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Eva Vincze - One of the best experts on this subject based on the ideXlab platform.
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Effects of Zn Fertilization on Hordein Transcripts at Early Developmental Stage of Barley Grain and Correlation with Increased Zn Concentration in the Mature Grain
2016Co-Authors: Mohammad Nasir Uddin, Agnieszka Kaczmarczyk, Eva VinczeAbstract:Zinc deficiency is causing malnutrition for nearly one third of world populations. It is especially relevant in cereal-based diets in which low amounts of mineral and protein are present. In biological systems, Zn is mainly associated with protein. Cereal grains contain the highest Zn concentration during early developmental stage. Although Hordeins are the major storage proteins in the mature barley grain and suggested to be involved in Zn binding, very little information is available regarding the Zn fertilization effects of Hordein transcripts at early developmental stage and possible incorporation of Zn with Hordein protein of matured grain. Zinc fertilization experiments were conducted in a greenhouse with barley cv. Golden Promise. Zn concentration of the matured grain was measured and the results showed that the increasing Zn fertilization increased grain Zn concentration. Quantitative real time PCR showed increased level of total Hordein transcripts upon increasing level of Zn fertilization at 10 days after pollination. Among the Hordein transcripts the amount of B-Hordeins was highly correlated with the Zn concentration of matured grain. In addition, protein content of the matured grain was analysed and a positive linear relationship was found between the percentage of B-Hordein and total grain Zn concentration while C-Hordein level decreased. Zn sensing dithizone assay was applied to localize Zn in the matured grain. The Zn distribution was not limited to the embryo and aleurone layer but was also present in the outer part of the endosperm (sub-aleurone layers) which known to be rich in proteins including B-Hordeins. Increased Zn fertilization enriched Zn even in the endosperm
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Lysine metabolism in antisense C-Hordein barley grains.
Plant physiology and biochemistry : PPB, 2014Co-Authors: Daiana Schmidt, Eva Vincze, Vanessa Rizzi, Salete Aparecida Gaziola, Leonardo Oliveira Medici, Marcin Kozak, Peter J. Lea, Ricardo Antunes AzevedoAbstract:The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the Hordeins, especially in the C-Hordein subgroup. Previously produced antisense C-Hordein transgenic barley lines have an improved amino acid composition, with increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise) and five antisense C-Hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-Hordein suppression did not only alter the metabolism of at least one aspartate-derived amino acid (threonine), but major changes were also detected in the metabolism of lysine and methionine. Modifications in the activities and regulation of aspartate kinase, dihydrodipicolinate synthase and homoserine dehydrogenase were observed in most transgenic lines. Furthermore the activities of lysine α-ketoglutarate reductase and saccharopine dehydrogenase were also altered, although the extent varied among the transgenic lines.
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Effects of Zn fertilization on Hordein transcripts at early developmental stage of barley grain and correlation with increased Zn concentration in the mature grain.
PloS one, 2014Co-Authors: Mohammad Nasir Uddin, Agnieszka Kaczmarczyk, Eva VinczeAbstract:Zinc deficiency is causing malnutrition for nearly one third of world populations. It is especially relevant in cereal-based diets in which low amounts of mineral and protein are present. In biological systems, Zn is mainly associated with protein. Cereal grains contain the highest Zn concentration during early developmental stage. Although Hordeins are the major storage proteins in the mature barley grain and suggested to be involved in Zn binding, very little information is available regarding the Zn fertilization effects of Hordein transcripts at early developmental stage and possible incorporation of Zn with Hordein protein of matured grain. Zinc fertilization experiments were conducted in a greenhouse with barley cv. Golden Promise. Zn concentration of the matured grain was measured and the results showed that the increasing Zn fertilization increased grain Zn concentration. Quantitative real time PCR showed increased level of total Hordein transcripts upon increasing level of Zn fertilization at 10 days after pollination. Among the Hordein transcripts the amount of B-Hordeins was highly correlated with the Zn concentration of matured grain. In addition, protein content of the matured grain was analysed and a positive linear relationship was found between the percentage of B-Hordein and total grain Zn concentration while C-Hordein level decreased. Zn sensing dithizone assay was applied to localize Zn in the matured grain. The Zn distribution was not limited to the embryo and aleurone layer but was also present in the outer part of the endosperm (sub-aleurone layers) which known to be rich in proteins including B-Hordeins. Increased Zn fertilization enriched Zn even in the endosperm. Therefore, the increased amount of B-Hordein and decreased C-Hordein content suggested that B-Hordein upregulation or difference between B and C Hordein could be one of the key factors for Zn biofortification of cereal grains due to the Zn fertilization.
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Effects of Zn treatments on percentage of different Hordeins of matured grain measured from image analyses of SDS-PAGE gel.
2014Co-Authors: Mohammad Nasir Uddin, Agnieszka Kaczmarczyk, Eva VinczeAbstract:In the figure B, C, D, γ and TI refers as B-Hordein, C-Hordeins, D-Hordeins, γ- Hordeins and A-Hordeins/Trypsin inhibitors/alpha amylase inhibitors respectively. The calculations are based on the biological replicates mentioned in table 1 and presented as means ± SE. A) Percentage of different Hordeins after different Zn fertilization; Zn treatment labels: low - blue; medium - red; high - green. B) % of [(B+γ+D)–C]-Hordein after different Zn fertilization.
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Effects of Zn fertilizations (low - blue; medium - red; high – green) on total amount of Hordein transcripts (the sum of all Hordeins) measured in amol of Hordein/amol of actin.
2014Co-Authors: Mohammad Nasir Uddin, Agnieszka Kaczmarczyk, Eva VinczeAbstract:Effects of Zn fertilizations (low - blue; medium - red; high – green) on total amount of Hordein transcripts (the sum of all Hordeins) measured in amol of Hordein/amol of actin.
Adela Olmedilla - One of the best experts on this subject based on the ideXlab platform.
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Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development
Journal of Experimental Botany, 2006Co-Authors: Ángel Pulido, Ferenc Bakos, Martine Devic, Alberto Hernando, Beáta Barnabás, Enrique Méndez, Adela OlmedillaAbstract:Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of Hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time Hordein expression during microspore embryogenesis. Surprisingly, these Hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of Hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.
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Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development
Journal of Experimental Botany, 2006Co-Authors: Ángel Pulido, Ferenc Bakos, Martine Devic, Alberto Hernando, Beáta Barnabás, Enrique Méndez, Adela OlmedillaAbstract:Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of Hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time Hordein expression during microspore embryogenesis. Surprisingly, these Hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of Hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.
Crispin A. Howitt - One of the best experts on this subject based on the ideXlab platform.
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Proteome Analysis of Hordein-Null Barley Lines Reveals Storage Protein Synthesis and Compensation Mechanisms.
Journal of agricultural and food chemistry, 2020Co-Authors: Utpal Bose, Keren Byrne, Crispin A. Howitt, Malcolm Blundell, James A. Broadbent, Michelle L. ColgraveAbstract:Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the Hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single Hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in Hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the Hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.
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Hordein Accumulation in Developing Barley Grains.
Frontiers in plant science, 2019Co-Authors: Gregory J. Tanner, Michelle L. Colgrave, Crispin A. Howitt, Malcolm Blundell, Antony BacicAbstract:The temporal pattern of accumulation of Hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from six days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of Hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic Hordein peptides representing all classes of Hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-Hordeins decreased significantly by 21-51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterised Hordeins, two novel barley B-Hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-Hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins are due to the Hordein family and the final Hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of Hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.
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Data_Sheet_3_Hordein Accumulation in Developing Barley Grains.xlsx
2019Co-Authors: Gregory J. Tanner, Michelle L. Colgrave, Crispin A. Howitt, Malcolm J. Blundell, Antony BacicAbstract:The temporal pattern of accumulation of Hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of Hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic Hordein peptides representing all classes of Hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-Hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized Hordeins, two novel barley B-Hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-Hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the Hordein family and the final Hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of Hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.
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Data_Sheet_1_Hordein Accumulation in Developing Barley Grains.pdf
2019Co-Authors: Gregory J. Tanner, Michelle L. Colgrave, Crispin A. Howitt, Malcolm J. Blundell, Antony BacicAbstract:The temporal pattern of accumulation of Hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of Hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic Hordein peptides representing all classes of Hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-Hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized Hordeins, two novel barley B-Hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-Hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the Hordein family and the final Hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of Hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.
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Liquid Chromatography–Mass Spectrometry Analysis Reveals Hydrolyzed Gluten in Beers Crafted To Remove Gluten
Journal of Agricultural and Food Chemistry, 2017Co-Authors: Michelle L. Colgrave, Keren Byrne, Crispin A. HowittAbstract:During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values 30 kDa in size. Barley gluten (Hordeins) were detected in all beers analyzed with peptides representing all Hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion...
Ángel Pulido - One of the best experts on this subject based on the ideXlab platform.
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Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development
Journal of Experimental Botany, 2006Co-Authors: Ángel Pulido, Ferenc Bakos, Martine Devic, Alberto Hernando, Beáta Barnabás, Enrique Méndez, Adela OlmedillaAbstract:Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of Hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time Hordein expression during microspore embryogenesis. Surprisingly, these Hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of Hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.
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Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development
Journal of Experimental Botany, 2006Co-Authors: Ángel Pulido, Ferenc Bakos, Martine Devic, Alberto Hernando, Beáta Barnabás, Enrique Méndez, Adela OlmedillaAbstract:Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of Hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time Hordein expression during microspore embryogenesis. Surprisingly, these Hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of Hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.
Michel Laurière - One of the best experts on this subject based on the ideXlab platform.
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Study of IgE antigenic relationships in hypersensitivity to hydrolyzed wheat proteins and wheat-dependent exercise-induced anaphylaxis.
International Archives of Allergy and Immunology, 2006Co-Authors: Jacques Snégaroff, Isabelle Bouchez-mahiout, Catherine Pecquet, Gerard Branlard, Michel LaurièreAbstract:BACKGROUND: Wheat is involved in different forms of respiratory, food and contact allergy. The IgE of patients generally reacts with various flour proteins. It is not known if antigenic relationships could explain some of these reactions and if proteins could be involved in different pathologies. METHODS: Two sera were selected as representative of patients with either wheat-dependent exercise-induced anaphylaxis (WDEIA) or hypersensitivity to hydrolyzed wheat proteins (HHWP). Their IgE specificity was studied with wheat, barley and rye proteins, using immunoblot, and immunoblot inhibition with recombinant gamma-3 Hordein. This protein was chosen for its cross-reactivity with omega-5 gliadin, a major allergen in WDEIA. RESULTS: The IgE from both sera strongly reacted with natural and recombinant gamma-3 Hordein but displayed different patterns of reactivity with wheat, barley and rye proteins. Those from the WDEIA patient showed expected reactions with omega-5 gliadin, gamma-35 and gamma-75 secalins, but also with wheat low-molecular-weight glutenin subunits (LMW-GS), and not with C Hordeins. On the contrary, IgE from a HHWP patient reacted with C Hordeins, various omega gliadins, and gamma-75 secalin, but very weakly with gamma-35 secalin and LMW-GS. Recombinant gamma-3 Hordein inhibited strongly but not totally the WDEIA patient's IgE binding to prolamins. No such inhibition could be observed for the HHWP patient's IgE. CONCLUSIONS: At least part of the reactions of prolamins with the IgE from the WDEIA patient was due to antigenic homologies. The occurrence of cross-reacting carbohydrates was unlikely. These common IgE epitopes were not involved in the pathology of the HHWP patient.
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Study of IgE antigenic relationships in hyper sensitivity to hydrolyzed wheat proteins and wheat-dependent exercise-induced anaphylaxis
International Archives of Allergy and Immunology, 2006Co-Authors: Jacques Snégaroff, Isabelle Bouchez-mahiout, Catherine Pecquet, Gerard Branlard, Michel LaurièreAbstract:Background: Wheat is involved in different forms of respiratory, food and contact allergy. The IgE of patients generally reacts with various flour proteins. It is not known if antigenic relationships could explain some of these reactions and if proteins could be involved in different pathologies. Methods: Two sera were selected as representative of patients with either wheat-dependent exercise-induced anaphylaxis (WDEIA) or hypersensitivity to hydrolyzed wheat proteins (HHWP). Their IgE specificity was studied with wheat, barley and rye proteins, using immunoblot, and immunoblot inhibition with recombinant -3 Hordein. This protein was chosen for its cross-reactivity with -5 gliadin, a major allergen in WDEIA. Results: The IgE from both sera strongly reacted with natural and recombinant -3 Hordein but displayed different patterns of reactivity with wheat, barley and rye proteins. Those from the WDEIA patient showed expected reactions with -5 gliadin, -35 and -75 secalins, but also with wheat low-molecular-weight glutenin subunits (LMW-GS), and not with C Hordeins. On the contrary, IgE from a HHWP patient reacted with C Hordeins, various gliadins, and -75 secalin, but very weakly with -35 secalin and LMW-GS. Recombinant -3 Hordein inhibited strongly but not totally the WDEIA patient's IgE binding to prolamins. No such inhibition could be observed for the HHWP patient's IgE. Conclusions: At least part of the reactions of prolamins with the IgE from the WDEIA patient was due to antigenic homologies. The occurrence of cross-reacting carbohydrates was unlikely. These common IgE epitopes were not involved in the pathology of the HHWP patient.