Abdominal aortic aneurysm (AAA) is a vascular disease characterized by progressive dilation of the abdominal aorta. Previous studies have suggested that dietary components are closely associated with AAA. Among those dietary components, eicosapentaenoic acid (EPA) is considered to have suppressive effects on AAA. In the AAA wall of AAA model animals bred under EPA-rich condition, the distribution of EPA-containing phosphatidylcholine (EPA-PC) has been reported to be similar to that of the markers of mesenchymal stem cells (MSCs) and M2 macrophages. These data suggest that the suppressive effects of EPA on AAA are related to preferential distribution of specific cells in the aortic wall. However, the distribution of EPA-PC in the AAA wall of AAA model animals fed a diet containing small amounts of EPA, which has not been reported to inhibit AAA, has not yet been explored. In the present study, we visualized the distribution of EPA-PCs in the AAA wall of AAA model animals fed a diet containing small amounts of EPA (1.5% EPA in the fatty acid composition) to elucidate the vasoprotective effects of EPA. Positive areas for markers of MSCs were significantly higher in the region where EPA-PC was abundant compared to the regions where EPA-PC was weakly detected, but not for markers of M2 macrophages, matrix metalloproteinase (MMP)-2, and MMP-9. The distribution of MSC markers was similar to that of EPA-PC but not that of M2 macrophages and MMPs. These data suggest preferential incorporation of EPA into MSCs under the conditions used in this study. The incorporation of EPA into certain cells may differ according to dietary conditions, which affect the development of AAA.

Abdominal aortic aneurysm (AAA) is a vascular disease that involves aortic wall dilation. Cigarette smoking is an established risk factor and rupture, and nicotine may be a major contributor to the onset of AAA. In humans the condition is associated with stenosis of the vasa vasorum (VV), which may be caused by nicotine. In this study, we evaluated the effects of nicotine on VV pathology. After 4 weeks of nicotine administration to rats using an osmotic pump, the VV patency rate in the nicotine administration group was significantly lower than that in the control group. The levels of Ki-67, a cell proliferation marker, were significantly increased in the regions containing VV in the nicotine group, as were hypoxia inducible factor-α levels. Collagen levels around VV were significantly lower in the nicotine group than in the controls. Our data suggest that nicotine can cause VV stenosis by inducing abnormal proliferation of smooth muscle cells in the VV. The increased risk of AAA development due to cigarette smoking may be partially explained by nicotine-induced VV denaturation and collagen fiber degradation.

BACKGROUND: We have identified and reported a novel antigen, nonprotein-specific secreted EP1-like glycoprotein (51 kDa), for lettuce-related respiratory allergy. OBJECTIVE: We aimed to identify a novel antigen for lettuce-related respiratory allergy that is different from epidermis-specific secreted EP1-like glycoprotein. METHODS: Immunoblotting was performed using an immunoglobulin E-specific antibody. The antigen-antibody reaction was confirmed by means of enzyme-linked immunosorbent assaying. LC-MS/MS analysis was carried out to detect a novel protein found in sera from 3 of 13 patients with lettuce-related respiratory allergy. Finally, we purified a novel protein from Escherichia coli. RESULTS: Immunoblotting assays showed common bands of 17 kDa in the sera of 3 of 13 patients. An enzyme-linked immunosorbent assay confirmed that the patient sera reacted with lettuce latex juice. A 17 kDa protein band that showed antigenic reactivity in 3 of 13 patient sera was identified as a kirola-like protein by LC-MS/MS. In addition, although we purified this protein, we failed to show the inhibitory effect. CONCLUSION: A 17 kDa protein that is a potentially novel antigen of lettuce-associated respiratory allergy was identified. In further studies, we will focus on purifying this novel protein to diagnose lettuce allergy.

A 56-year-old female lettuce farmer was admitted to the hospital with a low-grade fever, worsening cough, and dyspnoea. A blood test revealed eosinophilia and a high serum IgE concentration. The 3-year follow-up showed that her total IgE level increased in December, peaked in May, and suddenly decreased in August. This result was consistent with the lettuce harvest season. A chest x-ray taken on admission showed an infiltrative shadow in the upper lung field. Chest CT revealed patchy ground glass opacity on the upper lung field and thickening of the bronchial wall. The bronchoalveolar lavage fluid contained 8% eosinophils. She was treated with prednisolone, and her symptoms and radiological findings improved. The 37 kDa protein that reacted with the patient's sera was identified by immunoblot analysis.

Medical therapeutic options to prevent rupture of abdominal aortic aneurysm (AAA), a critical event, must be developed. Moreover, further understanding of the process of AAA development and rupture is crucial. Previous studies have revealed that aortic hypoperfusion can induce the development of AAA, and we successfully developed a hypoperfusion-induced AAA animal model. In this study, we examined the effects of medium-chain triglycerides (MCTs), tricaprylin (C8-TG) and tricaprin (C10-TG), on hypoperfusion-induced AAA rat model. We estimated the effects of MCTs on aortic pathologies, mechanical properties of the aorta, and development of AAA. C10-TG, but not C8-TG, significantly suppressed AAA development and completely prevented the rupture. We observed that C10-TG prevented the development and rupture of AAA, but not C8-TG. Additionally, regression of AAA diameter was observed in the C10-TG group. Pathological analysis revealed C10-TG improved the hypoperfusion-induced increase in hypoxia-inducible factor-1α levels, medial smooth muscle cells (SMCs) loss, degeneration of aortic elastin and collagen fibers, and loss of aortic wall elasticity. In addition, regression of the formed AAA was observed by administration of C10-TG after AAA formation. C10-TG administration after AAA formation improved degeneration of AAA wall including degradation of aortic elastin and collagen fibers, stenosis of vasa vasorum, and loss of medial SMCs. These data suggest C10-TG can prevent AAA by attenuating aortic hypoperfusion and degeneration. Considering the clinical safety of C10-TG, C10-TG can be a promising AAA drug candidate.

Abdominal aortic aneurysm (AAA) is a vascular disease that involves asymptomatic progressive expansion of the abdominal aorta. Aneurysm rupture is associated with a high mortality rate. Dietary conditions may be associated with the development and rupture of AAA. However, the relationship between nutrition and AAA is not completely understood. In this study, a nutrition survey was conducted using a brief self-administered diet history questionnaire (BDHQ) to evaluate the relationship between AAA and dietary habits. One-hundred and twenty-six Japanese people participated in the nutrition survey. Food intake status was analyzed in four groups: the analyzed group-1 (all men), analyzed group-2 (men with smoking history), analyzed group-3 (all women) and analyzed group-4 (women without smoking history). In group-2 and group-3, the intake of citrus fruits was significantly lower in the AAA group than in the non-AAA group. In group-2, the intake of soybean and soybean products was significantly lower in the AAA group than in the non-AAA group. In analyzed group-3, the intake of other vegetables (vegetables except for green and yellow vegetables and soybeans) and seafood was significantly lower in the AAA group than in the non-AAA group. This study suggests that AAA onset may be associated with low intake of fruits, soybeans, vegetables, and seafood.

β-caryophyllene (BCP) is a volatile bicyclic sesquiterpenoid found in essential oils obtained from several spices such as black pepper, oregano, basil, rosemary, cinnamon, and clove. BCP is a selective agonist of cannabinoid receptor 2 (CB2 receptor), and orally administered BCP exhibits various biological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, it is still unclear how volatile BCP affects living organisms. We previously reported that inhaled BCP is transferred to sera and organs in mice; additionally, metabolomic analysis revealed inhaled BCP affect the dynamics of metabolites in the livers of mice. These data suggest that inhaled BCP may affect several biological activities by stimulating biological systems. In this study, we evaluated the effects of BCP inhalation on nicotine-induced degeneration of the aortic wall. In the group of mice which inhaled volatile BCP, nicotine-induced increases in elastic fiber degradation and matrix metalloproteinase-2 (MMP-2)-positive areas were attenuated. In addition, BCP improved the nicotine-induced stiffness of aortae and vulnerability to aortic rupture. In cultured aortae, the suppressive effects of BCP were inhibited by the CB2 receptor inhibitor AM630. These results suggest that inhaled BCP is incorporated into the aortic wall and prevents nicotine-induced degeneration of the aorta via a CB2 receptor-dependent pathway.

We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1β and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by progressive dilation of the aorta which is reportedly associated with inflammation. Previous studies suggested that eicosapentaenoic acid (EPA) has suppressive effects on AAA development via anti-inflammatory activities. However, relationships between the anti-inflammatory effects and the cells in the AAA wall are poorly understood. In this study, we visualized the distribution of EPA-containing phosphatidylcholine (EPA-PC) in the AAA wall. EPA-PC was not ubiquitously distributed in both animal (hypoperfusion-induced AAA model) and human AAA walls, suggesting the preferential incorporation of EPA into certain cells. In the EPA-PC-high region of both animal and human AAAs, mesenchymal stem cell (MSC) marker positive areas were significantly higher than those in the EPA-PC-low region. Matrix metalloproteinase-positive MSCs were significantly lower in the AAA wall of the animal model which was administered EPA-rich fish oil. These data suggest that EPA is associated with the attenuation of MSC dysfunctions, which result in the suppression of AAA development.

Turmerones (α-turmerone, β-turmerone, and ar-turmerone) are the major volatile compounds in turmeric (Curcuma longa), a perennial herb of the ginger family. We previously reported that inhaled volatile turmerones could be transferred in the blood and organs. However, the difference between the two pathways, oral administration and inhalation, and the effect of inhaled turmerones on biological activities remain unknown. In this study, we compared the distribution patterns of turmerones after oral administration and inhalation. The relative levels (concentrations of turmerones in each organ/serum) in the lung, olfactory bulb, brain, heart, kidney, and epididymal fat in the inhalation group tended to be, or are significantly, higher than in the oral administration group. The relative levels of brown adipose tissue in the inhalation group were lower than in the oral administration group. Long-term (50 days) inhalation to volatile turmerones suppressed weight gain and hypertrophy of adipocytes in the epididymal fat of mice fed a high-fat diet. These results suggest that inhaled turmerones can be incorporated into the organs of mice via different pathway from as to those from oral administration and can affect the biological function of the organs under certain conditions.

Women are more resistant to vascular diseases; however, the resistance is reduced after menopause. It has been reported that the risk of vascular diseases such as atherosclerosis and abdominal aortic aneurysm is increased in postmenopausal women. Currently, methods to prevent vascular disease in postmenopausal women have not been established. Isoflavones are promising functional food factors that have a chemical structure similar to estrogen. In this study, we investigated the effects of isoflavones on ovariectomized (OVX)-induced degeneration of the aortic wall in mice. Increased destruction of elastic fibers in the thoracic and abdominal aorta was observed in the OVX group, and isoflavones attenuated the destruction of elastic fibers. The positive areas of matrix metalloproteinase (MMP)-2 and MMP-9 in the OVX group were higher than those in the control group. Isoflavones decreased the positive areas of MMP-2 and MMP-9 compared to those in the OVX group. These data suggest that isoflavones have a suppressive effect on OVX-induced degeneration of the aortic wall by inhibiting the increase in MMP-2 and MMP-9.

Transdermal sensitization to allergens is of great concern as a sensitization route for food allergies. This skin-mediated invasion and sensitization to allergens is involved in skin barrier breakdown and inflammation, followed by the production of several kinds of cytokines. Cytokines such as thymic stromal lymphopoietin and thymus and activation-regulated chemokine are also involved. In this study, we investigated the suppressive effect of tannic acid (TA) on transdermal sensitization using ovalbumin (OVA), a major egg-white allergen. We also analyzed the mechanisms associated with the inhibitory effects of TA. The results showed that the co-application with TA prevents transdermal sensitization to OVA. As possible mechanisms, its anti-inflammatory and astringent effect on the skin and binding ability with the protein were considered. These results indicate that TA could be applied to cosmetics and lotions, which could suppress the transdermal sensitization to allergens.

Dietary habit is closely associated with healthspan. Functional food factors are key to maintaining a health metabolism in our bodies. Because functional food factors are main components to determine the quality of foods, many technologies have been established to analyze functional factors in foods. High-performance liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry is a solid approach to detect functional food factors with high sensitivity and specificity. Findings obtained from these mass spectrometric approaches play essential roles in estimating the quality of foods. However, these technologies are not available for the analysis of the spatial distribution of molecules of interest in foods. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is considered an ideal approach to visualize distribution of molecules in foods. MALDI-MSI is a two-dimensional MALDI-MS technology that can detect compounds in a tissue section without purification, separation, or labeling. MALDI-MSI can be used to visualize the spatial distribution of wide range of food components including protein, peptides, amino acids, lipids, carbohydrate, and vitamins. Although the methodology of MALDI-MSI in food science is not yet fully established, the versatility of MALDI-MSI is expected to open a new frontier in food science. In this mini review, we briefly summarized the applications of MALDI-MSI in the field of food science.

A 73-year-old woman complaining of cough and dyspnea was admitted to our hospital. High-resolution computed tomography chest revealed patchy ground-glass attenuation in the upper lung field. The patient suffered an asthma attack and was diagnosed with allergic pneumonitis; prednisolone was administered for treatment. Bird-related hypersensitivity pneumonitis was suspected, as she had a gray parrot (Psittacus erithacus) and a budgerigar (Melopsittacus undulatus) at home. An immunoblotting analysis with the patient's serum demonstrated IgG-binding fractions to the gray parrot's feathers only; no binding was noted with the budgerigar antigens. The patient was conclusively diagnosed with hypersensitivity pneumonitis related to exposure to a gray parrot.

植物由来の食物アレルゲンの多くは感染特異的タンパク質(PR-P)ファミリーに属するものが多いことが知られている。これらは植物間で構造が類似しており、また植物内で比較的存在量が多いことや、分子内S-S結合に起因する消化抵抗性や吸収性、熱安定性などのアレルゲンとなりやすい分子特性を有する。このPR-Pファミリーは病虫害や環境ストレスなどによって発現が増加するため、植物の栽培環境によってこれらの分子が増減し、アレルゲン性に影響を及ぼす可能性がある。本稿では、食物アレルゲンとしての植物由来生体防御タンパク質について概説し、新たに果物由来の経皮感作抗原として同定されたアレルゲン候補分子も、これらの生体防御タンパク質に属することを紹介する。(著者抄録)

Vascular diseases such as atherosclerosis and aneurysms are associated with diet. Perivascular adipose tissue (PVAT) was reportedly involved in the regulation of vascular functions. It is suggested that imbalanced diets can cause PVAT inflammation and dysfunction as well as impaired vascular function. However, the association between diets and PVAT are not clearly understood. Here, we showed that a high-fat and a high-sucrose diet affected PVAT at different sites. A high-fat diet induced increased number of large-sized lipid droplets and increased CD (Cluster of differentiation) 68+ macrophage- and monocyte chemotactic protein (MCP)-1-positive areas in the abdominal aortic PVAT (aPVAT). In addition, a high-fat diet caused decreased collagen fibre-positive area and increased CD68+ macrophage- and MCP-1-positive areas in the abdominal aorta. In contrast, a high-sucrose diet induced increased number of large-sized lipid droplets, increased CD68+ macrophage- and MCP-1-positive areas, and decreased UCP-1 positive area in the thoracic aortic PVAT (tPVAT). A high-sucrose diet caused decreased collagen fibre-positive area and increased CD68+ macrophage- and MCP-1-positive areas in the thoracic aorta. These results could be attributed to the different adipocyte populations in the tPVAT and aPVAT. Our results provide pathological evidence to improve our understanding of the relationship between diet and vascular diseases.

Abdominal aortic aneurysm (AAA) involves the degradation of vascular fibres, and dilation and rupture of the abdominal aorta. Hypoperfusion in the vascular walls due to stenosis of the vasa vasorum is reportedly a cause of AAA onset and involves the induction of adventitial ectopic adipocytes. Recent studies have reported that ectopic adipocytes are associated with AAA rupture in both human and hypoperfusion-induced animal models, highlighting the pathological importance of hypoperfusion and adipocytes in AAA. However, the relationship between hypoperfusion and AAA remains unknown. In this study, we investigated the changes in inflammation-related factors in adipocytes at low glucose and serum levels. Low glucose and serum levels enhanced the production of AAA-related factors in 3T3-L1 cells. Low glucose and serum levels increased the activation of protein kinase B (also known as Akt), extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, and nuclear factor (NF) кB at the protein level. The inflammatory factors and related signalling pathways were markedly decreased following the return of the cells to normal culture conditions. These data suggest that low glucose and serum levels increase the levels of inflammatory factors through the activation of Akt, mitogen activated protein kinase, and NF-κB signalling pathways.

近年、食物アレルギーの感作経路として、皮膚から侵入したアレルゲンによる「経皮感作」が注目されている。経皮感作には、ランゲルハンス細胞や皮膚上皮細胞由来のTSLP、好塩基球などが関与する。私たちは、マウスモデル系を用いて、様々な食品抽出物を皮膚に塗布することで、マウスにおけるTh2型抗体であるIgE、IgG1の産生を指標にして経皮感作抗原の同定を進めている。本稿では、経皮感作に関する総論とともに、果物由来の経皮感作抗原の同定例として、チェリー及びキウイに関する我々の研究成果を簡単に紹介する。(著者抄録)

Women are more resistant than men to the development of vascular diseases. However, menopause is a factor leading to deterioration of female vascular integrity, and it is reported that the risk of vascular diseases such as atherosclerosis and abdominal aortic aneurysm is increased in postmenopausal women. Although it is suggested that perivascular adipose tissue (PVAT) is deeply involved in the increased risk of vascular disease development, the effect of menopause on PVAT integrity is unknown. In this study, we aimed to elucidate the effect of menopause on PVAT in ovariectomized (OVX) rats. PVAT was divided into 4 regions based on characteristics. Hypertrophy and increased inflammation of adipocytes in the PVAT were observed in the OVX group, but the effects of OVX were different for each region. OVX induced matrix metalloproteinase (MMP) -9 which degrade extracellular matrix such as elastin and collagen fibers in PVAT. Degeneration of the arterial fibers of the thoracic and abdominal aorta were observed in the OVX group. These results indicate that OVX can cause dysfunction of PVAT which can cause degradation of arterial fibers. Appropriate management of PVAT may play an important role in the prevention and treatment of diseases originating from ovarian hypofunction.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ± 3.3 vs. 8.3 ± 2.4, P < 0.05; 7.9 ± 1.1 vs. 5.9 ± 1.6, P < 0.05; 19.0 ± 4.2 vs. 14.1 ± 3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ± 8.3 vs. 43.9 ± 12.2 mg/dL P < 0.05) and adiponectin (7.7 ± 0.3 vs. 9.5 ± 0.6 μg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

Alzheimer's disease (AD) is the most common neurodegenerative disease. Garlic reportedly has various physiological effects, including a role in protecting against dementia. However, the action mechanisms of garlic on AD are not entirely clear. In this study, we investigated the inhibitory activity of garlic essential oil (GEO) against AD-related enzymes and evaluated the distribution of active substances in GEO to the brain. We found that several sulfur compounds in GEO significantly inhibited AD-related enzymes. Sulfur compounds were detected in the serum and brain 6 h post administration. The ratios of allyl mercaptan (24.0 ± 3.9%) and allyl methyl sulfide (49.8 ± 15.6%) in the brain were significantly higher than those in GEO, while those of dimethyl trisulfide (0.89 ± 34.8%), allyl methyl trisulfide (0.41 ± 19.0%), and diallyl trisulfide (0.43 ± 72.8%) in the brain were significantly lower than those in GEO. Similar results were observed in the serum, suggesting that the organosulfur compounds were converted to allyl mercaptan or allyl methyl sulfide in the body. Although allyl mercaptan and allyl methyl sulfide are not the main components of GEO, they might be key molecules to understand the bioactivities of GEO in the body.

The effect of ellagic acid (EA), a naturally occurring polyphenolic compound, on the secretion of apolipoproteins from human hepatocytes, HepG2, was investigated. The levels of apoB and apoA-1 secreted in the cell culture medium were determined by sandwich ELISA. EA did not affect cell viability at the tested concentrations (up to 50 µM). EA suppressed the secretion of apoB and enhanced that of apoA-1 from HepG2 cells. However, cellular apoB levels were increased, suggesting that EA inhibited the trafficking of apoB during the process of secretion. In contrast, the increase in the cellular levels of apoA-1 was consistent with its secreted levels. These results indicate that EA inhibits the secretion of apoB from hepatocytes and increases the secretion of apoA-1. Both of these effects are beneficial for lipoprotein metabolism in the prevention of lifestyle-related diseases. The detailed mechanism underlying these effects of EA on lipoprotein metabolism should be elucidated in the future, but this naturally occurring polyphenolic compound might be antihyperlipidemic. Based on these results, EA is suggested as a candidate food-derived compound for the prevention of hyperlipidemia.

Abdominal aortic aneurysm (AAA) is an aortic disease in which the aortic diameter is ≥3.0 cm; if left untreated, the aortic wall continues to weaken, resulting in progressive dilatation. Effective therapeutic drugs for AAA patients have not been discovered. Eicosapentaenoic acid (EPA) reportedly attenuates the development of AAA in experimental AAA animal models. However, the underlying mechanism of action is still not totally clear. To understand the mechanism, we visualized the distribution of EPA-containing phosphatidylcholine (PC) in the AAA wall by matrix-assisted laser desorption ionization-mass spectrometry imaging. EPA-containing PC was characteristically distributed in the AAA wall, and the positive area for the M2 macrophage marker was significantly higher in the region where EPA-containing PC was highly detected (region 2) than in the region where EPA-containing PC was poorly detected (region 1). The M1 macrophage marker levels were not different between regions 1 and 2. A comparative observation showed a similar distribution of the M2 macrophage marker and EPA-containing PC. These data suggest the preferential incorporation of EPA into M2 macrophages. Positive areas for matrix metalloproteinase 2 and malondialdehyde in region 2 were significantly lower than those in region 1. The reported suppressive effect of EPA on the development of AAA may be partly attributed to the increased anti-inflammatory property of M2 macrophages.

In this study, we fed obese model mice black soybean seed coat powder (BSCP) and evaluated the antiobesity effects. As a control, normal yellow soybean seed coat powder (YSCP) was used. C57BL/6J, a high-fat diet-induced obesity model mouse, was fed a high-fat diet containing BSCP or YSCP (20% fat) to induce obesity. The results showed that in the BSCP group, it caused significant suppression of body weight gain and suppression of white adipose tissue weight compared with the YSCP group. Moreover, it significantly decreased serum leptin levels, which correlated with visceral fat mass, and increased antidiabetic adipocytokine and adiponectin levels. Therefore, this suggests the pigmented components contained in BSCP have an antiobesity effect in obese model mice. It is suggested that this material, which can be prepared without extraction with an organic solvent and is suitable for use as a food material, could be a functional food material with a practicable antiobesity effect.

Hypoperfusion due to vasa vasorum stenosis can cause wall hypoxia and abdominal aortic aneurysm (AAA) development. Even though hypoperfusion is an important contributor toward pathological changes in AAA, the correlation between hypoperfusion and AAA is not fully understood. In this study, a time-dependent semi-quantitative pathological analysis of hypoperfusion-induced aortic wall changes was performed to understand the mechanisms underlying the gradual degradation of the aortic wall leading to AAA formation. AAA-related factors evaluated in this study were grouped according to the timing of dynamic change, and five groups were formed as follows: first group: angiotensin II type 1 receptor, endothelin-1 (ET-1), and malondialdehyde (MDA); second group: matrix metalloproteinase (MMP)-2, -9, -12, M1 macrophages (Mac387+ cells), and monocyte chemotactic protein-1; third group: synthetic smooth muscle cells (SMCs); fourth group: neutrophil elastase, contractile SMCs, and angiotensinogen; and the fifth group: M2 macrophages (CD163+ cells). Hypoxia-inducible factor-1α, ET-1, MDA, and MMP-9 were colocalized with alpha-smooth muscle actin cells in 3 h, suggesting that hypoperfusion-induced hypoxia directly affects the activities of contractile SMCs in the initial stage of AAA. Time-dependent pathological analysis clarified the cascade of AAA-related factors. These findings provide clues for understanding complicated multistage pathologies in AAA.

β-caryophyllene (BCP), an essential oil component of many herbs and spices, has various biological activities as a functional food factor. A distinct feature of BCP is its volatile double-ring sesquiterpene structure. Orally administered BCP is reportedly detected in its intact form in mice serum; however, the distribution of inhaled volatile BCP throughout the body remains unknown. This study aimed to estimate the distribution properties of inhaled volatile BCP and to investigate its effects on metabolism. After mice were exposed to volatile BCP, it was detected in the lung, olfactory bulb, brain, serum, heart, liver, kidney, epididymal fat, and brown adipose tissue. BCP was further detected in the brain, liver, and brown adipose tissue 24 h after exposure. Metabolites related to glutathione metabolism were significantly altered in the liver. These results suggest that inhaled volatile BCP is widely distributed in murine tissues and affects the dynamics of metabolites in the liver.

Numerous recent studies have suggested that food allergens enter the skin and predispose individuals to food allergies through the production of IgE antibodies in the body. Cherries are a popular fruit eaten worldwide. However, cherries are an allergenic food and percutaneous sensitization with cherry allergens through the perioral region may occur while ingesting cherries. The identity of the cherry protein that triggers percutaneous sensitization in humans or animal models remains unknown. In this study, the backs of BALB/c mice were shaved and crude cherry extracts containing sodium dodecyl sulfate were applied to the skin. Thereafter, the cherry-specific IgE and IgG1 antibodies generated and secreted in response to the epidermal application were measured using an enzyme-linked immunosorbent assay or immunoblotting. Skin exposure to cherry extracts elevated cherry-specific IgG1 levels. Application of fractionated and purified cherry proteins (antigen candidates for percutaneous sensitization) that bound to the IgG1 antibodies led to the identification of a thaumatin-like protein (Pru av 2). This molecule is known as the major cherry allergen that affects humans. In conclusion, our study identified Pru av 2 as a cherry allergen that triggers percutaneous sensitization in mice for the first time.

Background: Kiwifruit is a popular fruit consumed worldwide and is also used as a cosmetic ingredient. However, it is known to cause allergic reactions in humans. Recent studies have suggested an association between food allergy and food allergens entering the body via the skin. However, percutaneously sensitizing kiwifruit allergens have not been identified in human studies or in animal models. Objective: This study aimed to identify kiwifruit proteins that percutaneously sensitized mice through the epidermal application of crude extracts from green and gold kiwifruit on the dorsal skin, and serum IgE and IgG1 levels were used as sensitization markers. Design: BALB/c mice were back-shaved and their skin was exposed to crude extracts from green and gold kiwifruit that contained sodium dodecyl sulfate. Specific IgE and IgG1 antibodies generated and secreted in response to antigens were measured using enzyme-linked immunosorbent assay or immunoblotting. Results: Skin exposure to kiwifruit extract induced an increase in the levels of kiwifruit-specific IgE and IgG1, which are helper T cell 2-related allergenic antibodies in mice. These antibodies reacted with 18, 23, and 24 kDa proteins found in both green and gold kiwifruits. Thus, three percutaneously sensitizing allergens were identified and purified. Their amino acid sequences partially matched with that of kiwellin (Act d 5). Discussion and conclusion: Kiwellin has been identified as a plant defense-related protein. Interestingly, many plant allergens are biodefense-related proteins belonging to the pathogenesis-related protein family. Kiwellin, which was discovered to be a transdermal sensitizing antigen, might also be categorized as a biodefense-related protein. This study is the first to identify kiwellin (Act d 5) as a percutaneously sensitizing kiwifruit allergen in a mouse model.

BACKGROUND: Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms. METHODS: We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry. RESULTS: A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P < .05). Lettuce-specific IgE (ImmunoCAP® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51 kDa). CONCLUSION: The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy.

近年、大豆アレルギーの多様性が注目されている。特に、花粉症と関連する大豆アレルギーが成人を中心に増加したことも記憶に新しい。この原因抗原はGly m 4及びGly m 3である。一方、小児での重篤な大豆アレルギーの場合は、大豆の2SアルブミンであるGly m 8の重要性が指摘されている。他にも、新たな大豆アレルゲンとして、ビオチン結合タンパク質であるGly m 7が発見された。また、大豆の経皮感作抗原の同定や、変動解析についても新たな知見が得られている。(著者抄録)

近年、大豆アレルギーの多様性が注目されている。特に、花粉症と関連する大豆アレルギーが成人を中心に増加したことも記憶に新しい。この原因抗原はGly m 4及びGly m 3である。一方、小児での重篤な大豆アレルギーの場合は、大豆の2SアルブミンであるGly m 8の重要性が指摘されている。他にも、新たな大豆アレルゲンとして、ビオチン結合タンパク質であるGly m 7が発見された。また、大豆の経皮感作抗原の同定や、変動解析についても新たな知見が得られている。(著者抄録)

Several analyses of allergen levels have been reported as part of the safety assessment of genetically modified (GM) soybean; however, few comprehensive analyses have included new allergens. Thus, in this study the levels of eight major soybean allergens, including Gly m 7 (a newly reported soybean allergen), were semi-quantitatively detected in six GM soybeans and six non-GM soybeans using antigen-immobilized ELISA and immunoblotting. We also analyzed the IgE-reactivity to these soybeans through immunoblotting, using sera from three soybean-allergic patients. The results showed that there were no significant differences in the levels of the major soybean allergens in the GM and non-GM soybeans. Moreover, there were no significant differences in the serum IgE-reactive protein profiles of the patients, as analyzed using immunoblotting. These results indicate that, in general, CP4-EPSPS-transfected GM soybeans are not more allergenic than non-GM soybeans.

Oral allergy syndrome(OAS)は花粉症患者が新鮮な果物や生野菜を食べた際にみられるIgE抗体の関与する即時型アレルギーである。通常、症状は口腔や喉に限局し、原因食品であっても加熱すれば食べられる。こういった現象は、花粉アレルゲンに対するIgE抗体が、花粉抗原と共通の構造を有する食物抗原に対して交差反応を起こすことによって生じる。このことから花粉-食物アレルギー症候群(Pollen-associated Food Allergy Syndrome:PFAS)とも呼ばれる。また、PFASのアレルゲンコンポーネントの特徴として、熱に弱く、消化されやすい特徴を有する。しかし、豆乳など大豆製品では例外的に全身症状を伴うことがある。本稿では、PFASに係るコンポーネントの特徴をわかりやすく解説し、シラカンバ花粉症でみられる豆乳アレルギーが重症化する機序に関して仮説も加えて記述する。また最近ヒノキ科の花粉とモモGibberellin-regulated protein(GRP)との関連が海外で報告されており、これについても若干の検証を加える。(著者抄録)

Existing animal models do not replicate all aspects of abdominal aortic aneurysms (AAAs), including the rupture mechanisms. From histopathological analyses conducted in humans, it has been found that the vasa vasorum of the AAA wall is the starting point of circulatory failure and that bulging and dilatation of the abdominal aorta occurs through inflammation and tissue degeneration. We created a new animal model (the hypoperfusion-induced model) of AAAs. In this study, we describe the current animal models of AAAs and present the utility of our new model of AAAs.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by weakening of vascular walls and progressive dilation of the abdominal aorta. Nicotine, the main component of tobacco, is reportedly associated with the development and rupture of AAA. It is desirable to attenuate the destructive effect of nicotine on vascular walls, using dietary food components. However, effective methods for preventing AAA progression using dietary food components remain unestablished. This study focuses on proanthocyanidins, well known for their potent antioxidant activity. We speculated that proanthocyanidins can suppress nicotine-induced weakening of vascular walls. To estimate the effect of black soybean seed coat extract (BSSCE), rich in proanthocyanidins, on nicotine-induced weakening of the aortic wall, mice were divided into four groups: the control diet and distilled water group (named C), BSSCE solution diet and distilled water group (named B), control diet and 0.5 mg/mL nicotine solution group (named CN), and BSSCE solution diet and 0.5 mg/mL nicotine solution group (named BN). Nicotine-induced degradation of elastin and collagen fibers were significantly suppressed in BN group. The positive areas for matrix metalloproteinase (MMP)-2 and oxidative stress in BN group were significantly decreased compared to those in CN group. These results suggest that proanthocyanidins-rich BSSCE can prevent the weakening of the aortic wall via inhibiting MMP-2 upregulation.

β-Conglycinin is the major storage protein in soybeans. Pre-clinical animal models and human clinical studies have demonstrated the triglyceride-lowering effect of this protein, suggesting that it could be put into practical use as a functional food material. To date, however, there are no accurate and simple assays for quantification of β-conglycinin. In this study, samples were pretreated by mixing them with rice flour powder prior to extraction of proteins. Then, we used commercially available ELISA kits for detection of allergens that could be present in any contaminating soybean residue. This enabled accurate and highly reproducible quantitation of β-conglycinin content in several processed soybean foods.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by weakening of the vascular walls. Male sex is a risk factor for AAA, and peak AAA incidence occurs in men 10 years earlier than in women. However, the growth rate of AAA is faster in women, and women have a higher mortality due to AAA rupture. The mechanisms underlying sex-related differences in AAA remain unknown. Herein, we evaluated the effects of ovariectomy (OVX) on AAA in rats. Upon evaluation of the effects of OVX and AAA induction, AAA incidence rate and the aneurysm diameter increased in the OVX group. However, the histopathology in the developed AAA wall was not different between groups. When the effects of OVX on the vascular wall without AAA induction were evaluated, elastin and collagen levels were significantly decreased. Furthermore, the level of matrix metalloproteinase-9 significantly increased in the OVX group. According to our results, it is speculated that decreased levels of collagen and elastin fibers induced by OVX might be involved in increased incidence rate and diameter of AAA. Weakening of the vascular wall before the onset of AAA might be one reason for the faster rate of AAA growth in women.

Nicotine has been linked to the development of abdominal aortic aneurysms. Isoflavones, a group of polyphenolic compounds, reportedly exhibit antioxidant and anti-inflammatory properties and facilitate cardiovascular protection. However, the effects of isoflavone on nicotine-induced abdominal aortic aneurysms have not yet been elucidated. The objective of the current study was to evaluate the inhibitory effect of isoflavone on nicotine-induced weakening of the aortic wall in mouse models. Nicotine reportedly increases the occurrence of abdominal aortic aneurysms by activating endothelin-1 (ET-1), angiotensinogen and the angiotensin II type 1 (AT1) receptor, leading to an increase in neutrophil elastase, oxidative stress, and matrix metalloproteinase (MMP)-2 expression, which causes vascular wall weakness and damage. Immunohistological analyses have indicated that isoflavone significantly inhibits the activation of ET-1, angiotensinogen and the AT1 receptor in nicotine-administered mice. Additionally, isoflavone suppressed elastic fiber destruction and decreased areas positive for MMP-2, neutrophil elastase, and malondialdehyde in the vascular wall of nicotine-administered mice. Considered together, these findings suggest that isoflavone shows potential for preventing vascular wall injury induced by nicotine administration, and that food containing isoflavone may protect against abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by the dilation of the abdominal aorta, resulting in a high mortality rate caused by vascular rupture. Previous studies have suggested that the abnormal appearance of adipocytes in the vascular wall is associated with the development of AAA. However, the mechanisms underlying the appearance of the ectopic adipocytes remain unknown. In this study, we showed that CD44+CD90+ MSCs express adipogenic transcription factors in the AAA wall of a hypoperfusion-induced AAA model. The number of CD44+CD90+ cells and adipocytes in the AAA wall significantly decreased in the perivascular adipose tissue (PVAT)-removed vascular wall. The AAA diameter significantly decreased in the PVAT-removed vascular wall compared with that in the vascular wall with PVAT. These data suggested that PVAT plays important roles in the differentiation of MSCs into adipocytes in response to vascular hypoperfusion. The decreased number of adipocytes in the PVAT-removed vascular wall might be associated with the decreased AAA diameter.

食物アレルゲンの新たな感作経路として、皮膚を介した経皮感作が注目されている。現在明らかにされている経皮感作のメカニズムとしては、ランゲルハンス細胞や上皮細胞由来のサイトカインであるTSLPやIL-33などが主に作用して引き起こされることが明らかになってきた。経皮感作の関与が疑われる症例報告も含め、経皮感作に関する研究は増加している。そこで本稿では、総説として経皮感作の研究例やメカニズム、さらに経皮感作が報告されているアレルゲン・コンポーネントについて簡単に紹介する。(著者抄録)

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.

Ginkgo biloba extract (GBE) is widely used as herbal medicine. Preventive effect of GBE against dementia, including Alzheimer's disease, has been reported. The bioactive compounds in GBE that impart these beneficial effects, flavonoids and terpene lactones, have poor bioavailability. Our previous study found distribution of bioactive compounds of sesame extract in mice brain after mixing it with turmeric oil. Here, we evaluate the distribution of bioactive compounds of GBE by combining it with the mixture of sesame extract and turmeric oil (MST). The content of terpene lactones in mice serum was significantly increased in a dose-dependent manner after administration of GBE. However, the contents of terpene lactones in mice brain were not significantly changed. Concentration of ginkgolide A in mice brain increased significantly when GBE was co-administrated with MST than when GBE was administered alone. These results suggest that MST may be effective in enhancing the bioavailability of ginkgolide A in GBE.

Accumulation of amyloid-β peptide is associated with Alzheimer's dementia. Previously, we reported that sesamin and sesamolin inhibited β-secretase activity in vitro, and each was transported to the serum and brain in mice after oral administration. However, the bioavailability of sesamin and sesamolin was poor in mice. In this study, we aimed to improve the bioavailability of sesamin and sesamolin. We found that the levels of sesamin and sesamolin in mouse serum and brain were higher after the administration of a mixture of sesame extract and turmeric oil (MST) than those after administering sesame extract alone. Serum sesamin and sesamolin contents in the MST-treated group were 23-fold and 15-fold higher, respectively, than those in the sesame extract-treated group. Brain sesamin and sesamolin contents in the MST-treated group were 14-fold and 11-fold higher, respectively, than those in the sesame extract-treated group. These results suggest that turmeric oil is an effective solvent to enhance the bioavailability of sesamin and sesamolin.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by the weakening of the vascular walls and the progressive dilation of the abdominal aorta. Nicotine, a primary component of cigarette smoke, is associated with AAA development and rupture. Nicotine induces AAA development by weakening vascular walls. However, little is known about preventive methods using functional food factors for nicotine-induced vascular destruction. Sesamin and sesamolin are functional food factors that are fat-soluble lignans found in Sesamum indicum seeds. Previous reports indicated that sesamin and sesamolin have anti-oxidative and anti-inflammatory effects. In this study, we evaluated the effects of sesamin and sesamolin-rich sesame extract on the weakening of vascular walls in nicotine-administered mice. Sesame extract attenuated the degradation of collagen and elastin fibers caused by nicotine. In addition, sesame extract decreased the area positive for matrix metalloproteinase 12 (MMP-12) and oxidative stress in the vascular walls. These results suggest that sesame extract may decrease the weakening of vascular walls by suppressing the nicotine-induced degradation of collagen and elastin fibers. Sesame extract may be effective in preventing AAA development by decreasing both, MMP-12 expression and oxidative stress in vascular walls.

Soyasapogenol is a soyasaponin aglycone, which has been suggested to exert a more potent function than the glycoside form. In this study, the effect of soyasapogenol A and B on cultured adipocyte cell function was investigated using mouse 3T3-L1 adipocyte cells. 3T3-L1 cells were treated with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine for differentiation to adipocytes, and the cells were then cultured in the presence of soyasapogenol A or B (6.25 or 12.5 µM). The media were harvested and refreshed every 2 d. After a 10 d culture, the cells were harvested and the triglyceride content of the cells was determined. The triglyceride content of soyasapogenol B-treated cells was significantly lower than those of vehicle-treated cells. Glycerol and free fatty acid levels in the soyasapogenol-treated cell media were higher than those in vehicle cells. However, there was no difference in the level of adipose triglyceride lipase among soyasapogenol A-, soyasapogenol B-, and vehicle-treated cells. The secreted adiponectin and resistin levels of soyasapogenol-treated cell media were also different compared with those of vehicle-treated cells. Especially, the secreted resistin level in soyasapogenol B-treated cell media was obviously reduced compared with that of vehicle-treated cells. Taken together, these results suggest that soyasapogenol B exerted an anti-obesity and anti-diabetic effect on adipocytes by lowering the cellular triglyceride level by accelerating triglyceride lipolysis with reduced resistin secretion.

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin β subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model.

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30 K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.

Abdominal aortic aneurysm (AAA) is a vascular disease that involves the gradual dilation of the abdominal aorta followed by its rupture. AAA is closely associated with weakening of the vascular wall due to oxidative stress, chronic inflammation, and degradation of the extracellular matrix. No effective drug therapy is currently available for preventing aneurysm progression or rupture. Adipocytes in the vascular wall are reportedly closely associated with AAA development and rupture. Fiber degradation in the aneurysm wall is enhanced by increased numbers of adipocytes, and rupture risk may increase as well. Recent studies suggested that appropriate control of adipocytes in the vascular wall may be an important strategy to prevent AAA rupture, and further studies may aid in the establishment of a method for preventing AAA rupture by therapeutic drugs or functional foods. In this review, we summarize adipocyte function and the correlation between AAA and adipocytes.

Abdominal aortic aneurysm (AAA) is a vascular disease that results in rupture of the abdominal aorta. The risk factors for the development of AAA include smoking, male sex, hypertension, and age. AAA has a high mortality rate, but therapy for AAA is restricted to surgery in cases of large aneurysms. Clarifying the effect of dietary food on the development of AAA would be helpful for patients with AAAs. However, the relationship between dietary habits and the development of AAA is largely unknown. In our previous study, we demonstrated that adipocytes in vascular wall can induce the rupture of AAA. Therefore, we focused on the diet-induced abnormal triglyceride metabolism, which has the potential to drive AAA development. In this study, we have evaluated the effects of a high-sucrose diet on the development of AAA in a vascular hypoperfusion-induced animal model. A high sucrose diet induced high serum TG level and fatty liver. However, the AAA rupture risk and the AAA diameter were not significantly different between the control and high-sucrose groups. The intergroup differences in the elastin degradation score and collagen-positive area were insignificant. Moreover, matrix metalloproteinases, macrophages, and monocyte chemoattractant protein-1-positive areas did not differ significantly between groups. These results suggest that a high-sucrose diet does not affect the appearance of vascular adipocyte and AAA development under the vascular hypoperfusion condition.

In this study, we isolated and characterized the acid-soluble skin collagen of Pacific bluefin tuna (PBT, Thunnus orientalis). The PBT skin collagen was composed of two α chains (α1 and α2) and one β chain. The denaturation temperature of PBT collagen was low although it was rich in proline and hydroxyproline. The primary structure of PBT skin collagen was almost identical to that of calf and salmon skin collagen however, it differed with respect to the epitope recognition of the antibody against salmon type I collagen. These results suggest that the primary structure of skin collagen was highly conserved among animal species, although partial sequences that included the epitope structure differed among collagens.

Rationale: Anthocyanins, which belong to a class of molecules called flavonoids, are known to have beneficial effects for both humans and animals. Many physiological functions have been attributed to anthocyanins since ancient times. The most important function is the relief of eyestrain, but the biodistribution of anthocyanins remains unknown. In this study, we analyzed the kinetics of anthocyanin species in mice eyeballs and surrounding tissues. Methods: We used mice that were administered bilberry extract solution intraperitoneally. After harvesting eyeballs, cross-sections were prepared using a cryostat and analyzed using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Results: Various ions of anthocyanin species, m/z 419, 449, 463, 465, 479, and 493, were observed in MALDI-MSI spectra. Most of these peaks corresponded to places considered to be extraocular muscles with the outer layer of the retina. Conclusions: Through MALDI-MSI and MALDI-MS/MS analyses, we demonstrated that anthocyanin species are distributed at muscle tissues with the outer layer of the retina. It is speculated that anthocyanin species directly improve eyestrain at the extraocular muscles.

Group B soyasaponins, found in soy, have various health-promoting properties, but it is unclear whether they have an anti-obesity effect. The aim of this study was to evaluate the anti-obesity effect of group B soyasaponin glycosides and aglycone in mice fed a high-fat diet. Six-week-old C57/BL6 mice were divided into three groups (each n=10) and orally administered a high-fat diet for 35 d; two of the groups also received group B soyasaponin glycosides or aglycone. Although there was no significant difference among the three groups in consumption, the weight of fat adipose tissue at autopsy was more than 30% lower in the group B soyasaponin aglycone group than in the control group, but X-ray computed tomography showed no significant difference in muscle weight between these two groups. The ratio of muscle to whole body weight was higher in the group B soyasaponin aglycone group than in the control group. These results suggest that group B soyasaponin aglycone has a stronger anti-obesity effect than group B soyasaponin glycosides, without a loss in muscle weight, and that it increases the ratio of muscle to whole body weight. To our knowledge, this is the first report showing the anti-obesity effect of soyasaponin aglycone in vivo using animal models.

Thoracic aortic aneurysm (TAA) is a lethal vascular disease that involves localized dilation of the thoracic aorta. The detailed mechanisms of TAA development and rupture are not fully understood. Recent reports have shown that the abnormal appearance of adipocytes in the vascular wall is associated with abdominal aortic aneurysm (AAA) progression or rupture. However, the presence of adipocytes in the TAA wall remains unknown. In this study, we observed the pathology of thoracic aortae to investigate whether adipocytes abnormally appear in the TAA wall. Abnormal appearance of adipocytes was mainly observed in the adventitia in the TAA vascular walls. The adipocyte area in the vascular wall was significantly increased in the TAA wall compared to the control wall. Destruction of collagen fibers, and increase in areas positive for matrix metalloproteinase (MMP) -2, MMP-9, and Mac387+ macrophages were observed in the area around adipocytes in the vascular wall. This study demonstrated the appearance of adipocytes in the TAA wall. The accumulation of adipocytes in AAA wall reportedly facilitates the destruction of fibers surrounding adipocytes, and thereby, leads to vascular wall weakness. Therefore, adipocytes in the TAA wall can be associated with the weakening of the vascular wall as well as the AAA wall. The appropriate control of adipocytes in the vascular wall may prevent weakening of the vascular wall in TAA.

Oral tolerance prevents allergic responses, but cutaneous exposure to food allergens predisposes individuals to food allergies. Soybean, a major allergenic food, is also an ingredient in various cosmetic products. However, it remains to be determined whether oral tolerance prevents percutaneous sensitization to soybean proteins in humans or animal models. In this study, BALB/c mice were divided into three groups; the SS group fed a soybean-containing diet, and the CS and control (C) groups fed a soybean-free diet. After being dorsally shaved, the CS and SS groups were epicutaneously exposed to a soybean extract while the control group was exposed to only the carrier. Specific IgE and IgG1 immunoglobulins secreted in response to the soybean proteins were measured using enzyme-linked immunosorbent assays. Exposure to the soybean extract elicited the secretion of IgE and IgG1 specific for Gly m 5 and Gly m 6, and trypsin inhibitor. Oral soybean consumption attenuated the secretion of all the soybean-specific IgEs and IgG1s, suggesting that percutaneous sensitization to soybean proteins is attenuated by oral tolerance.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Most cases of AAA remain asymptomatic until rupture, and the mortality rate of patients with AAA rupture is very high. Currently, the relation between dietary habits and AAA development remains unknown. In this study, we evaluated the effects of a high-fat diet on the development of AAA in a vascular hypoperfusion-induced animal model. The risk of AAA rupture and AAA diameter in the high-fat group significantly increased compared with those in the control group. The number and size of adipocytes in the vascular wall in the high-fat group significantly increased as compared with those in the control group. Additionally, the collagen-positive sections in the areas with adipocytes significantly decreased as compared with those without adipocytes. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12, and macrophage-positive areas in the parts with adipocytes also significantly increased as compared with those without adipocytes. These data suggested that AAA rupture risk increased through accelerating chronic inflammation due to the accumulation of adipocytes in the vascular wall in the high-fat group.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Epidemiologic data have clearly linked tobacco smoking to aneurysm formation and a faster rate of expansion. It suggested that nicotine, one of the main ingredients of tobacco, has been suggested to be associated with AAA development and rupture. In the condition where no established drugs are available; therefore, an effective approach to prevent the vascular damage from nicotine consumption may be the use of dietary functional food factors. However, little is known about the relationship between dietary components and AAA. In this study, we estimated the effect of dietary deoxyribonucleic acid (DNA) on the vascular wall. After habituation for 5 d, the mice were divided into four groups: control diet and distilled water group (C), DNA-Na diet and distilled water group (DNA), control diet and 0.5 mg/mL nicotine solution group (C-Nic), DNA-Na diet, and 0.5 mg/mL nicotine solution group (DNA-Nic). The dietary DNA attenuated the degradation of elastin fibers induced by nicotine administration. The areas stained positive for MMP-2 in the DNA-Nic group were significantly suppressed compared to C-Nic mice. These data suggest that the dietary DNA may prevent the weakening of the aortic wall via inhibition of the MMP-2-dependent pathway. In conclusion, we have revealed the protective effect of dietary DNA on the vascular pathology of nicotine-administrated mice. A nucleic acid-rich diet might be useful for people who consume nicotine via smoking, chewing tobacco, or nicotine patches.

2002年〜2014年にフキノトウアレルギー患者11例を対象に、検討した。診断はフキノトウ摂取後に蕁麻疹や口腔アレルギー症候群(OAS)などの症状を呈し、フキノトウによるプリックテスト(PT)陽性の場合とした。一部の患者では雄株・雌株それぞれの茎、小花の部位で分けテストを行った。PT判定は15分後、膨疹の大きさ「(最長径+その中点に垂直な径)/2)」を測定し、陽性コントロール(10mg/mlの二塩酸ヒスタミン水溶液)の2倍以上を4、同等以上を3、1/2以上を2+、1/2より小さく陰性コントロール(生理食塩水)より大きいものを1+、陰性コントロールと同等を-とし、3mm以上の膨疹で、2+以上を陽性とした。健常者2名で同様にPTを行い陰性を確認した。患者年齢は21〜58歳、平均39歳、男女比は男性3名、女性8名、全例で蕁麻疹を認め、OASが次いで多く、5例でアナフィラキシー症状を認めた。検討した11例において、臨床症状、花粉待機的IgE抗体、ウエスタンブロットで検出されたIgEバンドの分子量に関連性は見いだせなかった。

In recent years, the number of patients with osteoporosis has risen with the increase in average longevity. Therefore, the chemoprevention of osteoporosis using food materials or food components has become an increasingly important target. Syringic acid (SA) is a phenolic compound present in the fruit of the a double dagger ai palm Euterpe oleracea and the mycelium of the shiitake mushroom Lentinula edodes. This compound has no affinity for estrogen receptors and is potentially useful for disease prevention. However, little is known about the effects of a SA diet on bone metabolism, particularly bone resorption in vivo. Here, we demonstrated the effects of a SA diet on bone loss and uterine weight loss in ovariectomized (OVX) mice. Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/day) for 10 weeks. After 10 weeks of dietary SA, the body weight, food intake, and uterine weight of the OVX mice were unaffected; however, femoral bone mineral density (cortical bone density, cancellous bone density, and total bone density) was higher in the SA-fed groups than in the OVX-control group. Furthermore, histomorphometric analysis revealed that the number of osteoclasts and osteoblasts was decreased and increased, respectively, in the SA-fed groups. These results suggest that a SA diet suppresses bone loss by downregulating bone resorption and upregulating bone formation without affecting the uterus in OVX mice. Although further studies are needed, SA may be a compound that can be used to prevent or retard osteoporosis.

Abdominal aortic aneurysm (AAA) is a vascular disease involving gradual dilation of the abdominal aorta. Recent studies suggest that nicotine, which is a primary component in cigarette smoke, is closely associated with the development and rupture of an AAA. Nicotine accelerates AAA development through the weakening of the vascular wall by increasing oxidative stress and matrix metalloproteinase (MMP)-2 expression. However, little is known about preventing the AAA induced by nicotine. A non-surgical means of preventing the weakening of the vascular wall before the onset of AAA by functional food factors would be a valuable option over surgery. Fish oil is a functional food that is rich in n-3 polyunsaturated fatty acids that have an anti-inflammatory effect. In this study, we evaluated the effect of dietary fish oil on the weakening of the aortic wall due to nicotine administration in a mouse model. Histological analysis showed that the dietary fish oil suppressed the degradation of elastin fibers in the nicotine-administered mice. Additionally, the dietary fish oil suppressed the protein level of MMP-12, macrophage infiltration, and the oxidative stress in the vascular wall. These results suggest that fish oil could suppress the weakening of the vascular wall by suppressing the elastin fiber degradation caused by nicotine. By suppressing the nicotine induced weakening of the vascular wall, fish oil might help prevent the development of AAA.

Abdominal aortic aneurysm (AAA) is a vascular disease involving gradual dilation of the abdominal aorta. Recent studies suggest that nicotine, which is a primary component in cigarette smoke, is closely associated with the development and rupture of an AAA. Nicotine accelerates AAA development through the weakening of the vascular wall by increasing oxidative stress and matrix metalloproteinase (MMP)-2 expression. However, little is known about preventing the AAA induced by nicotine. A non-surgical means of preventing the weakening of the vascular wall before the onset of AAA by functional food factors would be a valuable option over surgery. Fish oil is a functional food that is rich in n-3 polyunsaturated fatty acids that have an anti-inflammatory effect. In this study, we evaluated the effect of dietary fish oil on the weakening of the aortic wall due to nicotine administration in a mouse model. Histological analysis showed that the dietary fish oil suppressed the degradation of elastin fibers in the nicotine-administered mice. Additionally, the dietary fish oil suppressed the protein level of MMP-12, macrophage infiltration, and the oxidative stress in the vascular wall. These results suggest that fish oil could suppress the weakening of the vascular wall by suppressing the elastin fiber degradation caused by nicotine. By suppressing the nicotine induced weakening of the vascular wall, fish oil might help prevent the development of AAA.

Abdominal aortic aneurysm (AAA) is a vascular disease that results in the gradual dilation of the abdominal aorta and has a high rupture-related mortality rate. However, the mechanism of AAA rupture remains unknown. In our previous study, we established a novel AAA animal model (hypoperfusion-induced AAA rat model) with spontaneous AAA rupture. Using the hypoperfusion-induced AAA rat model, we demonstrated that the abnormal appearance of adipocytes in the vascular wall is associated with AAA rupture. However, pathological analysis of the rupture area has not been performed because it is particularly difficult to identify the rupture point. In this study, we succeeded in obtaining samples from the rupture point and performed a histological analysis of the ruptured area in the vascular wall in the hypoperfusion-induced AAA rat model. Adipocytes were observed along the AAA-ruptured area of the vascular wall. In the areas around the adipocytes, macrophage infiltration and protein levels of matrix metalloproteinases 2 and 9 were significantly increased and collagen-positive areas were significantly decreased, as compared with areas without adipocytes. The AAA diameter was correlated with the number of adipocytes in the vascular wall of the hypoperfusion-induced AAA rat model. On the other hand, serum triglyceride levels and serum total cholesterol levels were not correlated with the number of adipocytes in the vascular wall. These results suggest that local adipocyte accumulation in the vascular wall, not serum lipids, has an important role in AAA rupture.

花粉症に関連するクラス2食物アレルギーにおいては、主な症例として豆乳や野菜・果物などの摂取時のOASが一般的である。この原因抗原として、汎アレルゲンであるPR-10やプロフィリンがよく知られている。そこで、本稿では、プロフィリン(Profilin)に注目し、各種野菜・果実飲料の可溶性画分における本アレルゲンを検出することで、プロフィリンによるOAS発症リスクを評価した。市販のこれら飲料では、加熱等による殺菌過程で、本アレルゲンは変性・不溶化しており、OASリスクは低いことが示唆された。(著者抄録)

There have been reports that hyperglycemia suppresses osteoclast (OCL) differentiation, although the underlying mechanism is poorly understood. Here we demonstrated that high glucose suppresses OCL differentiation through activation of liver X receptor (LXR) beta, a recently reported glucose-sensing nuclear receptor. The effect of hyperglycemia on osteoclastogenesis was tested in RAW264.7 cells, a murine macrophage cell line. Cells were treated with receptor activator of NF-kappa B ligand (RANKL) under normoglycemic (5.5 mM glucose), normoglycemic with high osmotic pressure (5.5 mM glucose + 10.0 mM mannitol), and hyperglycemic (15.5 mM glucose) conditions. RANKL-induced osteoclastogenesis was significantly suppressed by high-glucose treatment. Mannitol treatment also significantly suppressed osteoclastogenesis, but the inhibitory effect was lower than for high-glucose treatment. The suppression of mRNA expression of Lxr beta by RANKL was significantly restored by high glucose, but not mannitol. Additionally, the deactivation of Lxr beta by siRNA attenuated high-glucose-induced suppression of osteoclastogenesis. Although further validation of the underlying pathway is necessary, targeting LXR beta is a potential therapeutic approach to treating osteoporosis.

There have been reports that hyperglycemia suppresses osteoclast (OCL) differentiation, although the underlying mechanism is poorly understood. Here we demonstrated that high glucose suppresses OCL differentiation through activation of liver X receptor (LXR) beta, a recently reported glucose-sensing nuclear receptor. The effect of hyperglycemia on osteoclastogenesis was tested in RAW264.7 cells, a murine macrophage cell line. Cells were treated with receptor activator of NF-kappa B ligand (RANKL) under normoglycemic (5.5 mM glucose), normoglycemic with high osmotic pressure (5.5 mM glucose + 10.0 mM mannitol), and hyperglycemic (15.5 mM glucose) conditions. RANKL-induced osteoclastogenesis was significantly suppressed by high-glucose treatment. Mannitol treatment also significantly suppressed osteoclastogenesis, but the inhibitory effect was lower than for high-glucose treatment. The suppression of mRNA expression of Lxr beta by RANKL was significantly restored by high glucose, but not mannitol. Additionally, the deactivation of Lxr beta by siRNA attenuated high-glucose-induced suppression of osteoclastogenesis. Although further validation of the underlying pathway is necessary, targeting LXR beta is a potential therapeutic approach to treating osteoporosis.

Abdominal aortic aneurysm (AAA) is the progressive dilation of the abdominal aorta. Nicotine is reported to be associated with the development and rupture of AAA, but the pathological effects of nicotine on normal rat aorta have not been determined. We investigated pathological changes in the aortic wall of rats caused by the administration of nicotine. Nicotine administration weakened the vascular wall, increased gelatinolytic activity and promoted the destruction of elastin and collagen in the rat abdominal aorta. There were no differences in the areas positive for matrix metalloproteinase (MMP)-2 and MMP-9 between the control and nicotine treated groups. The areas positive for MMP-12 in the nicotine group were significantly greater than for the control group. Gelatinolytic activity in the aortic wall was increased significantly in the nicotine group. Our findings suggest that MMP-12 is sensitive to nicotine exposure in rats.

Aneurysm is characterized by balloon-like expansion of the arterial wall and eventual rupture of the aorta. The pathogenesis of aneurysm is associated with the degradation of matrix proteins by matrix metalloproteinases (MMPs) produced by activated macrophages. Although aneurysm is associated with significant mortality and morbidity, surgical intervention is the only proven treatment strategy. Therefore, development of therapeutic agents for aneurysm is greatly anticipated. Here, we demonstrated the protective effects of the major isoflavone puerarin, which is found in kudzu roots and vines. Aneurysms were surgically induced in ten-wk-old male mice using CaPO4. Subsequently, animals were intraperitoneally injected daily with puerarin at 2.5 mg/kg body weight or with vehicle alone for 2 wk. CaPO4-induced aneurysm was significantly suppressed by puerarin administration. In subsequent macrophage activation assays using Tumor necrosis factor (TNFa) and CaPO4 crystals in vitro, puerarin decreased Mmp9 mRNA expression and secreted protein levels. Moreover, induction of IKB, ERK, and p38 phosphorylation by TNFa and CaPO4 in macrophages was suppressed by puerarin treatments. Finally, puerarin attenuated reactive oxygen species production, following induction by TNFa and CaPO4. Taken together, the present data demonstrate that puerarin suppresses macrophage activation by inhibiting IKB, ERK, and p38 activity and reactive oxygen species production in a CaPO4-induced mouse model of aneurysm.

The need for a preventive agent against dementia led us to screen natural plant resources. Among the herbs and spices tested, turmeric, from rhizomes of Curcuma longa, showed high potency against beta-secretase. The active principles were determined as alpha-turmerone, beta-turmerone and ar-turmerone, with IC50 values of 39, 62 and 92 mu M respectively. In this study, the efficiency of collecting the essential oil using steam distillation of the volatile substance was disclosed. The active principles were explored, and four sesquiterpenoids and five monoterpenoids were revealed as active principles against beta-secretase. On the other hand, alpha-turmerone, beta-turmerone and ar-turmerone were also investigated in a pharmacokinetic absorption experiment. After oral administration, these compounds were detected in an intact form in the brain and serum. These results suggest that consumption of turmeric constituents may prevent dementia.

Food contains various compounds that have an impact on our daily lives. Many technologies have been established to analyze these molecules of interest in foods. However, the analysis of the spatial distribution of these compounds in foods using conventional technology, such as high-performance liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry is difficult. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is considered an ideal complementary approach. MALDI-MSI is a two-dimensional MALDI-MS technology that can detect compounds in a tissue section without extraction, purification, separation, or labeling. MALDI-MSI can be used to visualize the spatial distribution of chemical compounds or biomolecules in foods. Although the methodology of MALDI-MSI in food science is not yet fully established, the versatility of MALDI-MSI is expected to open a new frontier in food science. Herein, we describe the principles and applications of MALDI-MSI in food science and related fields. (C) 2016 Elsevier Ltd. All rights reserved.

Postmenopausal diabetes is exacerbated by estrogen deficiency. Ovariectomized (OVX) animal models can be used to develop strategies for preventing or treating postmenopausal symptoms. We previously found that a diet containing kudzu (Pueraria lobata) vine ethanol extract (PVEE) suppressed weight gain in OVX mice. Therefore, this study further elucidated how PVEE affected OVX mice. Ten-week-old OVX or sham-operated mice were fed diets containing either no PVEE (control) or 20 mg.kg(-1).d(-1) PVEE for 8 wk, 5 mg.kg(-1).d(-1) PVEE for 24 wk, or 20 mg.kg(-1).d(-1) puerarin (daidzein-8-C-glucoside), a major isoflavone present in PVEE, for 10 wk. The effects of puerarin on glucose tolerance were also tested in OVX mice. The experimental diets were not associated with any abnormalities in any mice tested in the present study. Weight gain and serum glucose levels were increased in OVX mice and these effects were significantly attenuated in OVX mice that consumed PVEE (5 or 20 mg.kg(-1).d(-1)) or puerarin. Puerarin-treated OVX mice also showed reduced serum glucose levels following administration of 1,000 mg.kg(-1) glucose. These results suggested that puerarin contributed to PVEE-mediated improvements in glucose metabolism in OVX mice. Although further studies are needed to clarify the molecular mechanism underlying these observations, PVEE and puerarin could provide effective approaches to the amelioration of postmenopausal diabetes.

Estrogen deficiency-induced obesity has a high risk of visceral fat accumulation and body weight gain. It is also associated with many adverse health conditions. Taheebo extract from Tabebuia avellanedae has been recognized as playing several biological and pharmacological roles. Therefore, we investigated whether the intake of n-BuOH extract of Taheebo shows anti-obesity effect in ovariectomized (OVX) mice. After 16 weeks of feeding, the mice administrated with 0.5% n-BuOH extract of Taheebo showed significantly decreased body weight compared with that of the control mice, and the fat mass also showed a significant decrease. In 3T3-L1 cells, supplementation with n-BuOH extract of Taheebo significantly reduced the triglyceride ( TG) levels. Furthermore, bioassay-guided purification of the nBuOH extract based on the TG levels in 3T3-L1 cells led to the isolation of compound 2 ( 1-dehydroxy-3,4-dihydroaucubigenin). These results suggested that the anti obesity effect of Taheebo extract is due to its capability in preventing the accumulation of adipocyte in mice. Taheebo extract might be a promising functional food resources capable of protecting against OVX-induced obesity. (C) 2016 Elsevier Inc. All rights reserved.

Low levels of serum testosterone are characteristically associated with diabetes, coronary atherosclerosis, obstructive sleep apnea, rheumatoid arthritis, and chronic obstructive pulmonary disease. Testosterone replacement therapy is effective against many of these disorders, indicating the importance of maintaining a healthy testosterone level. In this study, we investigated the effects of fish oil on murine testosterone metabolism and analyzed the dynamics of relevant lipids in testes by matrix-assisted laser desorption ionization mass spectrometry imaging. Testosterone was upregulated in mice that received fish oil. In the testicular interstitium, eicosapentaenoic acid-containing phosphatidylcholine was distributed characteristically. These data suggest that eicosapentaenoic acid is involved in testosterone metabolism.

Abdominal aortic aneurysm (AAA) is a vascular disease involving the gradual dilation of the abdominal aorta. It has been reported that development of AAA is associated with inflammation of the vascular wall; however, the mechanism of AAA rupture is not fully understood. In this study, we investigated the mechanism underlying AAA rupture using a hypoperfusion-induced animal model. We found that the administration of triolein increased the AAA rupture rate in the animal model and that the number of adipocytes was increased in ruptured vascular walls compared to non-ruptured walls. In the ruptured group, macrophage infiltration and the protein levels of matrix metalloproteinases 2 and 9 were increased in the areas around adipocytes, while collagen-positive areas were decreased in the areas with adipocytes compared to those without adipocytes. The administration of fish oil, which suppresses adipocyte hypertrophy, decreased the number and size of adipocytes, as well as decreased the risk of AAA rupture ratio by 0.23 compared to the triolein administered group. In human AAA samples, the amount of triglyceride in the adventitia was correlated with the diameter of the AAA. These results suggest that AAA rupture is related to the abnormal appearance of adipocytes in the vascular wall.

Abdominal aortic aneurysm (AAA) is a vascular disease involving gradual dilation of the abdominal aorta and high rupture-related mortality rates. AAA is histologically characterized by oxidative stress, chronic inflammation, and extracellular matrix degradation in the vascular wall. We previously demonstrated that aortic hypoperfusion could cause the vascular inflammation and AAA formation. However, the preventive method for hypoperfusion-induced AAA remains unknown. In this study, we evaluated the effect of fish oil on AAA development using a hypoperfusion-induced AAA animal model. Dilation of the abdominal aorta in the fish oil administration group was smaller than in the control group. Collagen destruction and oxidative stress were suppressed in the fish oil administration group than in the control group. These results suggested that fish oil could prevent the development of AAA induced by hypoperfusion.

The aortic wall is perfused by the adventitial vasa vasorum (VV). Tissue hypoxia has previously been observed as a manifestation of enlarged abdominal aortic aneurysms (AAAs). We sought to determine whether hypoperfusion of the adventitial VV could develop AAAs. We created a novel animal model of adventitial VV hypoperfusion with a combination of a polyurethane catheter insertion and a suture ligation of the infrarenal abdominal aorta in rats. VV hypoperfusion caused tissue hypoxia and developed infrarenal AAA, which had similar morphological and pathological characteristics to human AAA. In human AAA tissue, the adventitial VV were stenotic in both small AAAs (30-49 mm in diameter) and in large AAAs (>50 mm in diameter), with the sac tissue in these AAAs being ischemic and hypoxic. These results indicate that hypoperfusion of adventitial VV has critical effects on the development of infrarenal AAA.

Food allergy is defined as an immune system-mediated adverse reaction to food components. Food allergic reactions are mostly IgE mediated and also known as immediate type hypersensitivity (type I reaction). There are several characteristic clinical types of food allergy, such as Anaphylaxis, Food-dependent exercise-induced anaphylaxis (FDEIA), and Oral allergy syndrome (OAS). In addition, food allergy is also classified into two types (class 1 and class 2) based on the pathophysiological mechanism. In the class 2 food allergy, pollen allergy causes plant food allergy; therefore this type of allergy is sometimes called Pollen-food allergy syndrome (PFAS). The risk of food allergy (allergenicity) may vary with the treatment of the food allergens. The formation or status of the causative food affects its allergenicity. Class 1 food allergens are generally heat-, enzyme-, and low pH-resistant glycoproteins ranging in size from 10 to 70 kD. Class 1 food allergens induce allergic sensitization via the gastrointestinal tract and are responsible for systemic reactions. Class 2 food allergens are generally heat-labile, susceptible to digestion, and highly homologous with pollen allergens. Taken together, it may be important to consider the diversity of food allergy in order to fight against food allergy.

The pathophysiology underlying abdominal aortic aneurysms (AAAs) remains unknown. In this study, we applied imaging mass spectrometry (IMS) to analyze the pathophysiology of the aneurysmal wall. Comparisons were performed between the tissue samples from the neck and the sac of the AAA, at a single time point, in 30 patients who underwent elective surgery of their AAAs. The localization of each lipid molecule in the aortic wall was assessed by IMS. Histopathological examination and IMS revealed a characteristic distribution of triglycerides (TGs) specifically in the aneurismal adventitia of the sac. This characteristic TG distribution was derived from an ectopic appearance of adipocytes in the adventitia. Furthermore, ectopic adipocyte accumulation in the aortic wall leads to the loss of the collagen fiber network subsequent to the wall rupture. The underlying mechanism of adipocyte accumulation involves the presence of adipose-derived stem cells (ADSCs) in the aneurismal adventitia and the expression of peroxisome proliferator-activated receptor gamma 2, a master regulator of adipocyte differentiation by some ADSCs. This study reveals new, previously overlooked aspects of AAA pathology. (C) 2015 S. Karger AG, Basel

RATIONALEVisualization of the spatial distribution of phosphatidylcholine (PC) in tissues by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) provides insights into key physiological and pathophysiological processes. In MALDI-IMS analysis, the heterogeneity of adduct ions formed from PC lowers the specificity of detection of PC molecular species and poses a challenge in the identification of these species. To solve this problem, modified matrix solution and desalting with ammonium acetate (NH4Ac) buffer have been employed. However, the utility of these methods is limited to the analysis of brain sections. METHODSThe MALDI signal intensities of [PC+H], [PC+Na] and [PC+K] were compared after three different pretreatments (modified matrix solution, desalting with 150mM ammonium acetate, treatment with 150mM potassium acetate). RESULTSPretreatment of tissue sections with 150mM potassium acetate resulted in an increase in the signal intensity of [PC+K] ions produced from cryosections of the pancreas, brain, and liver tissues. CONCLUSIONSPretreatment with potassium acetate can be a simple, improved, and highly useful method for the reliable analysis of PC in tissues. Copyright (c) 2014 John Wiley & Sons, Ltd.

RATIONALESake is made from fermented rice and has been drunk in Japan for more than 1000 years. The rice must be polished prior to fermentation to obtain high-quality sake. It is traditionally recognized that the quality of sake is improved as the rice polishing ratio (percentage removed in the polishing process) increases. However, the underlying chemistry of the rice polishing process is incompletely understood. Herein, we analyzed the distribution of lysophosphatidylcholine (LPC) molecular species with unsaturated fatty acids in rice, as their presence is thought to exert a negative effect on the flavor of sake. METHODSThe distribution of LPC molecular species in rice was visualized via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). RESULTSLPC (16:0) is ubiquitously present in the endosperm of rice while LPC (18:0) is localized in the core of the endosperm. In contrast, LPC (18:2) and LPC (18:1) are present in the outer region of the endosperm. CONCLUSIONSThe enhancement of the quality of sake as the polishing ratio of the rice increases might be explained in terms of the distribution of LPC with unsaturated fatty acids in the rice. Copyright (c) 2014 John Wiley & Sons, Ltd.

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles. © 2014 The Japanese Society of Fisheries Science.

The use of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) for the assessment of vascular pathology is attracting attention as a new valuable tool for diagnosing disease and finding new markers. MALDI-IMS is a molecular imaging technique whereby the simultaneous measurement of multiple samples directly from clinical tissue sections is possible. The versatility of MALDI-IMS has opened a new frontier in vascular pathology. In this review, we describe the principle and applications of MALDI-IMS. (C) 2014 S. Karger AG, Basel

The levels of food allergens in gamma-irradiated soybean (0, 2.5, 5, 7.5, 10, 20, and 30 kGy) were investigated by immunoblotting and ELISA, using allergen-specific antibodies and patient serum. After 3 months of storage, Coomassie brilliant blue (CBB) staining indicated similar total protein profiles among the treatments, but that some proteins were degraded by irradiation at high doses. Immunoblotting with specific antibodies for major soybean allergens (beta-conglycinin, Gly m Bd 30 K, soybean trypsin inhibitor, and Gly m 4) resulted in apparent band profiles and intensities that were not significantly changed by irradiation. Competitive inhibition ELISA analyses suggested that there were no significant changes in the allergen contents, except for a decrease in the soybean trypsin inhibitor. The patient IgE binding allergenic protein patterns were not changed by irradiation up to 30 kGy. ELISA using patient serum also revealed that the IgE reactivity to the irradiated soybean extract did not increase from the level of the control, but that the reactivity to some patient serum IgE was significantly decreased by irradiation.

Miso paste (miso), a fermented soybean food, is popular in Japan and other Asian countries. However, the soybean is known to induce an allergenic response in some individuals. In the present study, we evaluated the allergenicity of various kinds of miso available in Japan. Total proteins were extracted from Amakuti-kome miso, Karakuti-kome miso, Mugi-miso and Mame-miso, and the protein profiles were analyzed. The major protein bands detected in the intact soybean extract were not present in any of the miso samples, which instead showed various low molecular weight protein bands of approximately 10-25 kDa. The existence levels of six major soybean allergens were determined by Western blotting using specific antibodies. We found that the allergen levels varied among miso and allergen types; however, allergen levels were consistently lower in miso than in the soybean extract. We obtained similar results for IgE-ELISA experiments using serum IgE from soybean allergy patients. Taken together, these results indicate that compared to soybean extract, various types of miso contain small quantities of intact soybean allergens. Additionally, several lines of evidence indicated that the allergen levels were exceptionally low in the dark-colored Karakuti-kome miso and Mame-miso, which are produced with relatively long fermentation periods, suggesting that the duration of fermentation might be a key factor in the hypoallergenicity of miso.

Ellagic acid (EA) is a polyphenol found in a wide variety of plant foods that not only exhibits free radicalscavenging activity, but also confers protective effects against liver injury. Previously, we reported that pomegranate fruit extract (PFE) had an inhibitory effect on resistin secretion from differentiated murine 3T3-L1 adipocytes and identified EA contained in PFE as a potent suppressor of resistin secretion. Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. In this study, we explored whether EA supplementation reduces serum resistin and improves hepatic steatosis and serum lipid profile by using KK-A(y) mice fed high-fat diet as a model for obese type 2 diabetes. We found that EA supplementation improved serum lipid profile and hepatic steatosis, and reduced serum resistin levels without altering mRNA expression levels in adipose tissue. Moreover, EA supplementation' upregulated mRNA expression of apoa1, ldlr, cpt1a, and ppara genes in the liver. In conclusion, our findings indicate that EA is a potent suppressor of resistin secretion in vivo and a transcriptional activator of ppara in the liver, suggesting a possibility for improving obesity-induced dyslipidemia and hepatic steatosis in KK-A(y) mice. (C) 2013 Elsevier Inc. All rights reserved.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RI-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline proteasetreated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

We have shown that dietary bluefin tuna skin (TUS) protects against carbon tetrachloride (CCl4)-induced hepatic damage in mice. The CCl4-induced necrotic area was decreased in mice fed a TUS-containing diet. Consistent with the decreased necrotic area, dietary TUS markedly lowered the elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the thiobarbituric acid-reactive substance (TBARS) formation induced by CCl4 injection. TUS diets also decreased phosphorylation of inhibitory kappa B-alpha and blocked the translocation of nuclear factor-kappa B to the nucleus. TUS is composed mainly (80.7 %) of type I collagen, and our results revealed that dietary tuna collagen peptides (TUCP) attenuated the increased hepatic necrotic area, serum AST and ALT activities, and liver TBARS levels induced by CCl4, similar to TUS, thus enabling us to attribute the hepatoprotective action of TUS in CCl4-intoxicated mice to tuna collagen. Therefore, TUS and TUCP may be potential food resources that are capable of alleviating hepatitis symptoms.

Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)-IMS analysis, by tandem mass spectrometry (MALDI-IMS-MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei). The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI-IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits.

Puerarin, a daidzein-8-C glucoside, is the major isoflavonoid in Kudzu (Pueraria lobata), and is unique in that it contains C-C conjugated glucose at position 8 of the isoflavonoid structure. A puerarin diet at a dose of 5 mg/kg b.w./d to fed ovariectomized mice for 2 mo diminished the urinary deoxypyridinoline, a typical bone-degradation product. Since the bone absorption marker, serum tartarate-resistant acid phosphatase (TRAP) activity of puerarin-fed mice decreased but the bone formation marker, osteocalcin level, did not alter, the puerarin diet was proved to specifically depress the bone absorption, but not the overall bone metabolism. In accordance with that results, the femur structure of puerarin-fed mice was restored compared with that of puerarin-free diet mice. The atrophied uterine due to low estrogen (E2) level after ovariectomy was not restored by the puerarin diet, suggesting that puerarin exerted the anti-osteoporotic action through a non estrogen receptor (ER) mediated-pathway, in vivo. The growth of an ER-positive human breast cancer cell, MCF-70, was not enhanced by puerarin, suggesting that puerarin did not show estrogen-like action on MCF-7 cells, even at a ten thousand times higher concentration than that of E2. Furthermore, ICI182,780 (Id), an estrogen antagonist, suppressed the enhanced growth of MCF-7 cells by E2, but not that by puerarin. In an ER-binding assay, puerarin was proved not to bind to ER alpha or beta, or if all, extremely weakly, although daidzein, an aglycon of puerarin, showed a little stronger binding compared with puerarin. All these results strongly indicate that puerarin exerts its anti-osteoprotic action independently of the ER-mediated pathway.

Black rice (Oryza sativa L. Japonica) contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. Several studies have identified the molecular species of anthocyanins in black rice, but information about the localization of each anthocyanin species is limited because methodologies for investigating the localization such as determining specific antibodies to anthocyanin, have not yet been developed Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a suitable tool for investigating the localization of metabolites. In this study, we identified 7 species of anthocyanin monoglycosides and 2 species of anthocyanin diglycosides in crude extracts from black rice by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. We also analyzed black rice sections by MALDI-IMS and found 2 additional species of anthocyanin pentosides and revealed different localization patterns of anthocyanin species composed of different sugar moieties. Anthocyanin species composed of a pentose moiety (cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species composed of a hexose moiety (cyanidin-3-O-hexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp. These results indicate that anthocyanin species composed of different sugar moieties exhibit different localization patterns in the pericarp of black rice. This is the first detailed investigation into the localization of molecular species of anthocyanins by MALDI-IMS.

症例は62歳男性.過去に3回,サンマ,カツオ,イワシにてアレルギー症状を起こしたことがある.今回,イカ摂取により発症したと考えたアニサキスアレルギーの1例を報告する.イクラとサーモンの和え物,サバの焼き魚を摂取して1時間後にマグロ,イカの刺身を摂取したところ,直後に,膨疹,意識消失,血圧低下が出現した.種々の海産物等でのImmunoCAP(特異的IgE測定)およびプリックテストはすべて陰性であったが,アニサキス特異的IgEはクラス3,アニサキス粗抗原を用いたプリックテストは3+であった.アニサキス粗抗原を用いたイムノブロットでは,18kDa, 17kDaに強く,35kDaにやや強く,28kDa, 26kDaに弱くバンドが形成された.11種のアニサキスアレルゲン(Ani s 1〜6, 8, 9, 11, 12,トロポニンC)を用いたELISAでは,Ani s 1, Ani s 12に強い反応,Ani s 2,トロポニンCに弱い反応が認められた.イムノブロットによる17kDaおよび18kDaのバンドはそれぞれAni s 1およびトロポニンCのいずれかであり,35kDaはAni s 12と推定した.

Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes. (C) 2011 Elsevier Inc. All rights reserved.

To elucidate the inhibiting mechanisms of fat accumulation by catechins, caffeine, and epigallocatechin gallate (EGCG), ICR mice were fed diets containing either 0.3 catechins or 0.1 EGCG and/or 0.05 caffeine for 4 weeks. After the feeding, intraperitoneal adipose tissues weights were significantly lower in the caffeine, catechins + caffeine, and EGCG + caffeine groups compared to controls. Hepatic fatty acid synthase (FAS) activity in the catechins + caffeine group was significantly lower, and the activities of acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase-II (CPT-II) were significantly higher, compared to the control group. However, these activities were not observed in the other groups. FAS mRNA expression levels in the catechins + caffeine group were significantly lower than in the control group. ACO and CPT-II mRNA levels were not different among all of the treatment groups. These findings indicate that the inhibitory effects of fat accumulation via a combination of catechins, EGCG, or caffeine were stronger collectively than by either catechins, EGCG, or caffeine alone. Moreover, it was demonstrated that the combination of catechins and caffeine induced inhibition of fat accumulation by suppression of fatty acid synthesis and upregulation of the enzymatic activities involved in β-oxidation of fatty acid in the liver, but this result was not observed by combination of EGCG and caffeine. © 2012 Chikako Sugiura et al.

Bone-loss-improving action of kudzu vine ethanol extracts (PVEE) was clarified. PVEE was composed roughly of 80% fiber, 10% puerarin, 3.6% daidzin, 2.5% 6 ''-O-malonyldaidzin, and the other minor isoflavones. Ten-week-old ovariectomized (OVX) mice were fed diets containing PVEE (20 mg/kg body weight/day) for 8 weeks. The bone resorption markers (urinary deoxypyridinoline and tartrate-resistant acid phosphatase activity) was elevated in OVX mice and was significantly decreased in OVX mice that consumed PVEE for 8 weeks. Consistent with the decrease in the markers, the number of matured osteoclasts in the distal femur was diminished in OVX mice fed PVEE diets. PVEE diets also suppressed the decrease in femoral bone mineral density (BMD) by OVX. PVEE showed the affinity for estrogen receptor alpha and beta 3 nearly 1/10000 weaker than 17 beta-estradiol. No hypertrophy in the uterus by the PVEE diet was observed. These results suggest that PVEE could be a promising resource for a functional food that improves osteoporosis.

Activation of peroxisome proliferator-activated receptor (PPAR)-alpha which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPAR alpha activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPAR alpha activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPAR alpha agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO(2) and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPAR alpha activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPAR alpha activity is a novel target of PPAR alpha agonist for decreasing circulating levels of lipids under postprandial conditions. (C) 2011 Elsevier Inc. All rights reserved.

症例1,28歳女性.液状のローヤルゼリーを初めて一口試食した直後より,頸部の紅潮・気分不良・腹痛・下痢が出現.症例2,21歳女性.ローヤルゼリーグミを初めて食べた数時間後より鼻閉・両側眼周囲の腫脹が,さらに10日後に同じグミを摂取した4時間後より鼻閉・呼吸困難・全身の紅潮〜膨疹が出現.プリックテストで両者共にローヤルゼリー自体で陽性であったため,ローヤルゼリーアレルギーと診断した.初回摂取時に発症した理由としては,過去に知らず知らずの間にローヤルゼリーに対して既感作であったとの機序を考えたが,症例2ではヨモギの特異的IgEおよびプリックテストが共に陽性であり,ヨモギアレルギーとの交叉反応によって発症した可能性も疑われた.ローヤルゼリーは本来クラス1アレルゲンと考えられているが,過去にも初回摂取後の発症の報告例が多く認められており,花粉類などとの交叉反応により発症に至るクラス2アレルギーの可能性も考慮した上で検索を行う必要があると考えた.

Nonspecific lipid transfer protein 1 (nsLTP1) is widely distributed in plants and has been recognized as a widely cross-reacting plant pan-allergen. In the present study, a recombinant protein of rice nsLTP1 was prepared with an Escherichia coli expression system, and polyclonal antiserum was raised by immunizing rabbits with this recombinant nsLTP1. The antiserum specifically immunoreacted with rice nsLTP1 and homologous nsLTP1 proteins of other kinds of cereal grains. Moreover, the antiserum was applied to compare nsLTP1 content among rice cultivars and among rice fractions separated by grain-milling. Variation of nsLTP1 contents were observed among rice cultivars, and rice nsLTP1 was proved to be mainly located in the outer layer of rice grains, providing evidence for decreasing nsLTP1 content by grain-milling.

RAG2 is a member of the rice 14-16 kDa α-amylase/trypsin inhibitors, which are the main allergens in rice grains. Recombinant RAG2 protein was expressed in Escherichia coli, and polyclonal antisera were prepared against the protein. The polyclonal antisera specifically bound with 14-16 kDa a-amylase/ trypsin inhibitors from husked brown, red and black rice. No cross-reaction was observed between the polyclonal antisera and saline-soluble proteins from other tested cereal grains. By immunoblotting with the polyclonal antisera, 14-16 kDa α-amylase/trypsin inhibitors were found to be distributed throughout the endosperm of brown rice and their contents were estimated. Moreover, 14-16 kDa a-amylase/trypsin inhibitors were also detected in processed foods. Thus, the polyclonal antisera prepared against recombinant RAG2 protein could be used to compare the content of 14-16 kDa a-amylase/trypsin inhibitors in rice samples and probe the presence of the allergen in processed foods.

【症例1】蕁麻疹の既往がある53歳，男性．加熱調理したゴボウの摂食後にアナフィラキシーショックを呈した．Prick-prick testでは生ゴボウ，加熱ゴボウ共に陽性を示した．【症例2】モモとメロンの口腔アレルギー症候群（OAS）の既往がある31歳，女性．加熱調理したゴボウの摂食後に全身の蕁麻疹と消化器症状を呈した．Prick-prick testでは生ゴボウは陰性，加熱ゴボウが陽性を示した．【解析】2人の血清を用いたイムノブロットの結果，症例1では35 kDa付近に，症例2では17 kDa付近にバンドを認め，それぞれ原因抗原と推測された．35 kDaのタンパク質はアミノ酸配列解析によりperoxidase（POD）であることが，17 kDaのタンパク質は［Cu-Zn］superoxide dismutase（SOD）であることが判明した．

大豆アレルギーの多様性とそのリスク低減化戦略について解説した。特に低アレルゲン大豆加工食品の開発と流通システムの構築の試みに重点を置いて解説した。

The presence of Listeria monocytogenes in fresh produce is a growing concern because of the possibility of food-borne illness. Ionizing radiation is an effective non-thermal means of eliminating pathogenic bacteria in fresh produce; however, the effect of ionizing irradiation on the allergenic properties of the host commodities remains unknown. This study aimed (i) to determine the effective dose of gamma-irradiation in eliminating L. monocytogenes on whole cherry tomatoes and (ii) to evaluate the effect of gamma-irradiation on the allergenic properties of tomato proteins. Cherry tomatoes that were inoculated with a mixture of five L. monocytogenes strains were treated with gamma-rays from a (60)Co source. A 1.25 kGy dose of gamma-irradiation was found to be sufficient to eliminate L. monocytogenes on whole cherry tomatoes. The immunoblot profile of serum samples obtained from two patients with tomato allergy revealed that gamma-irradiation did not affect the allergenicity of tomato proteins for up to 7 days after irradiation when the tomatoes were stored at 20 degrees C. Additionally, the m-RNA levels of P-fructofuranosidase, polygalacturonase, pectin esterase, and superoxide dismutase, the main allergenic proteins in tomato, were not affected by the applied irradiation dose. Thus, this study demonstrated that a 1.25 kGy dose of gamma-irradiation effectively eliminates L. monocytogenes on cherry tomatoes without affecting the expression of allergenic proteins in the fruits. (C) 2009 Elsevier Ltd. All rights reserved.

食物アレルギー患者数は増加傾向にあり，特に先進国の消費者にとって大きな問題となっている．日本人の主食であるコメに対するアレルギーも報告されている．アナフィラキシーショック等の症状を示すことは非常にまれであるが，アトピー性皮膚炎の原因食物の一つとして考えられている．貯穀害虫防除を目的とした臭化メチル代替殺虫技術として開発された高圧二酸化炭素処理を玄米に対し施し，コメのアレルゲンタンパク質にどのような影響を及ぼすかについて検討した．また，従来使用されてきた，臭化メチルおよびリン化水素によるくん蒸処理も行った．さらに，貯穀害虫であるノシメマダラメイガ（Plodia interpunctella）の幼虫を玄米に投入し，摂食後のコメアレルゲンタンパク質への影響も検討した．その結果，これらの害虫防除処理，および貯穀害虫の摂食によるアレルゲンタンパク質への影響は認められなかった．

In this study, HepG2 cells were treated with short peptides (7S-peptides) derived from highly purified soybean beta-conglycinin (7S), which was free from lipophilic protein, and the effect of the peptide treatment on lipid metabolism was determined. 7S-peptide treatment suppressed the secretion of apolipoprotein B-100 from HepG2 cells into the medium. The 7S-peptides also suppressed the incorporation of (3)H-glycerol and (14)C-acetate into triacylglyceride but not into major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Additionally, the synthesis of cholesterol esters was dramatically decreased for 2 h after the addition of the 7S-peptides, whereas the synthesis of cholesterol remained unchanged by 4 h and increased by 8 h after the addition of the 7S-peptides. The cleaved nuclear form of SREBP-2 increased 8 h after the addition of the 7S peptides, suggesting a decrease in intracellular cholesterol levels. Analysis of changes in mRNA expression after 7S-peptide treatment suggested that the 7S-peptides lower the level of cholesterol in the endoplasmic reticulum, increase the mRNA of genes related to beta-oxidation of fatty acids, and increase the synthesis of cholesterol. From these results, it may be concluded that the peptides derived from 7S altered the lipid metabolism to decrease secretion of apolipoprotein B-100-containing lipoprotein from HepG2 cells.

Background: Food-dependent exercise-induced anaphylaxis (FDEIA) due to soybeans is a rare disorder. The allergen responsible for FDEIA due to soybeans has not yet been determined. Objective: We characterized the clinical features of a patient with FDEIA due to tofu, who was well tolerant to drinking soy milk. We then sought to identify the responsible soybean allergen(s) in that patient. We further studied whether different stabilities of the allergen(s) to pepsin digestion between two soybean products are related to their clinical allergenicity. Methods: Skin prick tests and provocation tests using soybean products were performed to detect the responsible food and other factors that induced the allergic symptoms. Specific IgE to various soybean allergens were examined by ImmunoCAP®, ELISA and protein microarray assays. Immunoblotting for soybeans and soybean products using the patient's serum was also performed. Soybean products were serially digested by pepsin to disclose the stability of the allergens. Results: Provocation with ingestion of tofu and exercise induced the allergic symptoms, while ingestion of soy milk and exercis e did not. Immunoblot analysis, ELISA and protein microarray assay revealed that β-conglycinin mainly reacts with IgE antibodies in the patient's serum. By immunoblot analysis, β-conglycinin in soy milk completely disappeared after pepsin digestion within 20 min, whereas β-conglycinin in tofu was almost intact after more than 120 min of pepsin digestion. Conclusion: We identified β-conglycinin as the causative allergen in a patient with FDEIA induced by tofu. The difference in resistance to pepsin digestion between tofu and soy milk suggests that the presence of undigested allergens in the digestive tract is a prerequisite for the development of FDEIA. © 2008 The Authors.

【症例1】35歳男性.30歳時,モモ1個を摂取後に口腔内〓痒と蕁麻疹が出現した.嘔吐,呼吸困難,血圧低下,意識低下も伴った.血液検査所見では血清総IgE値は436IU/ml,モモの特異的IgE測定(CAP)は陰性であった.プリックテスト(SPT)では,モモは生と缶詰共に陽性で,ハンノキ花粉は陰性であった.【症例2】33歳女性.27歳時,白桃のケーキを摂取後に手掌の〓痒から始まり,眼瞼浮腫,咽喉頭刺激感,蕁麻疹,鼻汁,嘔気,腹痛,下痢が出現した.血液検査所見では血清総IgE値が85IU/ml,モモのCAPは陰性であった.SPTで桃缶詰は陽性であったが,ハンノキ花粉は陰性であった.原因抗原検索では,2例共にrPru p 1,3,4のCAPは陰性であったが,IgEイムノブロットで約10kDaにバンドを検出した.モモによるアレルギーでは,CAPが陰性となっても重篤なアナフィラキシーを呈することがあり,またリンゴ抗原,アプリコット抗原,及びプラム抗原といったバラ科果物抗原と交叉抗原性を有しているとの報告があるので注意が必要である.

モモアレルギー5例について病歴聴取や皮膚テストとともにモモ主要抗原Pru p1(Bet v1ホモログ)、Pru p3(lipid transfer protein)、Pru p4(Bet v2ホモログプロフィリン)特異IgEをImmunoCAPで測定し、モモのイムノブロットも施行して原因抗原を検討した。その結果2群に分類された。モモ摂取によりショックを起こした3例では、Pru p1、Pru p3、Pru p4のImmunoCAPは全て陰性であった。イムノブロットでもPru p1やPru p4に相当する17kDa,14kDaのバンドは陰性、1例のみにPru p3と考えられる9kDAのバンドを検出したが、他に20kDaや28kDaなどのバンドが検出された。多種の果物のOASも合併していたモモOAS2例ではImmunoCAP、イムノブロットともにPru p1、Pru p4特異IgEや、Pru p1、Prup4に相当する17kDa,14kDaのバンドを検出した。後者の多くはハンノキ花粉症を有し、ハンノキからの交差反応が主であると推測された。本邦でもモモショック例と多種の果物OAS合併例では感作経路や反応抗原が異なると考えられたが、ヨーロッパとは異なる可能性がある。(著者抄録)

リンゴとダイズを例に、病害虫被害によって増加する感染特異的タンパク質関連の農作物アレルゲンの増加と農薬防除による抑制についてデータを含めて解説した。

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 mu g/g in foods) and 0.94 ng/mL (equivalent to 0.38 mu g/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human alpha(1)-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.

即時型アレルギーの原因となるアレルゲンの検出、抗原解析（in vitro）について、イムノブロッティング法を中心に解説した。

Background: Many cases of spice allergy have been reported especially from Scandinavian countries, but in contrast there are few reports in Japan. This time we experienced two cases of apiaceae spice allergy and practiced some kinds of examinations. We report here these two cases with the consideration concerning mechanizm of spice allergy. Methods and Subjects: We practiced 1) specific IgE of pollens and foods, 2) prick tests of spices and apiaceae vesitables, 3) immunoblot of spices, against two cases suspected spice allergy from their clinical courses. Clinically Case 1 32 y.o. male had been no history of pollinosis, in contrast Case 2 46 y.o. female had been suffered from pollinosis during spring and autumn seasons. Results: In Case 1 the scores of specific IgE of pollens were almost negative and immunoblot examination of spices revealed positive reaction at the site of 10∼12kDa and 60kDa. In Case 2 the scores of specific IgE of pollens were positive in many species and immunoblot examination of spices reacted positively at the site of 14kDa and 60kDa. Both of them showed positive reactions against many kinds of apiaceae spices in prick tests, so we diagnosed them as apiaceae spice allergy. Conclusion: According to these results we suspected Case 1 as class 1 allergy induced by the sensitization of spices themselvs and Case 2 as class 2 allergy caused by the cross reactions with pollinosis. So there may be some different mechanizms in the occurrence of spice allergy. In the future the occurrence of spice allergy will be supposed to increase and it will be necessary for us to pay much more attention to spice allergy even in Japan.

We aimed to detect the allergen proteins in food materials using recently developed near-infrared fluorescent probes. Sensitivities of this method were comparable to chemiluminescence detection methods, which are known to be sensitive. In addition, the sensitivities of this near-infrared fluorescent method were at least 10-50-times higher than those of the conventional visible fluorescent methods using Cy3 and Cy5 dyes. This method was effectively applicable to immunoblotting, dot-blotting and plate-assay (direct FLISA : fluorescence-linked immunosorbent assay) with ELISA plate. Allergen levels of the food sample were quantified by standard curves using standard allergen protein using the dot-blotting technique. This highly sensitive detection system also provided multiple detections of different allergens for different antibodies and dyes with distinct properties of wavelength. This enables high-throughput screening of characteristic allergen contents of target food materials, or cultivars. Generally, allergen proteins are recognized by patient's serum IgE. Therefore, we tried to detect patient's IgE-binding proteins, the putative allergens in foodstuffs. In this detection system, it was possible to detect IgE-binding proteins with sensitivity almost equivalent to a chemiluminescent detection system. Taken together, it was shown that this novel detection system was an effective technique for the sensitive detection and screening of food allergens.

A sensitive qualitative detection method for soybeans in foods by using the polymerase chain reaction (PCR) was developed. For specific detection of soybeans with high specificity, the primer pair of Gym 81/Gym 82 was designed on the gene encoding the Glycine max repetitive sequence. The trace amount of soybeans in commercial food products could be qualitatively detected by this method.

ローヤルゼリーのコレステロール低下作用に関連する肝臓の遺伝子発現変化をマウスを用いて解析した。（英文）

Our previous data showed that Gly m Bd 30K was absorbed from the gastrointestinal tract and circulated in blood in mice. This study was conducted to determine the mechanism and identify the inhibitor of such absorption. Using sandwich ELISA and immunoblotting, we found that intact Gly m Bd 30K was absorbed from apical to basolateral solutions and intracellularly accumulated by Caco-2 cells in a dose- and time-dependent manner. The absorption and intracellular accumulation of Gly m Ed 30K were significantly suppressed when Caco-2 cells were treated with sodium cromoglycate (SCG) (0-50 mmol/L) in a dose-dependent manner. In 24-d-old mice orally treated with SCG (10-1000 mg/kg body weight), plasma Gly m Bd 30K concentration decreased significantly 30-120 min after Gly m Bd 30K (2000 mg/kg body weight) administration. Moreover, inhibitors that suppress the clathrin-dependent endocytosis dansylcadaverine, the caveolae-dependent endocytosis nystatin and clathrin, and the caveolae-dependent endocytosis methyl-beta-cyclodextrin had inhibitory effects on the absorption and intracellular accumulation of Gly m Bd 30K by Caco-2 cells. These data indicate that Gly m 3d 30K is absorbed and intracellularly accumulated in Caco-2 cells via clathrin- or caveolae-dependent endocytosis. We propose that the absorption and intracellular accumulation of Gly m Bd 30K are inhibited by SCG via clathrin- or caveolae-dependent endocytosis.

The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that. the rin gene has the potential to regulate allergen accumulation in tomato fruit.

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide get electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AUPAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis. (c) 2005 Elsevier Inc. All rights reserved.

ヒト肝臓由来培養細胞からのＶＬＤＬ分泌評価系を構築し、この抑制作用からみた食品由来の脂質代謝調節因子の探索について解説した。

The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR alpha-specific activator. Phytol induced the increase in PPAR alpha-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR alpha. Moreover, the addition of phytol upregulated the expression of PPAR alpha-target genes at both mRNA and protein levels in PPAR alpha-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR alpha ligand and that it stimulates the expression of PPAR alpha-target genes in intact cells. Because PPAR alpha activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism. (c) 2005 Elsevier Inc. All rights reserved.

The mechanisms by which food allergens are absorbed and sensitized via the gastrointestinal tract have not been well characterized. In this study, the gastrointestinal absorption of a major soybean allergen, Gly m Bd 30K, in young and older mice, and the effects of dietary fat and exogenous emulsifier were investigated. In Expt. 1, Gly m Bd 30K [0, 500 or 2000 mg/kg body weight (BW)] was administered orally to 24-d-old mice, and blood was sampled at various time points over a 120-min period. Plasma Gly m Bd 30K was measured by sandwich ELISA and immunoblotting. Its concentration peaked at 30 min and was dose dependent. Intact Gly m Bd 30K and its 20-kDa fragments were identified in plasma after absorption. In Expt. 2, 24-d-old mice administered soy milk containing 1 mg Gly m Bd 30K showed a steady increase in plasma Gly m Bd 30K from 60 to 120 min that was significantly higher than that in 10-wk-old mice. In Expt. 3, when corn oil (5 or 30%) was coadministered with Gly m Bd 30K (2000 mg/kg BW) to 24-d-old mice, the plasma concentration increased significantly and generally reached a plateau after 30 min. The absorption after the coadministration of 30% corn oil and 3% sucrose fatty acid ester was higher than after the administration of 30% corn oil alone. Intact Gly m Bd 30K and its fragments that were < 20 kDa survived digestion and were absorbed into the blood. We propose that absorption was enhanced by fat carrier-mediated transport. J. Nutr. 135: 1738-1744, 2005.

We used a DNA microarray to compare the gene expression profiles in liver among three groups of mice fed a diet containing 5% royal jelly (RJ). a diet containing 59% RJ stored at 40 degrees C for 7 d (40-7d RJ) or a control diet which provides the same total energy as RJ. Expression of 267 genes was increased or decreased by 1.8-fold or more in animals given the RJ diet for 14 d as compared with control diet, though serum total cholesterol, triglyceride, phospholipid, glucose, insulin and leptin levels were unaffected. Many genes involved in cell growth, signal transduction, energy metabolism and transcription regulation were responsive to the RJ diet. Among the 267 genes whose expression was altered by RJ, 60% showed no change or a reduced change in response to 40-7d RJ diet. The 40-7d RJ diet contained little 57-kDa protein, identifled as a possible freshness marker of RJ. Furthermore, the RJ diet did not influence the gene expression of cytochromc P450 enzymes and detoxifying enzymes, whereas the 40-7d RJ diet increased the gene expression of glutathione S-transferase and glutathione peroxidase. Indeed, the RJ diet decreased the gene expression of cytochrome P450 4A14 (CYP4A14). which catalyzes peroxidation of endogenous lipids that is associated with nonalcoholic steatohepatitis and alcoholic liver disease, while the 40-7d RJ diet was not effective to decrease the gene expression of CYP4A14. The results indicate that the efficacy of RJ decreased and the toxicity of RJ increased during storage at high temperature. We suggest that application of DNA microarray technology to the biochemical evaluation of food safety may be effective for rapid and precise quality control.

Soybean (Glycine max L.) storage proteins are composed of two major components, beta-conglycinin and glycinin, corresponding to 7S and 11S globulins, respectively. Recently, soybean beta-conglycinin (7S globulin) has been reported to show beneficial functions in animals and human. To date, there is no method for the precise quantification of soybean beta-conglycinin in processed food products or soybean seeds. We report here a novel method for this purpose. At first, antibodies specifically reactive to the subunits of beta-conglycinin were prepared. And then. a direct enzyme-linked immunosorbent assay (ELISA) for the quantification of soybean P-conglycinin in processed foods and seeds was developed. In this assay, the sample was treated with sodium dodecyl sulfate sample buffer followed by dilution with phosphate-buffered saline. The diluted samples were poured and coated onto an ELISA plate and reacted with rabbit anti-beta-conglycinin antibody and peroxidase-labeled anti-rabbit IgG. Finally, the bound peroxidase-labeled antibody was detected by colorimetric reaction. By using this system, it has been possible to measure soybean beta-conglycinin concentrations in several processed food products. In addition, this simple quantification ELISA system was demonstrated to be adaptable for the quantification of beta-conglycinin contents of various soybean cultivars.

ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT). In this study, we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule. ER-60 was shown to be composed of at least four domains, named a, b, b', and a', by limited proteolysis. Recombinant fragments of ER-60, a, b', and a'c, were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient. These fragments each gave the circular dichroism (CD) spectrum of the folded protein. On the other hand, fragment b, which did not undergo the cooperative unfolding transition along a urea gradient gel, did not show any sign of the folded structure on the CD measurement. However, subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb'. Both a and a'c, which have a catalytic center CGHC motif, showed activity almost equivalent to half of that of wild-type ER-60. Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity, suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60. The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.

The purpose of this study was to discover the effects of soybean beta-conglycinin (7S-globulin) and glycinin (11S-globulin) on serum lipid levels and metabolism in the livers of normal and genetically obese mice. Male normal (ICR) and obese (KK-A(y)) mice were fed ad libitum high fat diets for two weeks, followed by a 2-week restriction of diet (2 g diet/mouse/day) containing 20% casein, soybean beta-conglycinin, or soybean glycinin, and then sacrificed immediately. Serum triglyceride (TG), glucose, and insulin levels of beta-conglycinin-fed mice were lower than in casein- and glycinin-fed mice of both strains. In order to analyze the related events to these effects, enzyme activities and relative mRNA levels of lipid metabolism-related proteins were measured. The activities of two enzymes related to fatty acid beta-oxidation were higher while that of fatty acid synthase was lower in livers of beta-conglycinin-fed mice than of casein-fed both mice. Messenger RNA levels of acyl-CoA oxidase (fatty acid beta-oxidation related enzyme) were significantly higher in livers of beta-conglycinin-fed mice than of both casein-fed mice. On the contrary, mRNA levels of SREBP-1 and 2 tended to be lowered in livers of soy protein-fed mice than of both casein-fed mice. Fecal excretion of TG was higher in beta-conglycinin-fed mice than in casein-fed mice. Our results demonstrated that the soy beta-conglycinin diet reduced serum TG levels by acceleration of beta-oxidation, suppression of fatty acid synthase and/or increased TG fecal excretion, and also diminished serum glucose and insulin levels. Some of these events might be caused at the transcriptional levels, judged from the result that relative messenger RNA levels of lipid metabolism-related proteins were altered. These results suggest that soy beta-conglycinin could be a potentially useful dietary protein source for the prevention of hypertriglyceridemia, hyperinsulinemia, and hyperglycemia, which are recognized as risk factors for atherosclerosis.

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid Substitution affects the electrophoretic mobilities of beta* and A3*. (C) 2003 Elsevier Ltd. All rights reserved.

Obesity is a major risk factor for insulin resistance. Resistin, an adipocyte-derived hormone-like molecule, is considered to serve as an important link between obesity and insulin resistance. However, the physiological role of resistin and the mechanism by which it neutralizes insulin action are still unclear. There are also conflicting reports that cast doubt on the cause of insulin resistance. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse resistin levels, analyzed in relation to insulin resistance. C57BL/6J mice fed high-fat diet compared with normal diet had low resistin levels (by 70%, P < 0.01) in epididymal adipose tissues. Genetically obese mice, db/db and KK-A(y), had hyperinsulinemia and hyperglycemia but low resistin levels (decreases by 83 and 90%, both P < 0.01) compared with C57/BL6J mice in epididymal adipose tissues. Serum resistin levels determined by Western blotting showed a similar pattern to those in adipose tissues. Resistin levels in adipose tissues correlated with serum adiponectin concentrations positively (r = 0.49). Our results indicate that the novel ELISA system is suitable for measurement of resistin levels in adipose tissues. The results do not support a role for resistin in insulin resistance. (C) 2003 Elsevier Science (USA). All rights reserved.

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had,peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.

The loaf volume decreased on the addition of soybean protein isolate (SPI) to bread. When phosphatidylcholine was added to the ingredients, including SPI, the adverse effect of SPI on the loaf volume could be counteracted. The addition of phosphatidylcholine showed little effect on the rheological properties of dough. It was shown by confocal microscopy that phosphatidylcholine was associated with gluten in the dough. The function of phosphatidylcholine could not be replaced by phosphatidylethanolamine, phospatidic acid, or lysophospatidylcholine. Phosphatidylcholine showed different effects depending on the phosphatidylcholine molecular species.

Human recombinant ER-60 was confirmed to have transglutaminase activity by a microtiter plate assay. Transglutaminase activity of ER-60 did not require calcium and was inhibited by cystamine, a substrate analogue. In addition, the transglutaminase activity of ER-60 was not inhibited by SH-blocking reagents. These results suggest that the properties of the transglutaminase activity of ER-60 are different from those in the cases of known mammalian transglutaminases of which the active site includes a cysteine residue.

A simple method for separation and quantification of neutral lipids was developed using thin-layer chromatography (TLC) and high-performance fluorescent scanning. Neutral Lipid classes were separated using the double-developing TLC method and detected by rhodamine 6G and a laser-excited fluorescent scanner. The amount of lipids applied correlated with scanned intensity volume in a dose-dependent manner. The mass of each neutral lipid band was determined by comparing band intensities of unknown samples to dilution curves of authentic standards. After scanning the dye-sprayed TLC, acyl chain species of triglyceride (TG) extracted from TLC could be determined by gas chromatography. Using this method, we quantified the amounts of TG in mouse liver and found that the measured total mass of TG correlated with that obtained by enzymatic methods. Our method should provide the basic technique for "lipidome" analysis, designed to determine and compare total lipid classes and mass present in biological samples. (C) 2001 Academic Press.

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease, Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells, While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin, Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage. (C) 2001 Academic Press.

Background: Plant food allergies have been associated with pollenosis, although most of the causative allergens are as yet undefined. It is important to elucidate the properties of plant food allergens in order to minimize a patient's risks in food selection. The purpose of the present study was to examine and characterize the IgE-binding proteins in carrots as possible allergens by using patients' sera. Method: IgE-binding proteins in carrot extract were screened by an immunoblot technique using sera of patients with atopic dermatitis (selected based upon a case history of food allergies). An allergenic protein was purified from carrot extract by chromatographic procedures. The N-terminal amino acid sequence of allergenic protein was determined and subjected to a computer homology search. Cross-reactivity between carrot and birch allergens was examined by immunoblotting. Results and Conclusion: A unique, approximately 20-kDa allergenic protein that reacted with about 14% of patients' sera was isolated and characterized. The N-terminal amino acid sequence of the purified protein was found to be homologous with those of plant cyclophilins. This allergen exhibited a peptidyl-prolyl cis-transisomerase activity, which was inhibited by the conjugation of cyclosporin A. These properties of the allergenic protein isolated from carrot identified it as a cyclophilin, a possible plant food allergen. No cross-reactivity between this 20-kDa carrot allergen and Bet v 7, a birch pollen cylcophilin, was observed. Copyright © 2001 S. Karger AG, Basel.

ER-60 protease contains two CGHC motifs that appear to include an active site cysteine residue(s). Its proteolytic activity was lost with a double mutation of the C-terminal cysteines of the two motifs to alanine, but not with a single mutation of the C-terminal cysteine of either of the motifs to alanine, This suggests that these C-terminal cysteines independently constitute the catalytic active site. A mutation of both histidine residues in the two CGHC motifs to serine did not abolish the proteolytic activity, suggesting these histidine residues in the CGHC motifs do not constitute the catalytic dyed of ER-60 protease, (C) 2000 Federation of European Biochemical Societies.

The accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non-ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH4Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy.

Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-β-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectrofocusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2, inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basaI cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 μM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50, < 5 μM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glyceroI, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation. © 1999, Taylor & Francis Group, LLC. All rights reserved.

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum, Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase, E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, ale inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase, This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.

The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b (+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine resldue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.

The mechanism of arachidonic acid (AA) release in collagen-activated human platelets was studied. An arachidonic acid metabolite, thromboxane B-2 (TXB(2)), was formed in parallel with the formation of phosphatidic acid (PA) without formation of lysophosphatidic acid (lysoPA) or lysophosphatidylinositol (lysoPI) in the absence of extracellular Ca2+, suggesting that AA was released from PI via a PI-specific phospholipase C (PI-PLC)/diacylglycerol (DG) lipase/monoacylglycerol (MG) Lipase pathway under the cytosolic low Ca2+ concentrations. Moreover, solubilized DG lipase and MG lipase could hydrolyze the substrates at basal cytosolic free Ca2+ concentrations. Subsequently, the relationship of cytosolic free Ca2+ concentrations and formation of AA metabolites was analyzed using Ca2+ ionophore, A23187. Collagen was able to induce a release of small amounts of AA under basal cytosolic Ca2+ conditions. However, a release of large amounts of AA was induced by phospholipase A(2) activated by both collagen-receptor occupancy and elevated Ca2+ levels. A TXA(2) mimetic agonist, STA(2) induced all the responses except for AA release. From these results, the mechanism of AA release and signal transduction in collagen-activated human platelets is discussed.

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C- α was purified to apparent homogeneity on sodium dodecyl sulfate- polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH 2 -terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-α. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.

Two types of cytosolic phospholipase C specific for phosphoinositides were purified from human platelets. The molecular masses of the purified enzymes were 440 and 290 kDa. These enzymes were concluded to be respectively a trimer and a dimer of homologous 146 kDa polypeptides. The 146 kDa polypeptide may be an immunologically novel isozyme among the 140-150 kDa PLC isozymes. Both enzymes hydrolyzed phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in a Ca 2+ -dependent manner. © 1990 Copyright, 1990 by the Journal of Biochemistry.

The Ca 2+ channel blocker, nifedipine, a dihydropyridine derivative, inhibited the Ca 2+ influx and release from internal stores caused by collagen or a low concentration of the thromboxane A 2 (TXA 2 analogue, 9,11-epithio-11,12-methano-TXA 2 (STA 2 (10 nM), but did not inhibit those caused by thrombin or a high concentration of STA 2 (100 nM). These results indicate the presence of two distinct, dihydropyridine-sensitive and insensitive, Ca 2+ channels dependent on the concentrations and classes of agonists in human platelets. © 1988 COPYRIGHT 1988 BY The Journal of Biochemistry.

The thromboxane A 2 antagonist, ONO-3708, completely inhibited the increase in cytosolic free Ca 2+ in human platelets during activation with collagen. Half-maximal Ca 2+ release and influx required about 3 and 4 nM STA 2 , a stable thromboxane A 2 mimetic, respectively. However, half maximal activation of phospholipase C required about 18 nM STA 2 . This suggests that thromboxane A 2 directly causes Ca 2+ mobilization without further activation of phospholipase C during activation of human platelets with collagen. © 1988 BY The Journal of Biochemistry.

ザクロは、肥満および糖尿病を予防・改善することが報告されている。そこで、我々はザクロ抽出物が脂肪細胞においてアディポサイトカイン、特にレジスチンおよびアディポネクチンの発現変動に影響を及ぼすか検討した。 【方法】ザクロ果汁は市販濃縮果汁(Wonderful種、全果搾汁)を用いた。分化10日目のマウス由来3T3-L1脂肪細胞にザクロ果汁(10、50、100 ？g/ml)を添加し、経時的に培地および細胞の全タンパク質を回収した。アディポサイトカイン量はELISA法およびWestern blotting法により測定、検出定量を行った。 【結果と考察】ザクロ果汁は、培地中へのレジスチンの分泌量を添加3時間後から濃度依存的に顕著に低下させた。一方、アディポネクチンは変化がみられなかった。細胞中のレジスチンタンパク質の存在レベルは12時間後より低下がみられた。以上の結果から、ザクロ果汁はレジスチンのタンパク質合成を抑制、もしくは分解を促進することによって、レジスチンの分泌を

ヒト培養肝細胞HepG2へアグリモニー及びアスナロエキスを添加することにより、アポＢ含有リポタンパク質（VLDL）分泌が抑制されることを見出した。 マウスを用いた動物実験においても、飼料中への0.5%(w/w)のアグリモニーエキス添加では内臓脂肪レベルが有意に低値を示した。 0.3%(w/w)のアスナロエキス添加では体重、内臓脂肪、いくつかの血中パラメータ（TG、レプチン、インシュリン）が有意に低値を示し、肥満や高脂血症に対して予防改善効果があることが示唆された。培養肝細胞からのアポＢ含有リポタンパク質の分泌抑制を指標にしたスクリーニングが、抗肥満物質の探索に有効である可能性が示唆された。

大豆によるクラス２食物アレルギーの主要な原因抗原と考えられる大豆アレルゲンGlym3、Glym4のクローニング、発現・精製、抗体作製を行った。 得られた抗体を用いて、市販豆乳や大豆加工食品におけるＧｌｙｍ3、Ｇｌｙｍ4の同時検出を行った。豆乳飲料ではGlym3が豆乳に比べて少ない傾向が見られた。 ＯＡＳ発症リスクに相関する可溶性のアレルゲンレベルでは、豆乳以外の加工食品ではGlym3がほとんど検出できず、本アレルゲンによるＯＡＳ患者では豆乳以外の加工食品ではリスクが少ないと考えられた。また、納豆や醤油などの発酵食品や水煮大豆、きなこ、豆乳ヨーグルトでは可溶性のGlym3、Glym4ともに検出されず、ＯＡＳ発症リスクは少ないと考えられた。 これらの結果から、様々な大豆加工食品においてＧｌｙｍ3、Ｇｌｙｍ4の存在パターンや存在量、存在形態に多様性が認められた。

ヒトAPO-AⅤを大腸菌にて発現させ、アフィニティー精製した。 発現タンパク質から作製したモルモット抗ヒトAPO-AV抗体は、マウスの肝臓ホモジネート、ヒトHepG2細胞ホモジネート中の内因性の本分子を感度良く検出できた。 作製抗体と市販抗体を組み合わせたサンドイッチELISA系によってHepG2細胞から経時的に分泌されるAPO-AⅤを半定量することが出来た。 フラボノイドなどの食品成分を添加した場合、イプリフラボンやイコール、ガレート型カテキン類などがAPO-AVの分泌を抑制することが示唆された。これらの食品成分が、APO-AVの分泌制御を介して中性脂肪代謝に影響を与える可能性が示唆された。

【目的】多くの農作物には、一部のアレルギー患者が反応するアレルゲンが含まれている。特に、野菜・果物のなかには、花粉症やラテックスアレルギーと交差反応するアレルゲンが含まれているものがある。このような農作物のアレルゲンレベルには品種間差違が存在することが示唆されている。そこで、本研究では、一般に消費の多い果物の代表としてリンゴに焦点をあて、より安全なリンゴを探索する目的でさまざまなリンゴのアレルゲンレベルを検証した。 【方法】市販リンゴまたは長野県産のリンゴから抽出液を得て電気泳動（SDS-PAGE）、ウエスタンブロッティングによってリンゴの主要アレルゲン（Mald1）レベルを相対比較した。Mald1に対する抗体は大豆でのホモログ分子であるGlym4に対して作製した抗体を用いた。また、リンゴに反応するアレルギー患者血清を用いてIgE結合性を評価した。 【結果】電気泳動後のCBB染色や蛍光染色によって検出された総タンパク質のパタ

【目的】花粉症に罹患した患者が、植物性食品を摂取した際に口腔内に発症するアレルギー症状（口腔内アレルギー症候群：OAS）が問題となっている。これは花粉抗原と交差反応を示す植物性食品中のアレルゲンによって惹起される。原因食材としては、果実や野菜などが多いが大豆においても発生することを見出した。そこで本研究では、大豆食品のアレルゲンリスク評価に資すため、主要な原因抗原と推察される大豆Glym3（プロフィリン）及びGlym4（Betv1ホモログ）のクローニング、抗体作製及び特性解析を試みた。 【方法】Glym4に関しては大豆葉cDNAよりPCRにて本遺伝子をT/Aクローニングしたのち、pET系発現ベクターにサブクローニングし、Hisタグタンパク質として可溶性状態にて発現させた。キレートカラムなどを用いて精製した標品を抗原としてウサギ抗血清(2種類)とマウス抗血清(4種類)を作製した。Glym3に関しても同様に実験を進めている。またGlym3に関しては長鎖合成ペプチド

【目的】大豆には様々な健康機能性成分が含まれている。個々の成分は、脂質代謝改善効果・抗肥満効果などの生活習慣病改善効果を有することはよく知られているが、大豆をほぼ丸ごと含む豆乳の食品としての機能性については十分には検証されていない。そこで、本研究ではマウスに豆乳を摂取させ、メタボリックシンドローム（MS）関連のパラメータを解析し、MSに対する豆乳の効果を検証することを目的とした。 【方法】AIG-93Gに準拠した基本飼料粉末を混合し、これに水を使って練った飼料を対照飼料とした。水の代わりに豆乳を使って練った飼料を豆乳飼料とした。添加された豆乳中の栄養素に関しては該当分を基本飼料から減らし、両飼料のカロリー及び主要栄養素含量を等しく揃えた。マウス(C57BL6J、雄性)を２群に分け、これらの飼料をそれぞれ自由摂取させた。約１ヶ月の飼育ののちに採血・解剖し、種々の解析に供した。 【結果】摂餌量、体重、内臓脂肪量、肝臓

【目的】ChREBP（炭水化物応答配列結合タンパク質）は、糖摂取のシグナルを受容し、解糖系や脂肪酸合成系に関わる酵素遺伝子の転写促進を司る転写因子である。飽食条件下では、この活性化を抑制することで脂肪酸合成系を抑えることができるため、メタボリックシンドローム改善のターゲット分子の一つと考えられる。そこで本研究ではヒト肝臓由来のHepG2細胞を用いて、ChREBPの量的変動解析及び活性化評価系の構築を行った。 【方法】量的変動解析では、培地中のグルコース濃度を変えてHepG2細胞を培養し、ChREBPタンパク質レベルをWestern blottingにて定量した。また、活性化評価のためには、近赤外蛍光標識のオリゴDNAプローブを用いたnon-RIでのEMSAにより解析した。 【結果】グルコース濃度の変化に応じて、細胞中の全ChREBPタンパク質レベルは変化し、特にグルコース枯渇条件下ではChREBP分子そのものが著しく減少することが判明した。 またEMSAによる活性化評価でも、グルコ

【目的】LRH-1は、ApoA-IやCYP7A1、アディポネクチンなどの脂質代謝やメタボリックシンドローム関連遺伝子の転写を促進する核内転写因子であり、メタボリックシンドローム改善の標的分子の一つと考えられる。本研究では、LRH-1の活性化の調節機構や食品成分による活性化への影響などを解明するため、本転写因子の活性化評価系の構築およびその変動解析を検討した。 【方法】まず始めにヒト肝臓cDNAライブラリーをテンプレートにしてLRH-1 cDNAのPCRクローニング、大腸菌での部分長の発現と精製および特異抗体の作製を行った。また、活性化評価のために、近赤外蛍光標識のオリゴDNAプローブを用いたnon-RIでのEMSAの構築を試みた。 【結果】作製した抗体を用いたWestern blottingによって、ヒト肝臓由来培養細胞HepG2の核抽出物中の本分子を検出することができた。また、核抽出物を用いたEMSAによって、本分子の活性化型レベルを相対定量することが可能であった。これらの系を用いて細

本研究では、マウスモデル系を用いて経皮感作しうる食品タンパク質について、その同定を行うことにより、経皮感作されやすいタンパク質の特性について検討するとともに、経皮感作に影響しうる要因を解明し、経皮感作を抑制しうる食品・栄養因子を明らかにすることを目的としている。また、このような食品中のアレルゲンについて、そのリスク変動要因の解明についても検討する。今年度は、チェリーやキウイを塗布した際の経皮感作抗原の同定に成功した（論文準備中）。また、遺伝子組換え大豆の経皮感作能に関しても検討を行っている（論文準備中）。その他、米ぬかが経皮感作することも見いだし、その原因抗原の同定を進めている。さらに、ソバやゴマなどの植物性食品に関しては現在、経皮感作抗原の同定を進めている（論文準備中）。 経皮感作に影響を与える因子の検討についても、抗原濃度、共存する界面活性剤の濃度、皮膚バリアの破壊度合い、共存する食品由来成分などによって影響を受けることを明らかにしつつある。今年度は特に、除毛クリームの経皮感作に及ぼす効果について検討した。これらの検討によって、安定的に経皮感作しうる条件の絞り込みを進めている。 また、これらの経皮感作抗原を含むアレルゲンの変動解析では、大豆について、遺伝子組換え大豆における主要アレルゲンの存在レベルを非組み換え大豆と比べることで検討したところ、遺伝子組換え大豆における主要アレルゲンレベルは非組み換え大豆と有意な差は見られないことを報告した。さらに、経皮感作に影響を及ぼす皮膚バリア関連タンパク質について、その分解をモニターしうる実験系を構築し、いくつかの食品成分がこれらの皮膚バリアタンパク質を分解しうることを見いだした。このように、研究は順調に進んでおり、次年度でも引き続き論文発表を進める予定である。

近年、食物アレルゲンの一部は、皮膚からの抗原浸入が感作源となることが示唆されている。これを経皮感作と呼ぶが、種々の食品に含まれる経皮感作抗原に関しては未だ不明な点が多い。そこで、マウスモデル系を構築し、食品抽出液を皮膚に塗布した際のIgE、IgG1産生を指標に経皮感作抗原を探索した。大豆や乳、卵黄、卵白、ソバ、ゴマなどの経皮感作抗原を同定した。また、塗布するタンパク質を経口摂取させると、経口免疫寛容が作動し、経皮感作を抑制しうることを大豆をモデルにして示した。さらに、経皮感作を抑制しうる食品成分についても同定することができた。また、これらの食物アレルゲンの各種処理における変動解析も行った。

皮膚からの食品抗原が侵入し、体内で特異的なIgE抗体の産生を検出する実験系の構築に成功した。この確立された評価系を用いて、大豆などの食品サンプルをマウス皮膚に塗布し、どのような大豆タンパク質に対する抗体が産生されたのか検討した。その結果、Gly m5と呼ばれる大豆アレルゲンに対して抗体が産生された。マウスを用いて、経皮感作を評価しうる系を構築し、大豆や果物などの食品を塗布することで特異的なIgE抗体が産生された。

花粉症に関連するクラス2食物アレルゲンとして、Betv1ホモログ、プロフィリン、ソーマチンライクプロテインをクローニングし、発現タンパク質から汎用性の高い抗体を作成した。これらを用いて、大豆や野菜、果物などの主要な農作物における品種間、栽培方法、加工法、調理法などの違いによるアレルゲン性の変化・変動を解析した。これらの条件によってアレルゲンレベルは大きく変動することが明らかとなった。また、農作物による食物アレルギーの原因抗原の探索も行った。

近年、生活習慣病が増えており、深刻な社会問題となっている。「インスリン抵抗性」を改善することが生活習慣病の予防・改善にとって大きな意義を持つ。このインスリン抵抗性に関わる分子としていくつかのホルモン様分子が発見されている。それらのうち、レジスチンと呼ばれる脂肪細胞が分泌する分子はこのインスリン抵抗性を引き起こす責任分子として数年前に報告された。そこで、本研究では、このレジスチンの生理作用やレジスチンの発現量,循環量に影響を与える食品成分の探索を行う。また、アディポネクチンに関しても同様に検討した。まずはじめに、レジスチンの特異抗体を作製し、この抗体を用いてサンドイッチELISA系を構築した。つぎに、培養脂肪細胞である3T3-L1細胞を十分に分化させるための培地の検討を行い、この細胞から実際にレジスチンタンパク質が培地へ分泌されることをサンドイッチELISA系を用いて明らかにした。アディポネクチンに関しても同様に分泌が確認された。そこで、これらの重要なアディポサイトカインの分泌に対して食品成分が影響を与えるかどうか検討した結果,ティーフラボン類などにレジスチンの分泌を抑制する効果があることが始めて明らかとなった。その他にもいくつかの食品成分に、これらのアディポサイトカインの分泌を調節する活性が見られた。レジスチンの分泌を調節しうる食品成分が明らかになったことについては重要な知見であると言える。

申請者は脂質性メディエーターの産生機構の解明や脂質代謝、リポタンパク質代謝に関わる酵素類及びその機能を調節しうる食品成分に関して研究を進めている。これまでに、新規な脂質性メディエーターである2-AG(2-アラキドノイルグリセロール)の産生に関わると推定されるDGリパーゼの精製や候補遺伝子である新規リパーゼ様遺伝子のクローニングに成功した。また、クローニングした新規脂質代謝関連遺伝子産物が生体内でどのような脂質分子を基質として、どのような触媒反応を担当するのかという点に関して、遺伝子を導入した細胞の脂質プロファイルの解析(リピドーム解析:生体中の脂質成分を一括定量比較解析する手法)を用いて解析した。さらに、遺伝子導入細胞または共存培養細胞(ターゲット細胞)のタンパク質リン酸化のプロファイルを抗リン酸化タンパク質抗体を用いたプロテオーム解析によって解析する基盤技術が確立できた。 さらに、本研究を発展させ、以下の2つの研究も行った。(1)脂肪細胞が分泌する代謝調節性ホルモンと推定されるレジスチンに関して、その生体内での役割を解明することをめざし、レジスチンの生理機能をトランスクリプトーム解析によって詳細に検討した。(2)肝細胞からのリポタンパク質分泌をモニターするELISAシステムを構築し、リポタンパク質(VLDL)分泌に影響を与える食品成分の探索に成功した。現在は、リピドーム解析とプロテオーム解析、トランスクリプトーム解析を駆使して、VLDL分泌抑制のメカニズムを解明することを試みている。

食物アレルギー患者のIgE抗体が認識する植物性食品素材のタンパク質の多くは、植物が病原体の侵入、外注の食害、環境ストレスに対して発現する感染特異的タンパク質(PR-P)あるいはディフェンスタンパク質(DP)と呼ばれているグループに帰属される物が多い。もし、この仮説が正しいとすると、厳しい環境下で生育した植物性食品素材は、植物アレルギー患者にとっては、アレルゲン性の高い食品となってしまう。食物アレルギー患者の健全な食生活を確保するためには、これらのリスクの小さい作物の供給される必要がある。従って、本研究では、食品素材のアレルゲン性(患者血清との反応性)が栽培条件・環境条件よってどのように変化するかを解明することを目的とした。 (1)温室栽培が容易な、二十日大根をストレスなしと、除草剤散布(エリシター作用のあるヒドロキシ安息香酸)によるストレス負荷による患者血清中のIgE抗体反応性を、個々のタンパク質について追跡した。 (2)大豆について、路地での栽培において、新しい農薬としてベンダゾンの散布が、大豆胚乳タンパク質アレルゲンに対してどのような影響を与えるかを検討した。 (3)いずれの場合のおいても、ストレス負荷によって、患者血清中のIgE抗体が認識するタンパク質成分の多くが、産生を促進され、コントロールに比して蓄積することを明らかにした。又逆に、全ての成分が増加する野でなく、一部のアレルゲン成分には、減少する物が観察された。 (4)これらの成分に内、増加する物についてN-末端アミノ酸シーケンスを解読し、相同性検索を行ったところ、明らかにPR-Pとして報告されているタンパク質に帰属される物が存在した。これらの多くは、過酸化酵素や、参加還元酵素類に属する物が多いことが示された。 (5)結論として、ストレス負荷が植物性食品素材の感染特異的タンパク質の産生を刺激し、アレルゲン性の増加につながること事実を立証した。

1,食物アレルギー患者が血清中に保有するIgE抗体の認識する食品素材中のタンパク質成分を、網羅的にイムノブロット法を用いて検出、特定すると共に、Nー末端アミノ酸配列の分析を行って得られた情報を基に、コンピューターデータベースに基づく解析を行い、既知タンパク質成分に帰属し、同定した。これらの情報を基に、植物に特有の感染特異的タンパク質(PR-P)ファミリーに属するものを選択した。 (1)患者血清の認識するタンパク質成分として、ニンジンより20kDaアレルゲンとして、cyclophilin(cyclospolin A binding protein)を同定した。 (2)ジャガイモより、18kDaアレルゲンとして、シラカバ花粉症の主要アレルゲンBet v 1 homologueを同定した。患者血清は、ピーマンやリンゴ由来の18kDaと交差する。 (3)トマトより45kDaアレルゲンとして、コルク質形成関連酸性ペルオキシダーゼを同定した。 (4)二十日大根より、37kDaアレルゲンとしてGlutathione S-transferase,25kDaアレルゲンとして,feredoxine/NADH oxidoreductaseを同定した。 (5)リコンビナントPR-5dを用いて患者血清をスクリニングしたが、認識抗体を保有する患者は確認することが出来なかった。この事実は、PR-5ファミリーに関しては感作能が低い(アレルゲン性が低い)と考えられる。 以上のアレルゲンタンパク質にはPR-Pに分類される物が多いことが判明した。その他の帰属不明なタンパク質も,PR-Pである可能性は高い。これらの事実は、PR-Pがヒトの食物アレルギーの罹患、発症に関わる感作、即ちIgE抗体の産生を特異的に誘導していることを強く示唆するものである。また、これらのアレルゲンは植物界に広く分布し、互いに相同性が70-80%以上あることからパンアレルゲンとして交差反応性が問題となることが示唆された。一方、これらは素材をストレス下で栽培することにより、その含量が変化する事実を確認に下。従って、生産条件の管理が植物性食品のアレルゲン性を大きく左右することを立証した。 2,植物性食品素材毎に抽出調製したタンパク質画分を二次元電気泳動を行って、一次元目の情報、等電点、二次元目の情報、分子量をセットとする画タンパク質の戸籍簿を作成する。更に、二次元のイムノブロットにより患者血清の認識するタンパク質成分をマッチングさせ、アレルゲンのとなるタンパク質成分を特定する。この特定成分上に、各種属性を三次元の情報として蓄積し、一食品素材一データベースシートを構築する。日本標準食品成分表と対比出来るようにする。更に、情報として、PR-Pの特徴であるストレス負荷によるアレルゲン性の増減をデータベース上に搭載し、食物アレルギー患者の治療、栄養指導における有効かつ緻密な情報源として提供することが可能である。又、低アレルゲン化の程度、加工食品における混合素材アレルゲンの網羅的解析が可能となるであろう。

2-アラキドノイルグリセロール(2-AG)は最近発見された機能性脂質分子であり、カンナビノイドレセプターに対する内因性のリガンドである。2-AGは記憶や免疫作用に関わる可能性が示唆されているが、その生理的意義は未だ明確ではない。ジアシルグリセロール(DG)リパーゼは生体内における2-AG産生酵素の有力な候補である。そこで、カビのDGリパーゼとホモロジーを有するヒト胎児脳由来のESTクローンを基に、5'-RACE法によってヒト脳由来cDNAライブラリーより完全長cDNAを得た。推定されるアミノ酸配列を解析したところ、本クローンはリパーゼの活性中心コンセンサス配列を有する672アミノ酸からなるタンパク質をコードし、N末端側に4つの膜貫通ドメインを有している膜結合型タンパク質であるとが示唆された。この遺伝子は脳や肝臓、胎盤、免疫細胞などの多くの組織で発現していた。本遺伝子をCOS1細胞に発現させたところ、本タンパク質は膜画分に発現した。そのホモジネートを用いてDGリパーゼ活性を測定したところ、コントロールに比べて約2〜4倍の活性上昇が認められ、この活性はDGリパーゼの特異的な阻害剤によって完全に阻害された。以上より、ヒト膜結合型DGリパーゼのクローニングに成功したと考えられる。さらに、本分子のアイソザイムをデータベースにて検索したところ、ホモログと考えられるクローン(KIAA0659)を見いだした。このクローンは完全長でなかったので、キャップサイトハンティング法によって5'-上流部を得た。こうしてこのホモログに関しても完全長の塩基配列を取得した。このクローンもリパーゼの活性中心コンセンサス配列を有し、N末端側に4つの膜貫通ドメインを有している膜結合型タンパク質であることが示唆された。分子サイズは115kDaであった。発現組織を解析したところ、脳に特に多いことが特徴的であった。

食物アレルゲンの探索、同定、検出定量法の開発、低減化の検討

植物性食品素材中にはlgE抗体の産生を誘導するタンパク質が広く存在する。本研究はそのlgE抗体結合性タンパク質をアレルギー患者の血清を用いてスクリーニングし、その特性を解明することを目的として行われたものであり、以下に示す研究成果をえた。 1, 植物性食品素材のタンパク質を抽出し、イムノブロット法によりlgE結合性タンパク質を特定した。その多くはタンパク質中のアスパラギン結合型糖鎖を認識するantiHRP抗体の添加で結合が阻害された。 2, 大豆主要アレルゲンのcDNAを利用してリコンビナントGly m Bd 30Kを大腸菌で調製した。このものは糖鎖を有せず、患者血清によるイムノブロットは極端に減退した。この事実より、患者血清中における糖鎖認識lgE抗体の存在を立証した。 3, 種々の植物性食品素材について糖鎖が認識されるタンパク質をスクリーニングした。ジャガイモ、ニンジン、カボチャ、小麦、タマネギ等ほとんど全ての素材においてその存在が確認された。 4, 患者血清の認識する主要タンパク質の代表としてジャガイモを材料にそのタンパク質を単離し、パタチンと同定した。これもantiHRPでその結合が阻害される高マンノース型の糖鎖含有タンパク質であることを明らかにした。 5, 植物性食品中の交差反応性を有する主要な共通抗原のエピトープは、タンパク質ペプチド鎖に由来せず、アスパラギン結合型糖鎖がエピトープとして機能していることが証明された。

低アレルゲン大豆食品を開発することによりアレルギー患者の食生活を改善することを目的として本研究は行われた。えられた成果は以下に示すとおりである。 1, 低アレルゲン化のターゲットアレルゲンを主要3アレルゲン、β-conglycininのα-subunit,Gly m Bd 30K およびGly m Bd 28Kとした。 2, まず放射線照射変異株から分子育種法により、β-conglycininのα-subunit欠失品種、東北124を確立した。 3, 東北124を用いて、酵素処理により手アレルゲン煮豆(試料1)を調製した。 4, 東北124を脱脂し、遠心法によりGly m Bd 30K、28Kを除去したSPIを調製した。これは低アレルゲン豆腐の作製原料となる。 5, 東北124を用いて豆乳を作り、これを酵素処理して低アレルゲン化したものに多糖類を添加し、ゼリー風、あるいはプリン風にした食品を調製した。 6, 医師、栄養士、患者によるアレルゲン性評価チームを組織した。 7, 綿密な評価手法を確立し、試料1を用いてチャレンジテストを実施した。 8, 試料1に関しては約80%の患者が有効であると判定した。 9, 試料2,3についても中間段階の判定としてその有効性を示すデータを得たが、続けて評価を実行中である。

メチル化タンパク質由来 内囚性N^GN^G-dimethylarginine(ADMA)N^G^NG-dimethylarginine(SDMA),N^G-monomethylarginine(MMA)の内、ADMA,MMAが一酸化窒素産生酵素(NOS)の阻害剤として働き、高血圧などの病態の要因となる可能性がある。内因性のみならず食餌タンパク質由来の,ADMAやMMA量について検討した。一方、ADMA,MMA分解酵素ジメチルアリギニナーゼ(DDAH)のcDNAをヒト腎臓よりクローニングした。 1{方法} タンパク質中のMMA,ADMA量は微量であり、その分析法を最初に確立した。ABMAとSDMAはアルギニンの少し前に両者が分離して溶出させる。MMAはイオン交換樹脂法ではアルギニンとの分離が不可能であり、アルギナーゼ前処理によるアルギニン分解を行って、残存MMAを測定するシステムを構築した。2{結果} 日本人の主要タンパク質摂取源について分析を行い、タンパク質1g当たり、牛肉ではADMA 340nmol,MMA180nmol,鶏肉ではADMA 160nmol,MMA700nmol程度含まれることを明らかにした。これを食餌タンパク質1g当たりの平均値と仮定すると、日本人の一日のタンパク質摂取量が約80gであるから、ADMAとMMAを合わせて約40μmol程度摂取していると推定される。3{考察}我々の過去の研究から体組織中のADMAとMMAのプールは約1nmol/gと推定され50kg体重の人の場合,50μmolのプールと食餌由来の40μmolを合計して約100μmolとなり、生体内濃度は約100μmol/50kg≒2μMと推定される。一般にNOSに対する阻害定数(Ki)は数μM-数十μMであり、十分影響を与える濃度に達し、従って、DDAHによるプール濃度の調節によるNO産生の制御が意義を持つと判断される。

申請者は、成人病に関与する脂質性の食品成分の分子レベルでの役割と作用機構の解明を目指している。一般に、脂質成分の関与する反応の多くは生体膜周辺に存在するタンパク質(酵素)によって達成されるので、これらの研究にとって膜タンパク質の構造と機能の解析は不可欠である。しかしながら、可溶性タンパク質に比べ、膜タンパク質では精製や機能解析の技術はまだまだ困難である。本研究では、膜タンパク質の機能解析法と電気泳動法を用いた精製法の開発と応用を目的とした。昨年度は、電気泳動法、メンブランブロット法及び活性測定を組み合わせた膜タンパク質の活性同定法(Blot-Assay)を開発し、小胞体膜上に存在する膜タンパク質であるHMG-CoAレダクターゼの調節的分解のメカニズムや関与する膜結合プロテアーゼのキャラクタライゼイションを行った。本年度はNative PAGE、等電点電気泳動、SDS-PAGEの3つの電気泳動法を連続的に行うことによって可溶化した膜タンパク質(膜酵素)を同定・精製する方法を開発し、その方法を用いてこれまで精製の不可能であった膜酵素であるジアシルグリセロールリパーゼをヒト血小板膜画分から精製することに成功した。精製した本酵素の分子サイズは33kDaで、等電点は6.0であった。本酵素の諸性質を調べたところ、至適pHは7-8、活性はカルシウム非依存的で、セリン残基阻害剤やシステイン残基阻害剤によって活性は阻害された。したがって、活性発現にこれらの残基の存在が重要な役割を担っていることが示唆された。さらに、興味深いことには、本活性は細胞質型ホスホリパーゼA2に対する阻害剤で効果的に阻害され、これら2つの脂質加水分解酵素の構造や触媒機構の類似性が示唆された。

申請者らはヒト血小板において、イノシトールリン脂質代謝回転が、アラキドン酸遊離反応とクロストークし、密接に関わりあっていることを示唆してきた。本研究では、そのクロストークの分子機構の解明、およびその中心となる酵素反応系の性格づけと酵素分子の単離精製を試みることを目的とした。ヒト血小板をコラーゲンで刺激したとき、刺激初期の低Ca^<2+>条件下ではイノシトールリン脂質のみが分解され、アラキドン酸が遊離することを明らかにしている。この遊離経路を同定するために^<32>Pでラベルした血小板のリン脂質代謝を解析した。その結果、この条件下では、イノシトールリン脂質からホスホリパーゼC、ジアシルグリセロール(DG)リパーゼ、モノアシルグリセロール(MG)リパーゼによってアラキドン酸が遊離されることが明らかになった。さらに、膜画分からDGリパーゼおよびMGリパーゼを可溶化することに成功し、これらの酵素活性のCa^<2+>依存性について検討したところ、共に細胞内基底Ca^<2+>濃度条件下でほぼ最大活性が得られることが判明し、この酵素系が刺激初期の低Ca^<2+>濃度条件下で作動しうることを確認した。つぎに、刺激後期の細胞内Ca^<2+>濃度が上昇した条件下でのアラキドン酸遊離機構について検討した。その結果、刺激後期には、コラーゲンレセプターからの情報と細胞内Ca^<2+>濃度の上昇という2つのシグナルによって協調的に活性化されるホスホリパーゼA_2によって大量のアラキドン酸が遊離されることが明らかとなった。また、コラーゲン刺激の終期には、アラキドン酸代謝物であるトロンボキサンA_2が静止血小板へと働きかけるが、その場合はイノシトールリン脂質代謝回転は引き起こすが、アラキドン酸遊離反応は引き起こさないことを明らかにした。このようにして、イノシトールリン脂質代謝回転とアラキドン酸遊離反応のクロストークの経路、機構およびその多様性を明らかにすることができた。

外部情報が細胞内へ伝達される過程で、高度不飽和脂肪酸を結合したリン脂質分子種が重要な役割を果たしている。ヒト血小板において、コラーゲンなどの外部刺激により活性化されたホスホイノシチド特異的ホスホリパーゼC(PIPLC)により、ホスホイノシチドより生成したジアシルグリセロールからアラキドン酸が遊離し、エイコサノイドへ転換される分子機構の研究を行って来た。特に、ジアシルグルセロールリパーゼ及びモノアシルグルセロールリパーゼの役割を明らかにした。その一方で、PIPLCアイソザイムのうちPIPLC-αをラット肝臓小胞体から純化することに成功したが、このものはPIPLC活性を有さず、プロテアーゼ活性を持つことを発見した。そしてER-60プロテアーゼと命名した。そして、さらにER-72プロテアーゼん発見した。これらの酵素は小胞体に局在するタンパク質を基質としており、小胞体におけるタンパク質の質的かつ量的調節を行っている可能性を示した。これらの酵素に共通することはCGHCモチーフを有すること及びシステインプロテアーゼ阻害剤で活性の抑制を受けることである。その上、非常に興味深いことには、ER-60プロテアーゼ及びER-72プロテアーゼはホスファチジルイノシトールなどの酸性リン脂質によって活性調節を受けることであり、小胞体におけるリン脂質代謝とタンパク質代謝とのクロストークが明らかとなった。

細胞内における脂質代謝の調節機構を解明する