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Food and Chemical Toxicology : An... May 2023Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate...
Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.
Topics: Animals; Female; Ovomucin; Chickens; Transcription Activator-Like Effector Nucleases; Allergens; Egg Hypersensitivity
PubMed: 36889429
DOI: 10.1016/j.fct.2023.113703 -
Poultry Science Oct 2021Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in...
Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.
Topics: Animals; Chickens; Fallopian Tubes; Female; Humans; Ovalbumin; Oviducts; Promoter Regions, Genetic; Transgenes
PubMed: 34375836
DOI: 10.1016/j.psj.2021.101365 -
Nutrients Feb 2023Food allergy is one of the major existing health problems, but no effective treatment is available. In the current work, a murine model that closely mimics pathogenesis...
A Murine Model of Food Allergy by Epicutaneous Adjuvant-Free Allergen Sensitization Followed by Oral Allergen Challenge Combined with Aspirin for Enhanced Detection of Hypersensitivity Manifestations and Immunotherapy Monitoring.
Food allergy is one of the major existing health problems, but no effective treatment is available. In the current work, a murine model that closely mimics pathogenesis of human food allergy and its quantifiable diagnostic parameter design, even for mild hypersensitivity reactions, were established. BALB/c mice were epicutaneously sensitized with 1 mg chicken egg ovomucoid (OVM) or cow's milk casein, free of adjuvants, five times a week for two consecutive weeks. Eleven days later, allergen-specific IgG1 and IgE in serum were measured by ELISA. On day 25, 20 mg OVM or 12 mg α-casein was administered orally, and allergic reactions such as the fall in rectal temperature, symptom scores during 90-120 min, serum mast cell protease-1 and cytokine levels were monitored. The detection of mild allergic reactions due to adjuvant-free allergen sensitization and oral allergen challenge routes was amplified by the combination of oral allergen and aspirin administration simultaneously or aspirin administration within 15-30 min before an allergen challenge. Quantification of the maximum symptom score and the frequency of symptoms during the monitoring period improved evaluation accuracy of food allergy signals. Based on these results, efficacy of casein oral immunotherapy for cow's milk allergies, which are generally difficult to detect, was monitored adequately.
Topics: Humans; Female; Cattle; Mice; Animals; Allergens; Caseins; Aspirin; Disease Models, Animal; Food Hypersensitivity; Milk Hypersensitivity; Adjuvants, Immunologic; Ovomucin; Immunotherapy
PubMed: 36771462
DOI: 10.3390/nu15030757 -
The Journal of Physical Chemistry. B Sep 2011The interaction among each of three dilute "tracer" proteins (bovine serum albumin, superoxide dismutase, and ovomucoid) at a concentration of 2 mg/mL and each of two...
The interaction among each of three dilute "tracer" proteins (bovine serum albumin, superoxide dismutase, and ovomucoid) at a concentration of 2 mg/mL and each of two "crowder" proteins (ovomucoid and BSA) at concentrations up to 100 mg/mL was characterized by analysis of dependence of the equilibrium gradients of both tracer and crowder upon the concentration of crowder. The equilibrium gradients of both crowder proteins were found to be independent of temperature over the range 5-37 °C. The equilibrium gradients of tracer BSA and ovomucoid in the complementary crowder species were likewise found to be independent of temperature over this range, indicating that interaction among these tracers and crowders is predominantly repulsive and essentially entirely entropic in nature. The equilibrium gradient of tracer SOD in BSA was also found to be independent of temperature over this range, but the gradient of tracer SOD in ovomucoid depended significantly upon temperature in a manner indicating a significant enthalpic (attractive) component of the overall interaction between SOD and ovomucoid. The experimental data are analyzed using model-free expansions of the thermodynamic activity coefficients of tracer and crowder in powers of the concentration of crowder and using approximate statistical thermodynamic models based upon highly simplified descriptions of molecular structure and interactions. Detailed analysis of the results indicates a relatively small contribution of nonspecific attraction to the total protein-protein interaction, which is dominated by steric repulsion.
Topics: Animals; Cattle; Fluorescein-5-isothiocyanate; Models, Chemical; Ovomucin; Protein Binding; Protein Interaction Mapping; Serum Albumin, Bovine; Solutions; Superoxide Dismutase; Temperature
PubMed: 21846103
DOI: 10.1021/jp2049266 -
Bioscience, Biotechnology, and... Nov 1997Sulfated glycopeptides in ovomucin, chalazae and yolk membrane were found to activate cultured macrophage-like cells, J774.1, and TGC-induced macrophages from the...
Sulfated glycopeptides in ovomucin, chalazae and yolk membrane were found to activate cultured macrophage-like cells, J774.1, and TGC-induced macrophages from the peritoneal cavity of male mice. The macrophage-stimulating activity was estimated by the growth and morphology of the cells, H2O2 generation, and interleukin-1 (IL-1) production from the cells. The in vitro culture assay with macrophages showed that the protease digests of ovomucin, yolk membrane, and chalazae induced morphologic alteration and increased H2O2 generation and IL-1 production in lower concentration (100 micrograms/ml). The isolation of the components having macrophage-stimulating activity was attempted to elucidate the molecular mechanism. The O-linked carbohydrate chains, consisting of N-acetylgalactosamine, galactose, N-acetylneuraminic acid and sulfate, in the sulfated glycopeptide were identified as a component having macrophage-stimulating activity.
Topics: Animals; Carbohydrates; Cell Division; Chickens; Eggs; Glycopeptides; Humans; Hydrogen Peroxide; Interleukin-1; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred Strains; Ovomucin; Sulfates; Yolk Sac
PubMed: 9404068
DOI: 10.1271/bbb.61.1883 -
Allergology International : Official... Oct 2022Early food introduction induces tolerance, but epicutaneous exposure, especially via eczema lesions, promotes IgE sensitization. Aiming for safe and effective primary...
BACKGROUND
Early food introduction induces tolerance, but epicutaneous exposure, especially via eczema lesions, promotes IgE sensitization. Aiming for safe and effective primary prevention of egg allergy, we examined several protease-digested egg-white (EW) products for three properties: 1) induction of oral tolerance that prevents IgE sensitization, 2) weak IgE binding that can prevent allergic reactions even in IgE-sensitized mice, and 3) minimal epicutaneous IgE sensitization even when in contact with inflamed skin.
METHODS
Heated EW was digested with several proteases under optimal conditions. First, three-week-old BALB/c female mice were intragastrically administered EW or each protease-digested EW product, followed by intraperitoneal ovalbumin (OVA) or ovomucoid (OVM) injection with alum. Serum OVA- and OVM-specific IgE titers were measured. Second, six-week-old mice were sensitized with OVA/OVM, and the rectal temperature was measured after intraperitoneal administration of EW or each protease-digested EW. Third, EW or each protease-digested EW product was applied to the tape-stripped skin for 3 days/week for 3 weeks. Serum OVA- and OVM-specific IgE titers were measured.
RESULTS
Orally administered pepsin-digested EW product (PDEW) and Thermoase PC10F-digested EW product (TDEW) significantly suppressed OVA-/OVM-specific IgE production. Neither product elicited a body temperature decline (anaphylaxis) in OVA-/OVM-sensitized mice. Serum OVA-/OVM-specific IgE levels were significantly lower in mice epicutaneously exposed to PDEW or TDEW than in EW-exposed mice.
CONCLUSIONS
Two protease-digested EWs showed potential as optimal EW products for early introduction for primary prevention of egg allergy.
Topics: Allergens; Animals; Egg Hypersensitivity; Eggs; Female; Immunoglobulin E; Mice; Mice, Inbred BALB C; Ovalbumin; Ovomucin; Pepsin A; Peptide Hydrolases
PubMed: 35443911
DOI: 10.1016/j.alit.2022.03.006 -
Journal of Animal Science Feb 2018The aim of this study was to investigate how dietary supplementation of tea polyphenols (TP) and tea catechins (TC) affect laying performance, albumen quality, ovomucin...
The aim of this study was to investigate how dietary supplementation of tea polyphenols (TP) and tea catechins (TC) affect laying performance, albumen quality, ovomucin composition, and magnum morphology of laying hens in the late phase of production. Two hundred seventy Hy-Line Brown laying hens (64 wk old) were assigned to a basal diet (the control), the basal diet supplemented with 200 mg/kg tea polyphenols (TP200) or 200 mg/kg tea catechins (TC200). Each treatment had 6 replicates with 15 hens each. The feeding trial lasted 10 wks. Over the course of the trial, dietary supplementation with TP200 significantly increased the egg production (EP) and improved the feed conversion ratio (FCR) in wk 6 to 10 and wk 1 to 10 (P < 0.05). The albumen height and the Haugh unit (HU) of hens fed TP200 were higher than those of hens fed the control diet at wks 8 and 10 (P < 0.05). However, there were no significant differences in the albumen height and the HU between the TP200 and TC200 groups (P > 0.05). The SDS-PAGE analysis indicated that bands of the ovomucin fractions in the TP200 group had the highest intensity compared with those of the control and TC200 groups. Compared with the control, there was a significant increase in protein sulfhydryl (SH) content of the albumen in the TP200 group at the end of experiment, while a significant decrease in protein carbonyl content and protein surface hydrophobicity (P < 0.05). There were also obvious increase in the height and width of the primary folds, epithelial cell height, and cilia height of the simple columnar epithelium in the TP200 group compared with the control and TC200 groups (P < 0.05). In conclusion, dietary supplementation with 200 mg/kg TP can improve performance, albumen quality, and magnum morphology of aged hens. In addition, TP rather than TC could improve the health status of the magnum for aged layers.
Topics: Animal Feed; Animals; Chickens; Diet; Dietary Supplements; Female; Ovum; Polyphenols; Protein Carbonylation; Random Allocation; Tea
PubMed: 29378003
DOI: 10.1093/jas/skx007 -
American Journal of Physiology.... Mar 2014We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based...
We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered.
Topics: Asialoglycoproteins; Biological Transport; Cell Line, Tumor; Endocytosis; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Kinesins; Ovomucin; Transport Vesicles; rab1 GTP-Binding Proteins
PubMed: 24407591
DOI: 10.1152/ajpgi.00118.2013 -
The Journal of Allergy and Clinical... Jun 2012Oral immunotherapy (OIT) is a promising treatment for food allergy. Studies are needed to elucidate mechanisms of clinical protection and to identify safer and...
BACKGROUND
Oral immunotherapy (OIT) is a promising treatment for food allergy. Studies are needed to elucidate mechanisms of clinical protection and to identify safer and potentially more efficacious methods for desensitizing patients to food allergens.
OBJECTIVE
We established a mouse model of OIT to determine how the dose or form of antigen may affect desensitization and to identify mechanisms of desensitization.
METHODS
Increasing doses of egg white or ovomucoid as OIT were administered orally to sensitized mice. The impact of OIT on anaphylaxis elicited by oral allergen challenge was determined. Allergen-specific antibody and cytokine responses and mast cell and basophil activation in response to OIT were measured. Gene expression in the small intestine was studied by microarray and real-time PCR.
RESULTS
OIT resulted in desensitization but not tolerance of mice to the allergen. OIT did not result in desensitization of systemic effector cells, and protection was localized to the gastrointestinal tract. OIT was associated with significant changes in gene expression in the jejunum, including genes expressed by intestinal epithelial cells. Extensively heated ovomucoid that does not trigger anaphylaxis when given orally to sensitized mice was as efficacious as native ovomucoid in desensitizing mice.
CONCLUSIONS
OIT results in clinical protection against food-induced anaphylaxis through a novel mechanism that is localized to the intestinal mucosa and is associated with significant changes in small intestinal gene expression. Extensively heating egg allergen decreases allergenicity and increases safety while still retaining the ability to induce effective desensitization.
Topics: Administration, Oral; Allergens; Anaphylaxis; Animals; Cytokines; Desensitization, Immunologic; Disease Models, Animal; Egg White; Epitopes; Female; Food Hypersensitivity; Gastric Mucosa; Gene Expression Profiling; Immunoglobulin A; Immunoglobulin E; Intestinal Mucosa; Mice; Mice, Inbred C3H; Ovomucin; Protein Denaturation
PubMed: 22554705
DOI: 10.1016/j.jaci.2012.04.009 -
Molecular Biology and Evolution Aug 2016The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying...
The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying mucins based on profile hidden Markov models, we have found a large number of such proteins in Metazoa, aiding in their classification and allowing evolutionary studies. Most vertebrates have 5-6 gel-forming mucin genes and the genomic arrangement of these genes is well conserved throughout vertebrates. An exception is the frog Xenopus tropicalis with an expanded repertoire of at least 26 mucins of this type. Furthermore, we found that the ovomucin protein, originally identified in chicken, is characteristic of reptiles, birds, and amphibians. Muc6 is absent in teleost fish, but we now show that it is present in animals such as ghost sharks, demonstrating an early origin in vertebrate evolution. Public RNA-Seq data were analyzed with respect to mucins in zebrafish, frog, and chicken, thus allowing comparison in regard of tissue and developmental specificity. Analyses of invertebrate proteins reveal that gel-forming-mucin type of proteins is widely distributed also in this group. Their presence in Cnidaria, Porifera, and in Ctenophora (comb jellies) shows that these proteins were present early in metazoan evolution. Finally, we examined the evolution of the FCGBP protein, abundant in mucus and related to gel-forming mucins in terms of structure and localization. We demonstrate that FCGBP, ubiquitous in vertebrates, has a conserved N-terminal domain. Interestingly, this domain is also present as an N-terminal sequence in a number of bacterial proteins.
Topics: Amino Acid Sequence; Animals; Cell Adhesion Molecules; Epithelial Cells; Evolution, Molecular; Genome; Humans; Markov Chains; Mucin-6; Mucins; Mucus; Ovomucin; Phylogeny; Sequence Analysis, RNA; Structure-Activity Relationship
PubMed: 27189557
DOI: 10.1093/molbev/msw066