Molecular cloning and epitope analysis of the peanut allergen Ara h 3.
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Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions. (+info)
Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice.
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To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans. (+info)
Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of aspergillus flavus along a transect within the United States.
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Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B1 and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 micrometer in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 micrometer in diameter. The S-strain isolates generally produced high levels of aflatoxin B1, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B1 production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B1 and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 microgram of aflatoxin B1 per ml. The percentages of isolates producing >10 microgram of aflatoxin B1 per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B1 or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect. (+info)
Definitive diagnosis of nut allergy.
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OBJECTIVE: To compare findings of tests for nut allergy in children. DESIGN: Retrospective survey of a clinical practice protocol. SETTING: Children's hospital paediatric outpatient clinic. SUBJECTS: 96 children referred by general practitioners and accident and emergency doctors over 27 months (1994-96). MAIN OUTCOME MEASURES: Allergic manifestations (generalised urticarial rash, facial swelling, bronchospasm, anaphylactic shock, vomiting on three occasions) related to specific nut IgE concentrations and following touch, skin prick, or oral ingestion of nuts. RESULTS: 16 children from a sample of 51 who were tested for nut allergy had no reaction to an oral challenge. Positive IgE against peanuts was found in nine of these 16 children. CONCLUSIONS: Skin prick testing and IgE measured by radioallergosorbent testing are inadequate tests for nut allergy. The definitive diagnostic test for nut allergy in the hospital setting is direct oral challenge. (+info)
Optical imaging of odorant representations in the mammalian olfactory bulb.
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We adapted the technique of intrinsic signal imaging to visualize how odorant concentration and structure are represented spatially in the rat olfactory bulb. Most odorants activated one or more glomeruli in the imaged region of the bulb; these optically imaged responses reflected the excitation of underlying neurons. Odorant-evoked patterns were similar across animals and symmetrical in the two bulbs of the same animal. The variable sensitivity of individual glomeruli produced distinct maps for different odorant concentrations. Using a series of homologous aldehydes, we found that glomeruli were tuned to detect particular molecular features and that maps of similar molecules were highly correlated. These characteristics suggest that odorants and their concentrations can be encoded by distinct spatial patterns of glomerular activation. (+info)
The first triple gene block protein of peanut clump virus localizes to the plasmodesmata during virus infection.
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The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata. (+info)
Response to influenza vaccine in adjuvant 65-4.
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A comparison was made of the antibody response and subjective reactions to zonally-purified influenza vaccine in aqueous suspension and in peanut oil adjuvant 65-4. Both preparations contained 700 CCA units of A/Aichi/2/68, and 300 CCA units of B/Mass/1/71. Subjective reactions were recorded by asking the volunteers to complete a record daily for 5 days. Pain at the injection site was recorded by 64 per cent of the recipients of the oil adjuvant vaccine compared with 35 per cent of the aqueous recipients, but local redness was more frequent after aqueous vaccine. Systemic symptoms was recorded a little more frequently after aqueous than oil adjuvant vaccine. When measured 71/2 weeks after a single dose of vaccine, the HAI geometric mean antibody titre (G.M.T) to the A/Hong Kong/1/68 antigen (antigenically similar to the A/Aichi/2/68 antigen in the vaccine) increased 2-7 fold after aqueous and 16-4 fold after adjuvant vaccine. Sixty-two weeks after vaccination the antibody titres remained higher in those given adjuvant vaccine. The G.M.T. to B/Mass/1/71 increased 1-9 fold 71/2 weeks after aqueous vaccine and 3-7 fold after adjuvant vaccine. The antibody response to both influenza A and B antigens was broader in the recipients of adjuvant vaccine. The G.M.T. to A/England/42/72 increased 2-8-fold after aqueous and 13-fold after adjuvant vaccine; and to B/England/847/73 it increased 1-3-fold after aqueous and 1-9-fold after adjuvant vaccine. (+info)
Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.
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Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites. (+info)