DA to inhibit LPS-induced nitric oxide (NO) production was investigated. LPS-induced NO production and inducible NO synthase (iNOS) gene transcription were inhibited by 10H2DA. LPS-stimulated interferon (IFN)-β production, IFN regulatory factor-1 induction and IFN-stimulated response element activation, which are required for iNOS induction, were unaffected by 10H2DA. IFN-β-induced NO production, however, was significantly inhibited by 10H2DA. Furthermore, IFN-β-induced nuclear factor (NF)-κB activation and tumour necrosis factor (TNF)-α production were significantly inhibited by 10H2DA, and TNF-α-induced NF-κB activation was also inhibited by 10H2DA. These results and our previous study suggest that 10H2DA inhibits LPS- and IFN-β-induced NO production via inhibition of NF-κB activation induced by LPS or IFN-β.
Galacto-trehalose (GT) is a novel class of 1,1'-linked nonreducing disaccharide having an α-galactoside epitope. In this study, a pair of α,α- and α,β-GT isomers were prepared in one pot with our α-glycosylation method, converted into vinyl monomers and then subjected to radical copolymerization with a second sugar (4-acrylamidophemyl β-Glc or β-GlcNAc) in the presence of acrylamide. The derived glycopolymers were assayed with α-galactoside-specific proteins (BSI-B(4) lectin and Shiga toxin-1) to show the results that both α,α- and α,β-isomers are recognized by these carbohydrate-binding proteins more strongly in forms of the GT polymers. Moreover, the glycopolymer carrying both α,α-GT and β-GlcNAc along the polymer chain showed an integrated detoxifying activity to the E. coli toxin as the result of a "module effect" of the second sugar.
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