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Objective: This population-based, cross-sectional study was performed to investigate the relationship between a history of glaucoma and subjective happiness., Methods and Analysis: We conducted a cross-sectional questionnaire-based survey of 92 397 Japanese men and women aged 40-74 who participated in the Japan Public Health Center-based Prospective Study for the Next Generation study. A multivariable logistic regression model was used to estimate the ORs of glaucoma associated with subjective happiness and their two-sided 95% CIs., Results: Among 40 727 men and 51 670 women, 1733 participants (635 men, 1098 women) had a history of glaucoma. The odds of unhappiness in male participants with a history of glaucoma were higher (OR 1.26; 95% CI 1.05 to 1.51) than in female participants (OR 1.05; 95% CI 0.90 to 1.23). In a subgroup analysis stratified by age, among participants with a history of glaucoma, males in the younger group (40-59 years) showed the most robust association with unhappiness (OR 1.40; 95% CI 1.04 to 1.88)., Conclusions: These findings suggest that a history of glaucoma is related with subjective unhappiness, especially in men., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

This study aimed to determine the reliability and validity of the Japanese version of the Ocular Pain Assessment Survey (OPAS-J) to measure ocular pain and quality of life. A multi-institutional cross-sectional study was conducted on participants with and without ocular pain. The Wong-Baker FACES® Pain Rating Scale served as the gold standard for measuring the intensity of ocular pain. Sixty-four participants who visited two clinics located in Japan between May 2019 and October 2019 were included in the study. The OPAS was translated and culturally adapted to Japanese. The internal consistency of the OPAS-J was assessed using Cronbach's alpha coefficient. Twenty-four (37.5%) and 40 (62.5%) participants were classified as having ocular pain and no ocular pain, respectively. All dimensions of the OPAS-J had good reliability, with a Cronbach's alpha coefficient of 0.870 for ocular pain intensity over the past 24 h and 0.874, 0.899, 0.874, 0.871, and 0.876 for ocular pain intensity over the past 2 weeks, non-ocular pain, interference with quality of life, aggravating factors, and associated factors, respectively. The OPAS-J is a reliable and responsive tool that can be used to quantify ocular pain intensity., (© 2023. The Author(s).)

Objective: To investigate the relation between body mass index (BMI) and dry eye disease (DED)., Methods: We conducted a cross-sectional questionnaire-based survey in 85,264 Japanese men and women aged 40 to 74 years who participated in the Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT Study). Dry eye disease was defined as the presence of severe symptoms or clinical diagnosis. A multivariable logistic regression model was used to estimate the odds ratios (ORs) of DED associated with BMI and their two-sided 95% confidence intervals (CIs). We adjusted for age, cohort area, visual display terminal time, smoking status, alcohol intake, education status, income status, as well as history of hormone replacement therapy for women., Results: Prevalence of DED was 23.4% (n = 19,985; 6,289 men, 13,696 women). Higher BMI was correlated with a lower prevalence of DED in a dose-response fashion, with an adjusted OR of DED (95% CI) per 1 kg/m2 increment of BMI of 0.98 (95% CI: 0.97-0.99) for men and 0.97 (95% CI: 0.97-0.98) for women., Conclusions: This large population-based study showed an inverse relationship between BMI and prevalence of DED in a Japanese population. Underestimation of DED is warned, especially for participants with high BMI., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Contact Lens Association of Ophthalmologists.)

Purpose: The continuous use of warming eye masks improves tear function. In this double-blind randomized controlled trial, we aimed to analyze the effects of warming eye mask use on the ocular surface and mental health., Methods: We enrolled 86 participants (age range: 23-89 years) from affiliated institutions who were divided into two groups: the warm group that used warming eye masks that generate moist heat at 40 °C for 10 min and the control group that used non-warming eye masks. The participants used the masks for 10 min once daily for 2 weeks. Before and after the intervention, in 79 participants (warm group 39, control group 40), we analyzed the tear break up time (TBUT) and corneal and conjunctival fluorescein staining results in the right eye and Hospital Anxiety and Depression Score (Anxiety: HADS-A, Depression: HADS-D), and Subjective Happiness Scale (SHS) questionnaires. The parameters before and after the intervention were compared via paired-t tests., Results: The following variables changed after the intervention: TBUT (warm group: 1.4 ± 2.1 s vs. control group: -0.01 ± 2.38 s), fluorescein staining score (-0.7 ± 1.1 vs.-0.2 ± 1.1), HADS-A (-0.8 ± 3.1 vs. -0.2 ± 2.2), and HADS-D (-1.0 ± 2.4 vs. -0.4 ± 1.9). Significant changes were observed in the TBUT (warm group), fluorescein staining score (warm group), and HADS-D (warm group)., Conclusions: Using a warming eye mask improves not only the ocular surface conditions but also the subjective depression scores., (Copyright © 2021. Published by Elsevier Inc.)

Objective: We examined the production of prostaglandin E 2 (PGE 2 ), which is the key prostaglandin involved in inflammatory disorders of the ocular surface. Tears and conjunctival fibroblasts were evaluated in order to assess allergic inflammation and the effect of specific drugs., Methods and Analysis: PGE 2 was measured in tears from both patients and normal volunteers. Primary cultures of human conjunctival fibroblasts were incubated with interleukin (IL)-4 and tumour necrosis factor (TNF)-α with or without ketotifen fumarate or dexamethasone. The culture supernatants were removed 24 hours after exposure and the concentrations of PGE 2 were quantified by ELISA., Results: Significantly higher levels of PGE 2 were observed in the tears of patients with severe allergic conjunctivitis than in those with post-surgical inflammation (p=0.02), and this production was reduced by eye drops. Stimulation with IL-4 and TNF-α induced the generation of PGE 2 in supernatants of conjunctival fibroblasts, and this production was significantly downregulated by ketotifen fumarate or steroids., Conclusion: PGE 2 may participate in the pathogenesis of severe ocular allergic disease, and both ketotifen fumarate and steroid reduce the production of PGE 2 ., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The present study aimed to compare the prevalence of dry eye disease (DED) among visual display terminal users using the revised and previous DED criteria., Methods: This cross-sectional study included 561 participants (187 women)., Results: The distribution of diagnostic status according to the previous DED criteria was as follows: definite DED (n = 65, 11.6%), probable DED (n = 303, 54.0%), and non-DED (n = 193, 34.4%). According to the revised criteria, 329 participants (58.6%) were classified as DED and 232 participants (41.4%) were classified as non-DED., Conclusions: The prevalence of DED among visual display terminal users has increased to 58.6% from 11.6%, according to the revised DED diagnostic criteria of the Asia Dry Eye Society.

Purpose: To evaluate the relationship between the severity of ocular and systemic pain., Methods: A cross-sectional study was conducted in patients with ocular pain symptoms who were observed in Keio University Hospital between April 2017 and September 2018. Evaluations consisted of the Dry Eye-Related Quality of Life Score (DEQS), Short-Form McGill Pain Questionnaire (SF-MPQ), visual analog scale (VAS), and ophthalmologic examination, including tear breakup time measurement and Schirmer I test. The revised Asia Dry Eye Society Dry Eye Disease (DED) diagnostic criteria were used., Results: The present study involved 41 participants (31 women; mean age, 57.6 ± 17.5 years). Thirteen participants had aqueous-deficient DED, 18 participants had short tear break-up time DED, and 10 participants had no DED. Multivariate regression analysis revealed that the DEQS was significantly associated with both the SF-MPQ total (Coefficient score β = 0.92, p < 0.01) and the VAS (β = 0.25, p = 0.01). In addition, correlation analysis revealed that the DEQS had a significant positive linear correlation with both the SF-MPQ total (Spearman correlation score rs = 0.70, p < 0.01) and the VAS (rs = 0.66, p < 0.01)., Conclusions: The findings of the present study suggested that the ocular pain score was significantly associated with systemic pain score. Participants with higher systemic pain scores have an increased risk of having ocular pain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We analyzed the effects of enzyme replacement therapy (ERT) on the visual acuity and visual fields of a patient with mucopolysaccharidosis type II, Hunter syndrome, with degeneration of the retina and abnormalities of the optic nerve. After the ERT, there was an improvement of the visual acuity and visual fields and an improvement of the activities of daily living. Despite the late onset of Hunter syndrome in this patient, ERT was still able to improve the visual function. We conclude that ERT should be considered regardless of the age of the manifestations of the signs and symptoms of Hunter syndrome., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2019 by S. Karger AG, Basel.)

Currently, the available treatment for dry eye disease (DED) varies. The present study aimed to investigate the characteristics of undiagnosed DED and patient-based self-care management for DED based on a web-based survey performed for Tear's Day in Japan; 1030 participants (301 women) responded; 155 participants (72 women) had a clinical diagnosis of DED. We defined undiagnosed DED ( n = 116; 54 women) as those with DED symptoms, as evaluated by a frequently used questionnaire despite not having a clinical diagnosis. A multivariate adjusted model indicated that younger age (odds ratio (OR), 0.97 for each one-year decrease; 95% confidence interval (CI), 0.95-0.99), female sex (OR, 2.12; 95% CI, 1.28-3.50), and prolonged visual display terminal usage (OR, 1.12; 95% CI, 1.04-1.21) were risk factors for undiagnosed DED. To investigate the efficacy of self-care management for DED, a sub-analysis was conducted. The number of self-care methods used was significantly higher among women than men. For undiagnosed DED, those with less than three self-care methods had a significantly worse Dry Eye-related Quality-of-Life Score compared with those with diagnosed DED. This study revealed risk factors for undiagnosed DED; individuals with those risk factors need to be clinically assessed and should not rely solely on self-care.

Scope: We evaluated the potential of retinol and retinoic acid (RA) to enhance intracellular glutathione (GSH) levels in a murine cultured macrophage cell line, RAW264, to investigate whether the RA signaling pathway is involved in the β-carotene-induced GSH enhancement., Methods and Results: We examined GSH levels in RAW264 cells cultured in media supplemented with β-carotene and various inhibitors (ER50891 for RA receptor (RAR)α, CD2665 for RARβ/γ, or HX531 for all subtypes of retinoid X receptor (RXR)), to verify each inhibitor's activity against β-carotene, as well as in media supplemented with various stimulants (AM80 for RARα, CD2314 for RARβ, CD437 for RARγ, or SR11237 for RXR), to compare their activity with that of β-carotene. We also examined the GSH level and glutamate-cysteine-ligase (GCL) expression in RAW264 cells cultured in all- trans RA- or retinol-supplemented media. Enhanced GSH production was not inhibited by any tested antagonist, and, apart from β-carotene, no agonist induced GSH production. Retinol, but not all- trans RA, enhanced GSH synthesis and increased GCL expression, similar to that observed with β-carotene., Conclusion: The RA signaling pathway may not be involved in the β-carotene-induced enhancement of GSH levels in RAW264 cells, whereas, like β-carotene, retinol can enhance the GSH level and GCL expression.

Scope: Glutathione (GSH) increases in RAW264 murine macrophage cells exposed to β-carotene or β-cryptoxanthin, however, the underlying mechanism has not been clarified. In the present study, we investigated the expression of glutamate-cysteine-ligase (GCL), the rate-limiting enzyme in GSH synthesis, in these cells., Methods and Results: Both the protein and mRNA expression of GCL increased in a β-carotene concentration-dependent manner. Buthionine sulfoximine, a GCL inhibitor, abolished the β-carotene-induced GSH increase without affecting the β-carotene-induced GCL protein expression. Both cycloheximide, a translation inhibitor, and actinomycin D, a transcription inhibitor, completely suppressed the β-carotene-induced GCL protein expression and the concomitant GSH increase. Actinomycin D inhibited the β-carotene-induced Gcl mRNA expression as well. Similarly to β-carotene, β-cryptoxanthin upregulated the GCL protein expression, but lutein did not. The c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed the β-carotene-induced GSH increase, whereas a p38 mitogen-activated protein kinase inhibitor or an extracellular signal-regulated kinase 1/2 inhibitor did not. The JNK inhibitor also suppressed the β-carotene-induced GCL protein expression, and consistently β-carotene induced JNK phosphorylation., Conclusion: These findings revealed that certain carotenoids induce the Gcl mRNA expression in RAW264 cells and subsequently the GCL protein expression, which concomitantly enhances the intracellular GSH level, in a JNK pathway-related manner., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases.

We found aporocotylid larval stages (sporocysts and cercariae) from five individuals of terebellid polychaete Terebella sp., which were collected from seabed substrate and ropes and floats attached to tuna cages in a tuna farm on the coast of Tsushima Island, Nagasaki, Japan. Nucleotide sequences of the regions of internal transcribed spacer 2 ribosomal DNA and 28S ribosomal DNA from these larval stages were 100% identical to those of Cardicola opisthorchis registered in GenBank. C. opisthorchis is a pathogen causing blood fluke infection of Pacific bluefin tuna Thunnus orientalis, which is considered to have a significant impact on the Japanese Pacific bluefin tuna aquaculture industry. This is the first description of the intermediate host of C. opisthorchis. This indicates that the life cycle of C. opisthorchis is completed within tuna farms in this area., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes., Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed., Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei., Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The effects of fish consumption and n-3 poly-unsaturated fatty acid (PUFA) levels on atopic disorders are inconsistent in previous reports, but few studies have investigated the effects of both fish and n-3 PUFA. The aim of the present study was to investigate whether erythrocyte fatty acids and the consumption of fish are associated with atopic diseases in pre- and early adolescents., Methods: A total of 135 students with eczema, 136 students with asthma, and 137 healthy control students were selected from fifth and eighth grades in Shunan, Japan. Atopic disorders and dietary intake were evaluated with questionnaires, and total serum IgE was measured using an enzyme-linked immunosorbent assay. In addition, erythrocyte membrane levels of PUFA were assessed via gas chromatography., Results: Total IgE was significantly elevated in the atopic subjects (P < 0.001). The intake of fatty and dried fish or seafood was significantly associated with eczema (odds ratios of the highest quartiles: 0.46, 95% confidence interval (95%CI): 0.22-0.94; 0.34, 95%CI: 0.16-0.71, respectively). Additionally, only erythrocyte eicosapentaenoic acid (EPA) level had a negative association with eczema (P= 0.048). For asthma, the effect of fish consumption was not significant., Conclusions: Fish consumption was related to a low prevalence of eczema, but not asthma in Japanese pre- and early adolescents. EPA may be involved in this mechanism., (© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.)

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivo techniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed via the gastric mucosa prior to its digestion in the intestines.

Beta-carotene is a carotenoid with a range of reported health benefits besides vitamin A activity. If the enzymatic conversion of beta-carotene to retinal is suppressed in the digestive tract, residual beta-carotene that reaches the tissues increases. We evaluated the function of quercetin and rutin (quercetin-3-rutinoside) to increase the accumulation of beta-carotene in vitro and in vivo in BALB/c mice. When the conversion of beta-carotene by a preparation of the murine small intestine was measured in vitro, the addition of quercetin or rutin considerably inhibited the conversion. When the levels of hepatic beta-carotene and retinoids were measured among three groups of mice fed a diet supplemented with beta-carotene plus quercetin or rutin or beta-carotene alone (four to six mice per group), quercetin increased the level of beta-carotene and decreased the level of retinol, whereas rutin did not. These results demonstrate that quercetin can suppress the conversion of beta-carotene which develops in the cytosol of small intestinal epithelial cells, and that rutin whose rutinose-moiety prevents being absorbed in the small intestine cannot suppress the conversion in vivo. This study offers a novel insight into the interaction between flavonoids and carotenoids with respect to the health benefits from the latter.

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.

To elucidate health benefits of beta-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of beta-carotene. In mice supplemented with beta-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of beta-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated beta-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.

beta-Carotene and/or supplemental alpha-tocopherol were fed to DO11.10 mice to investigate their effect on the immune function of naive splenocytes. A high secretion of interleukin-12 and interferon-gamma in response to the ex vivo primary antigen presentation occurred only when both were fed. This is consistent with the suppressed immunoglobulin E production under the similar condition described in our previous report.

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-beta-glucuronide (Q3GA) and related compounds. A 2',7'-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 microM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.

The activities of beta-carotene on redox status and the immune functions of RAW264 cells, a murine macrophage cell line, were investigated. Supplementation with beta-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 gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. alpha-Tocopherol, which can quench lipid peroxidation by free radical, neither inhibited that by beta-carotene nor influenced the intracellular redox status. Lipopolysaccharide-stimulated transcriptions of IL-1beta and IL-12 p40 in RAW264 were inhibited by beta-carotene but not by alpha-tocopherol. These results indicate that beta-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.

Oxidative stress is frequently mentioned in relation to the neurodegenerative diseases. This study examined the effect of three herb extracts, Hypericum perforatum, Ginkgo biloba L. and Apocynum venetum L., and their components on lipid hydroperoxide-induced oxidative stress in PC-12 cells. Among them, the extract of Apocynum venetum and its components showed the remarkable inhibitory effect, indicating that this herb extract serves as a protective agent against lipid peroxidation-related oxidative stress in CNS. Oxidative stress may be associated with the progress of depression, as this extract has been proposed to be an effective antidepresant herb.

In order to inspect the significance of the adsorbing property in the adjuvant activity to enhance IgE production, we immunized BALB/c mice against Kunitz-type soybean trypsin inhibitor (KSTI), the most potent experimental allergen among soybean proteins, associated with Aluminum hydroxide (alum) or DEAE-Sephadex particles. The production of immunoglobulin isotypes was analyzed at the various amounts, 3-3,000 microg per mouse, of the antigen dosages. In our experiments, although alum did not adsorb KSTI significantly, it augmented the total and the antigen-specific IgE without affecting the optimal range of the antigen dosage. On the other hand, alum did not effectively enhance the production of the other immunoglobulin isotypes. The production of immunoglobulin isotypes other than IgE increased dose-dependently on the antigen. These results ensured our previous finding that another protein, ovalbumin, was used as the antigen. We also demonstrated that the adsorption of KSTI by DEAE-Sephadex in the immunizing vehicle resulted in the requirement of more KSTI for accomplishing the equal immunity in BALB/c mice compared to the control. Moreover, we demonstrated that, regardless of the inability to adsorb KSTI, alum exerted its adjuvant activity only when it was co-injected with the antigen. These results showed that some biochemical effect, other than adsorptive activity, to enhance the production of the antigen-specific IgE resides in alum.

A diet containing different amounts of vitamin E (alpha-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 beta-carotene was supplemented to the basal diet containing vitamin E as alpha-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 beta-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus beta-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and beta-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and beta-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.

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.

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.

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 Man(3)GlcNAc(2) backbone with the beta1-->2 xylose and alpha1-->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., (Copyright 2000 S. Karger AG, Basel.)

A sandwich enzyme-linked immunosorbent assay for the soybean allergen, which consists of a monoclonal antibody (D4) as the fixing (first) antibody and another peroxidase-conjugated monoclonal antibody (C5) as the second, has been developed. Both D4 and C5 monoclonal antibodies strongly bound to the guanidine/HCl-denatured allergen, Gly m Bd 28K. Therefore the samples used in the present experiment were extracted with sodium phosphate buffer (pH 8.0) containing 6 M guanidine and 10 mM 2-mercaptoethanol, then completely dialyzed against phosphate-buffered saline (PBS). The dialyzed samples were subjected to the assay. Various soybean products were observed to contain the allergen at high concentrations, such as soybean protein isolate (SPI), tofu, kori-dofu, and yuba, but its content in soy milk and abura-age were found to be low. In fermented products such as natto, soy sauce, and miso, and even in the processed foods with soybean protein isolate (SPI), the allergen was not detected. These results were also confirmed by an immunoblotting technique with D4.

At least 15 allergenic proteins have been found in soybean using the sera of soybean-sensitive patients with atopic dermatitis [T. Ogawa et al., J. Nutr. Sci. Vitaminol., 35, 555-565 (1991)]. In the present study, a monoclonal antibody (mAb) against Gly m Bd 28K, one of the major allergens of soybean, was prepared, and Gly m Bd 28K was purified from defatted soybean flakes by five purification steps, including immunoaffinity chromatography with the mAb as a ligand. The purified allergen was found to be a glycoprotein with a molecular mass of 26 kDa. During the purification process the allergen was converted to more acidic proteins with the same molecular mass, suggesting that the allergen is unstable. The sugar composition and amino acid sequence of Gly m Bd 28K suggest that the allergen is a new glycoprotein with an Asn-linked sugar moiety. The distribution of the allergen in soybean products was examined by an immunoblotting technique with the mAb.

A micro-assay method for evaluating the allergenicity of soybean allergen was developed by using the mouse antiserum against Gly m Bd 30K, a major soybean allergen, and RBL-2H3, a rat mucosal mast cell line. The antiserum against Gly m Bd 30K was prepared by subcutaneously immunizing BALB/c mice with the allergen. The behavior by affinity-chromatography and the properties against heat treatment show that the reaginic activity of the antiserum resided in the IgE antibody specific for Gly m Bd 30K. The developed assay method is shown to be useful for simulating IgE mediated type-I allergy and to be highly sensitive for detecting the allergen.

Soybeans were treated with proteases to reduce allergenicity. By immunoblotting with a monoclonal antibody against a major soybean allergen (Gly m Bd 30K), two of eight proteases so far tested were selected to achieve a reduction in allergenicity. Both antigenicity to the monoclonal antibody and allergenicity to the sera from soybean-sensitive patients proved to be markedly reduced by processing with either protease. Thus, soybeans treated with an appropriate protease may possibly be supplied as a hypoallergenic foodstuff for patients.

A major soybean allergen, Gly m Bd 30K, is a glycoprotein. A peptide containing a sugar chain was isolated from an alpha-chymotrypic hydrolyzate of the allergen. The amino acid sequence analysis of the peptide showed that its sugar chain binds to the Asn170 residue. Furthermore, the sugar moiety of the allergen and peptide was shown to consist of mannose, N-acetylglucosamine, fucose, and xylose at a molar ratio of 3 : 2 : 1 : 1. These results indicate that Gly m Bd 30 K is a N-linked glycoprotein.

The allergenicity of a Kunitz-type soybean trypsin inhibitor (KSTI) was investigated by a micro-assay of beta-N-acetylhexosaminidase released from RBL-2H3 cells primed with the anti-KSTI serum. KSTI stimulated the release of beta-N-acetylhexosaminidase from RBL-2H3 cells primed with the antiserum. The response of RBL-2H3 cells to the reaginic activity of the mouse anti-KSTI serum correlates fairly well with that by the passive cutaneous anaphylaxis (PCA) test, the sensitivity of both assays appearing to be similar. These results suggest that measuring the beta-N-acetylhexosaminidase released from RBL-2H3 is a convenient way for studying the allergen or the reaginic activity of a murine serum in place of the PCA test.

By a sandwich enzyme-linked immunosorbent assay, a soybean major allergen, Gly m Bd 30K, in soybean products was measured. The allergen occurred at high concentrations in soy milk, tofu, kori-dofu, and yuba, but its content in kinako was small. No allergen was found in fermented foods such as miso, shoyu, and natto. The allergen was clearly shown to occur in meat balls, beef croquettes, and fried chicken that contained soybean protein isolate.

The monitor peptide receptor in the small intestine was investigated. The receptor is on the intestinal mucosal cells [Biochem. J., in press]. The specific binding of the (125)I labeled monitor peptide to dispersed rat small intestinal cells was inhibited by treatment with p-toluensulfonyl-L-lysine chloromethyl ketone (TLCK), an affinity labeling reagent for trypsin. Soybean trypsin inhibitor (SBTI) did not inhibit the binding. Analysis with reduced SDS electrophoresis-autoradiography indicated that an affinity-cross-linked complex of the (125)I labeled monitor peptide and the receptor was abolished by the TLCK treatment but was not affected by the presence of SBTI. Histochemical studies found a predominatnt binding of (125)I labeled monitor peptide on cholecystokinin (CCK)-immunoreactive cells in tissue sections of the rat upper intestine. The result suggests that the monitor peptide receptor is on the surface of CCK-producing cells.