Intranasal administration of ceramide liposome suppresses allergic rhinitis by targeting CD300f in murine models.

Allergic rhinitis (AR) is caused by type I hypersensitivity reaction in the nasal tissues. The interaction between CD300f and its ligand ceramide suppresses immunoglobulin E (IgE)-mediated mast cell activation. However, whether CD300f inhibits the development of allergic rhinitis (AR) remains elusive. We aimed to investigate the roles of CD300f in the development of AR and the effectiveness of intranasal administration of ceramide liposomes on AR in murine models. We used ragweed pollen-induced AR models in mice. Notably, CD300f deficiency did not significantly influence the ragweed-specific IgE production, but increased the frequency of mast cell-dependent sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in our models. Similar results were also obtained for MCPT5-exprssing mast cell-specific loss of CD300f. Importantly, intranasal administration of ceramide liposomes reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in AR models. Thus, CD300f-ceramide interaction, predominantly in mast cells, alleviates the symptoms and progression of AR. Therefore, intranasal administration of ceramide liposomes may be a promising therapeutic approach against AR by targeting CD300f.

Sudden-Onset Severe Back Pain Caused by Acute Gastric Anisakiasis.

Anisakiasis is a parasitic disease that usually causes acute abdominal pain, nausea, and vomiting after the ingestion of raw seafood. We present a case of anisakiasis in an 80-year-old man who complained of sudden-onset severe back pain that was reminiscent of aortic dissection. This case shows that anisakiasis should be considered as a possible differential diagnosis in patients with not only abdominal pain but also back pain. In addition, for "diagnostic excellence," it is essential to return to a comprehensive medical history that allows the reassessment of the diagnosis even when it differs from the initial differential diagnosis.

Murine model identifies tropomyosin as IgE cross-reactive protein between house dust mite and coho salmon that possibly contributes to the development of salmon allergy.

Background: Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Material and methods: Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Results: Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Conclusion: Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.

Staphylococcus aureus δ-toxin present on skin promotes the development of food allergy in a murine model.

Background: Patients with food allergy often suffer from atopic dermatitis, in which Staphylococcus aureus colonization is frequently observed. Staphylococcus aureus δ-toxin activates mast cells and promotes T helper 2 type skin inflammation in the tape-stripped murine skin. However, the physiological effects of δ-toxin present on the steady-state skin remain unknown. We aimed to investigate whether δ-toxin present on the steady-state skin impacts the development of food allergy. Material and methods: The non-tape-stripped skins of wild-type, KitW-sh/W-sh, or ST2-deficient mice were treated with ovalbumin (OVA) with or without δ-toxin before intragastric administration of OVA. The frequency of diarrhea, numbers of jejunum or skin mast cells, and serum levels of OVA-specific IgE were measured. Conventional dendritic cell 2 (cDC2) in skin and lymph nodes (LN) were analyzed. The cytokine levels in the skin tissues or culture supernatants of δ-toxin-stimulated murine keratinocytes were measured. Anti-IL-1α antibody-pretreated mice were analyzed. Results: Stimulation with δ-toxin induced the release of IL-1α, but not IL-33, in murine keratinocytes. Epicutaneous treatment with OVA and δ-toxin induced the local production of IL-1α. This treatment induced the translocation of OVA-loaded cDC2 from skin to draining LN and OVA-specific IgE production, independently of mast cells and ST2. This resulted in OVA-administered food allergic responses. In these models, pretreatment with anti-IL-1α antibody inhibited the cDC2 activation and OVA-specific IgE production, thereby dampening food allergic responses. Conclusion: Even without tape stripping, δ-toxin present on skin enhances epicutaneous sensitization to food allergen in an IL-1α-dependent manner, thereby promoting the development of food allergy.

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A encoding the voltage-gated Ca2+ channel Cav1.1 or the voltage-gated Na+ channel Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal that separates external fluid and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, based on HEK293T cells and by using the Sleeping Beauty transposon system, we generated HypoPP-model cell lines that co-express the mouse inward-rectifier K+ channel (mKir2.1) and HypoPP2-associated Nav1.4 channel. Whole-cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarizes the membrane potential to levels comparable to those of myofibers, and that some Nav1.4 variants induce notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants by using a ratiometric pH indicator. Our optical method provides a potential in vitro platform for high-throughput drug screening, not only for HypoPP but also for other channelopathies caused by VSD mutations.

Molecular characterization of a flavanone 3-hydroxylase gene from citrus fruit reveals its crucial roles in anthocyanin accumulation.

BACKGROUND: Flavanone 3-hydroxylase (F3H), a key enzyme in the flavonoid biosynthetic pathway, plays an important role in the regulation of flavonols and anthocyanidins accumulation. Citrus fruit is a rich source of flavonoids with varied flavonoid compositions among different varieties. To date, the study on F3H is limited in citrus, and its roles in regulating flavonoid accumulation in citrus fruit are still unclear. RESULTS: In this study, we isolated a CitF3H from three different citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Ponkan mandarin (C. reticulata Blanco) and blood orange 'Moro' (C. sinensis Osbeck). Functional analysis showed that CitF3H encoded a functional flavanone 3-hydroxylase. It catalyzed the hydroxylation of naringenin to yield dihydrokaempferol, which was a precursor of anthocyanins in flavonoid biosynthetic pathway. In the juice sacs, CitF3H was differentially expressed among the three citrus varieties, and its expression level was positively correlated with the accumulation of anthocyanins during the ripening process. In the juice sacs of Satsuma mandarin and Ponkan mandarin the expression of CitF3H kept constant at an extremely low level, and no anthocyanin was accumulated during the ripening process. In contrast, the expression of CitF3H increased rapidly along with the accumulation of anthocyanin in the juice sacs of blood orange 'Moro' during the ripening process. In addition, we found that blue light irradiation was effective to up-regulate the expression of CitF3H and improve anthocyanin accumulation in the juice sacs of blood orange 'Moro' in vitro. CONCLUSION: CitF3H was a key gene regulating anthocyanin accumulation in the juice sacs of citrus fruit. The results presented in this study will contribute to elucidating anthocyanin biosynthesis in citrus fruit, and provide new strategies to improve the nutritional and commercial values of citrus fruit.

Development of mouse model for oral allergy syndrome to identify IgE cross-reactive pollen and food allergens: ragweed pollen cross-reacts with fennel and black pepper.

Oral allergy syndrome (OAS) is an IgE-mediated immediate food allergy that is localized to the oral mucosa. Pollen food allergy syndrome (PFAS), a pollinosis-associated OAS, is caused by cross-reactivity between food and pollen allergens. However, we need to more precisely understand the underlying pathogenesis of OAS/PFAS. In the present study, we developed a method to comprehensively identify cross-reactive allergens by using murine model of OAS and protein microarray technology. We focused on lip angioedema, which is one of the most common symptoms of OAS, and confirmed that mast cells reside in the tissues inside the lower lip of the mice. Interestingly, when the food allergen ovalbumin (OVA) was injected inside the lower lip of mice with high levels of OVA-specific IgE followed by an intravenous injection of the Evans blue dye, we found immediate dye extravasation in the skin of the neck in a mast cell-dependent manner. In addition, the degree of mast cell degranulation in the oral cavity, reflecting the severity of oral allergic responses, can be estimated by measuring the amount of extravasated dye in the skin. Therefore, we used this model of OAS to examine IgE cross-reactive allergens in vivo. Protein microarray analysis showed that serum IgE from mice intraperitoneally sensitized with ragweed pollen, one of the major pollens causing pollinosis, bound highly to protein extracts from several edible plants including black peppercorn and fennel. We confirmed that the levels of black pepper-specific IgE and fennel-specific IgE were significantly higher in the serum from ragweed pollen-sensitized mice than in the serum from non-sensitized control mice. Importantly, analysis of murine model of OAS showed that the injection of black pepper or fennel extract induced apparent oral allergic responses in ragweed pollen-sensitized mice. These results indicate IgE cross-reactivity of ragweed pollen with black pepper and fennel. In conclusion, we developed mouse model of OAS to identify IgE cross-reactive pollen and food allergens, which will help understand the pathogenesis of OAS/PFAS.

Mature Myotubes Generated From Human-Induced Pluripotent Stem Cells Without Forced Gene Expression.

Human-induced pluripotent stem cells (hiPSCs) are a promising tool for disease modeling and drug screening. To apply them to skeletal muscle disorders, it is necessary to establish mature myotubes because the onset of many skeletal muscle disorders is after birth. However, to make mature myotubes, the forced expression of specific genes should be avoided, as otherwise dysregulation of the intracellular networks may occur. Here, we achieved this goal by purifying hiPSC-derived muscle stem cells (iMuSC) by Pax7-fluorescence monitoring and antibody sorting. The resulting myotubes displayed spontaneous self-contraction, aligned sarcomeres, and a triad structure. Notably, the phenotype of sodium channels was changed to the mature type in the course of the differentiation, and a characteristic current pattern was observed. Moreover, the protocol resulted in highly efficient differentiation and high homogeneity and is applicable to drug screening.

Exogenous Application of ABA and NAA Alleviates the Delayed Coloring Caused by Puffing Inhibitor in Citrus Fruit.

Combined spraying of gibberellin (GA) and prohydrojasmon (PDJ) was an effective method to reduce peel puffing in Satsuma mandarins. However, in the GA-and-PDJ combined treatment, fruit color development was delayed during the ripening process. In the present study, to improve the coloration of the GA and PDJ-treated fruit, the effects of exogenous application of 1-naphthaleneacetic acid (NAA) and abscisic acid (ABA) on chlorophyll and carotenoid accumulation were investigated. The results showed that both ABA and NAA treatments accelerated the color changes from green to orange in the GA and PDJ-treated fruit during the ripening process. With the NAA and ABA treatments, chlorophylls contents were decreased rapidly, and the contents of ß,ß-xanthophylls were significantly enhanced in the GA and PDJ-treated fruit. In addition, gene expression results showed that the changes of the chlorophyll and carotenoid metabolisms in the NAA and ABA treatments were highly regulated at the transcriptional level. The results presented in this study suggested that the application of NAA and ABA could potentially be used for improving the coloration of the GA and PDJ-treated fruit.

Molecular Insights into the Ligand-Based Six-Proton- and Six-Electron-Transfer Processes Between Tris-ortho-Phenylenediamines and Tris-ortho-Benzoquinodiimines.

The global demand for energy and the concerns over climate issues renders the development of alternative renewable energy sources such as hydrogen (H2 ) important. A high-spin (hs) FeII complex with o-phenylenediamine (opda) ligands, [FeII (opda)3 ]2+ (hs-[6R]2+ ), was reported showing photochemical H2 evolution. In addition, a low-spin (ls) [FeII (bqdi)3 ]2+ (bqdi: o-benzoquinodiimine) (ls-[0R]2+ ) formation by O2 oxidation of hs-[6R]2+ , accompanied by ligand-based six-proton and six-electron transfer, revealed the potential of the complex with redox-active ligands as a novel multiple-proton and -electron storage material, albeit that the mechanism has not yet been understood. This paper reports that the oxidized ls-[0R][PF6 ]2 can be reduced by hydrazine giving ls-[FeII (opda)(bqdi)2 ][PF6 ]2 (ls-[2R][PF6 ]2 ) and ls-[FeII (opda)2 (bqdi)][PF6 ]2 (ls-[4R][PF6 ]2 ) with localized ligand-based proton-coupled mixed-valence (LPMV) states. The first isolation and characterization of the key intermediates with LPMV states offer unprecedented molecular insights into the design of photoresponsive molecule-based hydrogen-storage materials.

[Study of care practices for patients with myotonic dystrophy in Japan-Nationwide patient survey].

We conducted a comprehensive anonymous questionnaire survey on medical care and treatment for patients with myotonic dystrophy, who registered in the Japanese national registry (Remudy) or were undergoing care in seven hospitals specializing neuromuscular diseases. The questionnaire consisted of 49 questions were distributed to 813 patients, and 342 valid responses were collected. Most prevalent symptoms or complaints were dysfunction of fingers and fatigue. One-third of the adult patients left the job, half of which was due to the disease. Twelve percent of the patients did not visit the specialist regularly, the main reason being distance. The most common reason that the patients did not follow the advice of using a ventilator by medical professionals was lack of feeling the need. One-fourth of the adult female patients had infertility treatment, 80% of which was before a diagnosis of this disorder. This first-time nationwide survey revealed the actual condition of Japanese patients with myotonic dystrophy and raised various care-related issues.

OsRap2.6 transcription factor contributes to rice innate immunity through its interaction with Receptor for Activated Kinase-C 1 (RACK1).

BACKGROUND: The rice small GTPase OsRac1 is a molecular switch in rice innate immunity. The Receptor for Activated Kinase C-1 (RACK1) interacts with OsRac1 to suppress the growth of the rice blast fungus, Magnaporthe oryzae. RACK1 has two homologs in rice, RACK1A and RACK1B. Overexpressing RACK1A enhances resistance to the rice blast fungus. However, RACK1A downstream signals are largely unknown. RESULTS: Here, we report the identification of OsRap2.6, a transcription factor that interacts with RACK1A. We found a 94% similarity between the OsRap2.6 AP2 domain and Arabidopsis Rap2.6 (AtRap2.6). Bimolecular fluorescence complementation (BiFC) assays in rice protoplasts using tagged OsRap2.6 and RACK1A with the C-terminal and N-terminal fragments of Venus (Vc/Vn) indicated that OsRap2.6 and RACK1A interacted and localized in the nucleus and the cytoplasm. Moreover, OsRap2.6 and OsMAPK3/6 interacted in the nucleus and the cytoplasm. Expression of defense genes PAL1 and PBZ1 as well as OsRap2.6 was induced after chitin treatment. Disease resistance analysis using OsRap2.6 RNAi and overexpressing (Ox) plants infected with the rice blast fungus indicated that OsRap2.6 RNAi plants were highly susceptible, whereas OsRap2.6 Ox plants had an increased resistance to the compatible blast fungus. CONCLUSIONS: OsRap2.6 contributes to rice innate immunity through its interaction with RACK1A in compatible interactions.

The intermediate domain defines broad nucleotide selectivity for protein folding in Chlamydophila GroEL1.

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding.

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.