Noriko Bando

Occurrence of IgE antibody-recognizing N-linked glycan moiety of a soybean allergen, Gly m Bd 28K.

Background: It has been reported that N-linked glycan moieties of glycoproteins function as IgE-reactive determinants. Gly m Bd 28K, a soybean allergen, was a glycoprotein with glycan moieties, which are supposed to be the Man3GlcNAc2 backbone with the β1→2 xylose and α1→3 fucose branches. The purpose of the present study was to examine the IgE-binding ability of the glycan moiety of Gly m Bd 28K in the binding reaction with patients’ sera. Methods: A peptide containing the glycan moiety was prepared from Gly m Bd 28K by digestion with lysyl endopeptidase. The binding site of the glycan moiety was determined by amino acid sequence analyses. The glycan moiety of the allergen was characterized using anti-horseradish peroxidase antibody (anti-HRP) recognizing the N-linked glycan moieties of glycoproteins. The binding of patients’ IgE antibodies with their glycan moiety was examined by an immunostaining technique using the glycopeptide and its deglycosylated peptide derived from Gly m Bd 28K. Results: The binding site of the glycan moiety in Gly m Bd 28K was shown to be its Asn20 residue. Gly m Bd 28K did react with anti-HRP and the sera of soybean-sensitive patients, but the binding of IgE antibodies was inhibited by the preincubation with anti-HRP. Moreover, the glycopeptide also reacted with the sera of soybean-sensitive patients, but its deglycosylated peptide did not react with any IgE antibodies of patients’ sera. Conclusions: The specific IgE antibodies recognizing the N-linked glycan moieties of Gly m Bd 28K and other glycoproteins with homologous glycan moieties occur in the sera of soybean-sensitive patients. It was indicated that the N-linked glycan moieties such as that of Gly m Bd 28K may be one of the common IgE-reactive determinants distributed in various plant food proteins.

Singlet molecular oxygen-quenching activity of carotenoids: relevance to protection of the skin from photoaging

Carotenoids are known to be potent quenchers of singlet molecular oxygen [O2 (1Δg)]. Solar light-induced photooxidative stress causes skin photoaging by accelerating the generation of reactive oxygen species via photodynamic actions in which O2 (1Δg) can be generated by energy transfer from excited sensitizers. Thus, dietary carotenoids seem to participate in the prevention of photooxidative stress by accumulating as antioxidants in the skin. An in vivo study using hairless mice clarified that a O2 (1Δg) oxygenation-specific peroxidation product of cholesterol, cholesterol 5α-hydroperoxide, accumulates in skin lipids due to ultraviolet-A exposure. Matrix metalloproteinase-9, a metalloproteinase family enzyme responsible for the formation of wrinkles and sagging, was enhanced in the skin of ultraviolet-A -irradiated hairless mice. The activation of metalloproteinase-9 and the accumulation of 5α-hydroperoxide, as well as formation of wrinkles and sagging, were lowered in mice fed a β-carotene diet. These results strongly suggest that dietary β-carotene prevents the expression of metalloproteinase-9 (at least in part), by inhibiting the photodynamic action involving the formation of 5α-hydroperoxide in the skin. Intake of β-Carotene therefore appears to be helpful in slowing down ultraviolet-A -induced photoaging in human skin by acting as a O2 (1Δg) quencher.

β-Carotene and β-cryptoxanthin but not lutein evoke redox and immune changes in RAW264 murine macrophages

The mechanism of immunological benefits induced by carotenoids has not been fully elucidated. Here, we investigated some of the immunity-related properties of beta-carotene and two other carotenoids, beta-cryptoxanthin, and lutein, on the murine macrophages cell line RAW264. beta-Carotene added to the culture medium accumulated in the cells in a time- and dose-dependent manner. The accumulation was positively correlated with cellular lipid peroxidation, demonstrating the pro-oxidative activity of beta-carotene, and also with the synthesis of glutathione, an intracellular antioxidant. Conversely, accumulation of beta-carotene was negatively correlated with the transcription of immune-active molecules, such as IL-1beta, IL-6, and IL-12 p40, in cells stimulated by LPS and INF-gamma. The transcription of the pro-inflammatory cytokines IL-1beta and IL-6 was more sensitive to the accumulation of beta-carotene than was IL-12 p40. The accumulation of beta-cryptoxanthin in cells resulted in effects similar to those of beta-carotene. However, lutein accumulated minimally and did not significantly affect the cells. These results demonstrate that beta-carotene, and beta-cryptoxanthin as well, can accumulate in RAW264 cells and induce changes in intracellular redox status, which in turn regulate the immune function of macrophages.

Inhibition of Immunoglobulin E Production in Allergic Model Mice by Supplementation with Vitamin E and β-Carotene

A diet containing different amounts of vitamin E (α-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of β-carotene was supplemented to the basal diet containing vitamin E as α-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with β-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus β-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and β-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and β-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.

Occurrence of singlet oxygen oxygenation of oleic acid and linoleic acid in the skin of live mice

To assess the contribution of singlet molecular oxygen [O2 (1Δg)] to lipid peroxidation in vivo, this study combined gas chromatography-mass spectrometry with thin layer chromatography to analyse peroxidized lipids in the skin of hairless mice. Hydroxyoctadecenoate isomers and unconjugated hydroxyoctadecadienoate isomers derived from peroxidized oleic acid and linoleic acid, respectively, which are specific to O2 (1Δg)-dependent oxygenation, were detected in the skin of live mice under ordinary feeding conditions. Short-term ultraviolet A (UVA)-irradiation of the skin in vivo elevated levels of the unconjugated hydroxyoctadecadienoate isomers significantly, whereas the irradiation of skin homogenate in vitro increased levels of all isomers derived from both O2 (1Δg) and free radical-dependent oxygenation to a much greater extent. This is the first report to demonstrate the occurrence of O2 (1Δg)-specific oxygenation of unsaturated fatty acids in living animals.

Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary β-carotene in photoaging of hairless mouse skin

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.

Combination of TLC Blotting and Gas Chromatography–Mass Spectrometry for Analysis of Peroxidized Cholesterol

We have established a sensitive and convenient method for analysis of cholesterol hydroperoxides (Chol-OOHs) as trimethylsilyloxyl derivatives using diphenylpyrenylphosphine (DPPP)-thin-layer chromatography (TLC) blotting and gas chromatography–electron ionization–mass spectrometry/selected-ion monitoring (GC–EI–MS/SIM). Chol-OOH standards were prepared by photosensitized oxidation and azo radical-induced peroxidation of cholesterol. Trimethylsilyloxyl derivatives of cholesterol 5α-hydroperoxide (Chol 5α-OOH), cholesterol 7α-hydroperoxide (Chol 7α-OOH), and cholesterol 7β-hydroperoxide (Chol 7β-OOH) could be separated from one another in the SIM chromatogram using a fragment ion with elimination of trimethylsilanol from the molecular ion. This method was used to characterize peroxidized cholesterol from azo radical-exposed human low-density lipoprotein and UVA-irradiated human keratinocytes in the presence of hematoporphyrin. Finally, we succeeded in the quantification of each Chol-OOH isomer present in hairless mouse skin with and without UVA irradiation by use of β-sitosterol hydroperoxide as internal standard. The accumulation of Chol 5α-OOH with Chol 7α/βOOH in the skin indicates that singlet molecular oxygen (1O2) participated in the peroxidation of skin cholesterol, because Chol 5α-OOH is known to be a 1O2 specific cholesterol peroxidation product. We concluded that the combination of DPPP-TLC blotting and GC–EI–MS/SIM is useful for quantifying peroxidized cholesterol in biological samples and confirming the participation of 1O2 in oxidative stress.

β-Carotene Modulates the Immunological Function of RAW264, a Murine Macrophage Cell Line, by Enhancing the Level of Intracellular Glutathione

The activities of β-carotene on redox status and the immune functions of RAW264 cells, a murine macrophage cell line, were investigated. Supplementation with β-carotene for RAW264 cells resulted in apparently inconsistent redox indices: lipid peroxidation was enhanced but intracellular oxidation was moderately attenuated. Attenuated intracellular oxidation was endorsed by an increase in glutathione accompanied by up-regulated transcription of a subunit of γ-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. α-Tocopherol, which can quench lipid peroxidation by free radical, neither inhibited that by β-carotene nor influenced the intracellular redox status. Lipopolysaccharide-stimulated transcriptions of IL-1β and IL-12 p40 in RAW264 were inhibited by β-carotene but not by α-tocopherol. These results indicate that β-carotene, which can modulate the intracellular redox status of macrophages by enhancing the level of intracellular glutathione, is related to the immune functions of macrophages.

Adjuvant activity of alum in inducing antigen specific IgE antibodies in BALB/c mice: a reevaluation.

The IgE production was compared in the presence and absence of aluminum hydroxide gel (alum). Without alum, the IgE production was induced within a suitable range of the antigen dosage; however, alum enhanced it. Alum did not affect the minimum requirement for the antigen dosage, indicating that alum may not take part in the efficiency of antigen presentation.

Association of serum carotenoids and tocopherols with atopic diseases in Japanese children and adolescents

Okuda M, Bando N, Terao J, Sasaki S, Sugiyama S, Kunitsugu I, Hobara T. Association of serum carotenoids and tocopherols with atopic diseases in Japanese children and adolescents. 
Pediatr Allergy Immunol 2010: 21: e705–e710.
© 2010 John Wiley & Sons A/S