Peach allergy is a common plant food allergy; specifically, sensitization to gibberellin-regulated protein (GRP; Pru p 7) and lipid-transfer protein (LTP; Pru p 3) is considered a predictor of severe allergic reactions in patients with peach allergy. However, the allergenic mechanisms in patients who are negative for these components remain unclear. Herein, we report the case of a 26-year-old Japanese man with a history of atopic dermatitis and pollinosis. The patient developed systemic allergic symptoms, including oral pruritus, pharyngeal swelling, respiratory discomfort, palpitations, and pallor, after ingesting a whole raw peach. Specific immunoglobulin E (IgE) to peach extract and Pru p 1 was positive, whereas specific IgE to Pru p 3, Pru p 4, and Pru p 7 was negative. Skin prick testing was positive for raw peach but negative for purified peach GRP. A proteomic approach was used to identify causative allergens. Two-dimensional electrophoresis and immunoblotting revealed an IgE-binding protein spot at approximately 25 kDa. Mass spectrometry identified the protein as peptidyl-prolyl cis-trans isomerase FKBP20-1 (FKBP20-1). Recombinant FKBP20-1 showed strong IgE reactivity with the patient’s serum but not with sera from nonallergic controls. Therefore, FKBP20-1 may represent a potential IgE-binding allergen involved in systemic reactions to peaches in patients who are not sensitized to GRP or LTP. Further studies are needed to clarify the clinical relevance of these findings.

Patients with peach allergy who experience severe symptoms, including anaphylaxis, reportedly have a higher positivity for peach gibberellin-regulated protein (GRP)-specific immunoglobulin (Ig) E than those with only oral symptoms. However, a study in Italy investigating apple allergy (another Rosaceae fruit) found no clear association between apple GRP-specific IgE levels and clinical disease types. This study aimed to evaluate the clinical utility of GRP-specific IgE measurement in Japanese patients with apple allergy. We collected sera from apple-allergic patients in Japan and measured their IgE levels specific to apple GRP. Apple-allergic patients (14 with oral reactions and 14 with systemic reactions) and seven non-allergic controls were examined. The specific IgE levels against apple, Mal d 1, Mal d 4, Japanese cedar, Japanese alder, Japanese white birch, Bet v 1, and Bet v 2 were also determined using 3gAllergy™. Positive results for apple-GRP-specific IgE by enzyme-linked immunosorbent assay were obtained in one patient with oral reactions and in seven cases of systemic reactions. Exercise as a cofactor was involved in cases with high apple GRP-specific IgE. GRP expression was considerably lower in apples than in peaches, as detected by reverse transcription-quantitative polymerase chain reaction testing. Thus, GRP-specific IgE may be an important marker for diagnosing systemic reactions triggered by exercise in fruits with low GRP expression, such as apples.

Bacteriophage T4 gene 32 protein (gp32) preferentially binds to single-stranded DNA (ssDNA) to facilitate DNA replication but shows weak binding to double-stranded DNA (dsDNA). Polyclonal and monoclonal antibodies against gp32 were raised, and an enzyme-linked immunosorbent assay was used to evaluate their reactivities against gp32. The reactivity of the monoclonal antibody MGP45 was diminished in the presence of 5 ng/mL dsDNA, suggesting a conformational change that reduces epitope availability. Notably, the same concentration of ssDNA had little effect; instead, 500 ng/mL ssDNA was required to elicit the same degree of inhibition. A decrease in MGP45 reactivity with gp32 was observed in the presence of NaCl at concentrations less than 100 m m under neutral conditions. These changes in antibody reactivity reflect differences in the gp32 conformation, which may underlie its different affinities for ssDNA and dsDNA.

It has been reported that patients with egg allergy can ingest baked eggs without the induction of allergy symptoms. Ovomucoid (OM) is a soluble protein that is resistant to heat; however, OM in baked eggs becomes insoluble. The aim of this study was to analyze the digestibility of OM and its properties (including solubility) to clarify the mechanism by which changes in its properties remove its capacity to induce allergic symptoms. We prepared purified OM, heated OM, non-baked OM, and baked OM and digested them using pepsin, trypsin, and chymotrypsin. We analyzed the digested samples using the Lowry method, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. The soluble protein concentrations of purified, heated, and non-baked OM remained unchanged following digestion. The soluble protein concentration in the supernatant of the digested, baked OM was low; however, in pepsin and chymotrypsin digestion, the soluble protein concentration increased during digestion. The immunoblotting results showed that the protein bands detected in the digested supernatant of baked OM were thinner than those of purified, heated, and non -baked OM, even though the protein concentrations were the same. Furthermore, intact OM was detected in the precipitate of baked OM. In trypsin digestion, little change was observed after digestion in either sample. This study suggests that baked OM may have lower antibody binding capacity than other samples, and intact OM remains in the precipitate, which may affect allergy symptom induction.Effect of changes in the properties of ovomucoid on its digestibility

Collagens are abundant structural proteins found in both mammalian and marine species, and attractive biomaterials used in various fields. Jellyfish collagen-based products have become increasingly popular because of their clinically proven health benefits such as the effects of skin wound healing and immune stimulation. To develop detection tools for jellyfish collagen, we generated four monoclonal antibodies, MCOL1, 2, 3, and 4, by immunizing mice with moon jellyfish collagen. The nucleotide and amino acid sequences of the variable regions of the monoclonal antibodies were determined. The antibody-binding kinetics toward collagens from moon jellyfish were evaluated using a surface plasmon resonance (SPR) biosensor, and the binding specificity was evaluated in comparison with binding to collagens from edible jellyfish, fish scales, and pig and cow skins. MCOL1, 3, and 4 specifically bound to moon jellyfish collagen, whereas MCOL2 bound to both moon and edible jellyfish collagens. Considering the results showing that the SPR responses of MCOL2 binding were greater than those seen with the other antibodies, MCOL2 could recognize the common and repetitive sequences of the two jellyfish collagens. Therefore, this monoclonal antibody will be most applicable for detecting jellyfish collagen.

Gibberellin-regulated protein (GRP) is a fruit severe allergen. The amounts of GRP expression normalized against actin in peach were determined by reverse transcription-quantitative PCR (RT-qPCR). The results were consistent with those determined by enzyme-linked immunosorbent assay (ELISA). The GRP expression was more evident in flesh than peel and increased rapidly in the maturing period. This approach is applicable to estimate the amount of GRP in other plants.

In this study, monoclonal antibodies against two major fruit allergens—gibberellin-regulated protein (GRP) and lipid transfer protein (LTP)—were established. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantification of peach GRP and LTP were constructed using these antibodies. Both ELISAs reacted with the respective antigens when heated at 100ºC for 20 min, but not when reduced with sodium sulfite, indicating that GRP and LTP are heat-stable, while disulfide bonds play an important role in their native steric structures. GRP and LTP in peaches and peach-containing foods were quantified by these ELISAs. In both cases, there were few differences among peach cultivars normally available on the market; however, concentrations were higher when the peach was ripe. GRP was localized in the pulp of the peach, while LTP was present in the peel. They could be quantified in peach-containing beverages, as well as in dried and canned peaches. GRP in Japanese apricots could also be determined using this ELISA, as its amino acid sequence is the same as that of peach GRP. Then, high concentrations of GRP were detected in umeboshi, a traditional Japanese pickled apricot. Peach leaves were found to have a high LTP content, accordingly, LTP was also observed in lotions containing peach leaf extract. The ability to quantitatively detect GRP and LTP in this study will, therefore, contribute to the improvement of component-resolved diagnoses and quality of life in patients allergic to peaches.

BACKGROUND:The Rosaceae family includes fruits, such as peach, apple, Japanese apricot, cherry (Prunoideae subfamily), and strawberry (Rosoideae subfamily). The allergens responsible for Rosaceae fruit allergies have been reported to include Bet v 1 and profilin, which mainly cause oral symptoms, and lipid transfer protein (LTP). Recently, gibberellin-regulated protein (GRP) has been identified as an allergen that induces generalized symptoms in peach-, orange-, and plum-related allergies. Most patients with food allergies induced by GRP show allergic symptoms accompanied by cofactors, such as exercise or drugs. To date, there are very few reports of generalized symptoms induced by strawberry.CASE PRESENTATION:We evaluated the reactivity of strawberry GRP in a 15-year-old boy who was confirmed to have generalized symptoms induced by strawberry with exercise using an oral food challenge test (OFCT). The patient's serum exhibited a strong positive reaction to strawberry GRP but not to peach GRP or peach LTP. The patient's basophils reacted to strawberry and peach GRP but not to peach LTP.CONCLUSIONS:Strawberry GRP may be a causative component for strawberry with exercise-induced generalized symptoms in this patient. This is the first study to investigate the role of GRP in strawberry with cofactor-induced allergic symptoms. Further epidemiological and clinical researches are necessary to improve diagnostic and therapeutic approaches for patients with strawberry allergy.

Pediatric Allergy and ImmunologyVolume 33, Issue 9 e13850 CLINICAL LETTERS A case of food-dependent exercise-induced anaphylaxis due to grape gibberellin-regulated protein Takae Kobayashi, Corresponding Author Takae Kobayashi takae0824@hotmail.co.jp orcid.org/0000-0002-6617-0227 Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, Japan Correspondence Takae Kobayashi, Department of Pediatrics, Kasugai Municipal Hospital, 1-1-1, Takaki-cho, Kasugai, Aichi 486-8510, Japan. Email: takae0824@hotmail.co.jpSearch for more papers by this authorNaoshi Shimojo, Naoshi Shimojo Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, Japan General Research and Development Institute, Hoyu Co., Ltd., Nagakute, JapanSearch for more papers by this authorMasashi Nakamura, Masashi Nakamura Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, Japan General Research and Development Institute, Hoyu Co., Ltd., Nagakute, JapanSearch for more papers by this authorKayoko Matsunaga, Kayoko Matsunaga Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, JapanSearch for more papers by this authorJoon Nakata, Joon Nakata Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, JapanSearch for more papers by this authorKazunori Tagami, Kazunori Tagami Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, JapanSearch for more papers by this authorKeiko Momma, Keiko Momma Department of Food and Nutrition, Kyoto Women's University, Kyoto, JapanSearch for more papers by this authorHiroshi Narita, Hiroshi Narita Department of Food and Nutrition, Kyoto Women's University, Kyoto, Japan Department of Food Science, Kyoto College of Nutritional & Medical Sciences, Kyoto, JapanSearch for more papers by this authorYasuto Kondo, Yasuto Kondo Department of Pediatrics, Fujita Health University School of Medicine, Nagoya, Japan Fujita Health University General Allergy Center, Bantane Hospital, Nagoya, JapanSearch for more papers by this author Takae Kobayashi, Corresponding Author Takae Kobayashi takae0824@hotmail.co.jp orcid.org/0000-0002-6617-0227 Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, Japan Correspondence Takae Kobayashi, Department of Pediatrics, Kasugai Municipal Hospital, 1-1-1, Takaki-cho, Kasugai, Aichi 486-8510, Japan. Email: takae0824@hotmail.co.jpSearch for more papers by this authorNaoshi Shimojo, Naoshi Shimojo Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, Japan General Research and Development Institute, Hoyu Co., Ltd., Nagakute, JapanSearch for more papers by this authorMasashi Nakamura, Masashi Nakamura Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, Japan General Research and Development Institute, Hoyu Co., Ltd., Nagakute, JapanSearch for more papers by this authorKayoko Matsunaga, Kayoko Matsunaga Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, Nagoya, JapanSearch for more papers by this authorJoon Nakata, Joon Nakata Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, JapanSearch for more papers by this authorKazunori Tagami, Kazunori Tagami Department of Pediatrics, Kasugai Municipal Hospital, Kasugai, JapanSearch for more papers by this authorKeiko Momma, Keiko Momma Department of Food and Nutrition, Kyoto Women's University, Kyoto, JapanSearch for more papers by this authorHiroshi Narita, Hiroshi Narita Department of Food and Nutrition, Kyoto Women's University, Kyoto, Japan Department of Food Science, Kyoto College of Nutritional & Medical Sciences, Kyoto, JapanSearch for more papers by this authorYasuto Kondo, Yasuto Kondo Department of Pediatrics, Fujita Health University School of Medicine, Nagoya, Japan Fujita Health University General Allergy Center, Bantane Hospital, Nagoya, JapanSearch for more papers by this author First published: 08 September 2022 https://doi.org/10.1111/pai.13850 Editor: Carmen Riggioni Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Supporting Information Filename Description pai13850-sup-0001-AppendixS1.docxWord 2007 document , 28.7 KB Appendix S1 pai13850-sup-0002-FiguresS1 and S2.docxWord 2007 document , 1.4 MB Figures S1 and S2 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume33, Issue9September 2022e13850 RelatedInformation

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.

BACKGROUND Pollen-food allergy syndrome (PFAS) usually manifests as an itching sensation in the mouth and throat immediately after eating fresh fruits and vegetables. However, some patients with PFAS experience systemic symptoms including anaphylaxis. In Europe, cypress gibberellin-regulated protein (GRP) has been noted to cause allergenicity and exhibit cross-reactivity with peach GRP. Japanese cedar (Cryptomeria japonica), classified in the cypress family, is the primary causative substance among all environmental allergens in Japan. However, studies on the prevalence of GRP sensitization in patients with cedar pollinosis are lacking. OBJECTIVE This study examined the prevalence of GRP sensitization in patients with Japanese cedar pollinosis. METHODS We enrolled 52 patients who had requested sublingual immunotherapy treatment with mild-to-severe rhinitis during spring, and had a JCP-specific immunoglobulin E (IgE) levels of >0.7 UA/mL. Peach GRP was purified using affinity chromatography with a monoclonal antibody column. Specific IgE levels to peach GRP were measured using an enzyme-linked immunosorbent assay. Samples exhibiting absorbance at 450 nm of over mean plus three standard deviations of the negative control value were defined as positive. Sera from three patients with severe peach allergy were used as positive controls. RESULTS Eleven sera from 52 patients with JCP-induced allergic rhinitis were positive for peach GRP. CONCLUSION Twenty percent of patients with cedar pollinosis were sensitized to peach GRP. Well-powered studies are needed to clarify whether these patients are at an increased risk for systemic symptoms or whether they primarily demonstrate only localized symptoms.

Melanocortin-4 receptor (MC4R) is a critical regulator of appetite and energy expenditure in rodents and humans. MC4R deficiency causes hyperphagia, reduced energy expenditure, and impaired glucose metabolism. Ligand binding to MC4R activates adenylyl cyclase, resulting in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a secondary messenger that regulates several cellular processes. Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cAMP and is reportedly involved in obesity. However, the precise mechanism through which CRTC1 regulates energy metabolism remains unknown. Additionally, there are no reports linking CRTC1 and MC4R, although both CRTC1 and MC4R are known to be involved in obesity. Here, we demonstrate that mice lacking CRTC1, specifically in MC4R cells, are sensitive to high-fat diet (HFD)-induced obesity and exhibit hyperphagia and increased body weight gain. Moreover, the loss of CRTC1 in MC4R cells impairs glucose metabolism. MC4R-expressing cell-specific CRTC1 knockout mice did not show changes in body weight gain, food intake, or glucose metabolism when fed a normal-chow diet. Thus, CRTC1 expression in MC4R cells is required for metabolic adaptation to HFD with respect to appetite regulation. Our results revealed an important protective role of CRTC1 in MC4R cells against dietary adaptation.

The melanocortin 4 receptor (MC4R) plays an important role in the regulation of appetite and energy expenditure in humans and rodents. Impairment of MC4R signaling causes severe obesity. MC4R mainly couples to the G-protein Gs. Ligand binding to MC4R activates adenylyl cyclase resulting in increased intracellular cAMP levels. cAMP acts as a secondary messenger, regulating various cellular processes. MC4R can also couple with Gq and other signaling pathways. Therefore, the contribution of MC4R/Gs signaling to energy metabolism and appetite remains unclear. To study the effect of Gs signaling activation in MC4R cells on whole body energy metabolism and appetite, we generated a novel mouse strain that expresses a Gs-coupled designer receptors exclusively activated by designer drugs [Gs-DREADD (GsD)] selectively in MC4R-expressing cells (GsD-MC4R mice). Chemogenetic activation of the GsD by a designer drug [deschloroclozapine (DCZ); 0.01∼0.1 mg/kg body wt] in MC4R-expressing cells significantly increased oxygen consumption and locomotor activity. In addition, GsD activation significantly reduced the respiratory exchange ratio, promoting fatty acid oxidation, but did not affect core (rectal) temperature. A low dose of DCZ (0.01 mg/kg body wt) did not suppress food intake, but a high dose of DCZ (0.1 mg/kg body wt) suppressed food intake in MC4R-GsD mice, although either DCZ dose (0.01 or 0.1 mg/kg body wt) did not affect food intake in the control mice. In conclusion, the current study demonstrated that the stimulation of Gs signaling in MC4R-expressing cells increases energy expenditure and locomotor activity and suppresses appetite.NEW & NOTEWORTHY We report that Gs signaling in melanocortin 4 receptor (MC4R)-expressing cells regulates energy expenditure, appetite, and locomotor activity. These findings shed light on the mechanism underlying the regulation of energy metabolism and locomotor activity by MC4R/cAMP signaling.

Several immunoassays for monitoring pork contamination have been developed, but they have limitations in accurate detection because they have been affected by protein denaturation and loss of extraction efficiency due to cooking. In this study, a sandwich enzyme-linked immunosorbent assay (s-ELISA) combined with an extraction method using sodium dodecyl sulfate (SDS) was developed for pork determination in raw and heated meats. To establish monoclonal antibodies (mAbs), SDS-denatured porcine myoglobin (Mb) and synthetic peptides with amino acid sequences of porcine Mb were used for immunization. The s-ELISA quantitatively detected the porcine Mb without cross-reaction to beef and chicken Mbs and lamb and goat meats. The 50% maximal effective concentration of porcine Mb for s-ELISA was 90 ng/mL, and the recoveries of porcine Mb spiked into raw and heated beef and chicken were 94-158%. This s-ELISA detected 1% (w/w) pork mixed with raw and heated beef. For the determination of Mbs in various parts of raw pork, including fatty bellies, the results correlated well with those obtained by high-performance liquid chromatography. Our s-ELISA could be available for authentication of meat products and prevention of mislabeling in pork products.

A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.