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Journal of Computational Chemistry Jan 2017Our Fuzzy-Border (FB) continuum solvent model has been extended and modified to produce hydration parameters for small molecules using POlarizable Simulations...
Our Fuzzy-Border (FB) continuum solvent model has been extended and modified to produce hydration parameters for small molecules using POlarizable Simulations Second-order Interaction Model (POSSIM) framework with an average error of 0.136 kcal/mol. It was then used to compute pK shifts for carboxylic and basic residues of the turkey ovomucoid third domain (OMTKY3) protein. The average unsigned errors in the acid and base pK values were 0.37 and 0.4 pH units, respectively, versus 0.58 and 0.7 pH units as calculated with a previous version of polarizable protein force field and Poisson Boltzmann continuum solvent. This POSSIM/FB result is produced with explicit refitting of the hydration parameters to the pK values of the carboxylic and basic residues of the OMTKY3 protein; thus, the values of the acidity constants can be viewed as additional fitting target data. In addition to calculating pK shifts for the OMTKY3 residues, we have studied aspartic acid residues of Rnase Sa. This was done without any further refitting of the parameters and agreement with the experimental pK values is within an average unsigned error of 0.65 pH units. This result included the Asp79 residue that is buried and thus has a high experimental pK value of 7.37 units. Thus, the presented model is capable or reproducing pK results for residues in an environment that is significantly different from the solvated protein surface used in the fitting. Therefore, the POSSIM force field and the FB continuum solvent parameters have been demonstrated to be sufficiently robust and transferable. © 2016 Wiley Periodicals, Inc.
Topics: Animals; Hydrogen-Ion Concentration; Models, Molecular; Ovomucin; Quantum Theory; Solvents; Turkeys
PubMed: 27785788
DOI: 10.1002/jcc.24519 -
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 -
Scientific Reports Apr 2016The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to...
The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.
Topics: Animals; Avian Proteins; CRISPR-Cas Systems; Cells, Cultured; Chick Embryo; Chickens; Female; Gene Targeting; Germ Cells; Male; Mutagenesis; Mutation; Ovalbumin; Ovomucin; Reproducibility of Results
PubMed: 27050479
DOI: 10.1038/srep23980 -
Journal of Investigational Allergology... 2016To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared...
OBJECTIVES
To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared before and after the children completed specific oral tolerance induction (SOTI) with egg.
PATIENTS AND METHODS
We studied 28 egg-allergic children who completed SOTI with egg. The basophil activation test and specific IgE determinations with egg white, ovalbumin, and ovomucoid were performed in all 28 children.
RESULTS
A decrease in antigen-specific activation with egg white, ovalbumin, and ovomucoid was observed only at the 2 lowest concentrations used (5 and 0.05 ng/mL). Baseline activation was higher in patients with multiple food allergies and in those who developed anaphylaxis during SOTI; this activation decreased in both groups after completion of SOTI. A significant decrease was also observed in specific IgE values for egg white, ovalbumin, and ovomucoid after tolerance induction.
CONCLUSIONS
Food tolerance induction is a specific process for each food that can be mediated by immunologic changes such as a decrease in specific IgE values and in specific and spontaneous basophil activation.
Topics: Anaphylaxis; Antigens; Basophils; Biomarkers; Child; Child, Preschool; Desensitization, Immunologic; Dose-Response Relationship, Immunologic; Egg Hypersensitivity; Egg White; Female; Humans; Immune Tolerance; Immunoglobulin E; Intradermal Tests; Male; Monitoring, Immunologic; Ovalbumin; Ovomucin; Predictive Value of Tests; Treatment Outcome
PubMed: 27012013
DOI: No ID Found -
PloS One 2016We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the...
We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring.
Topics: Bacterial Proteins; Cytoskeletal Proteins; Escherichia coli; Escherichia coli Proteins; Ficoll; Ions; Ovomucin; Polyethylene Glycols; Protein Multimerization; Protein Structure, Quaternary; Ribonuclease, Pancreatic; Solutions
PubMed: 26870947
DOI: 10.1371/journal.pone.0149060 -
Bioorganic & Medicinal Chemistry Jan 2016Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated...
The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion.
Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH2Cl2 extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies.
Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line; Cell Movement; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epithelial Cells; Female; Humans; Indole Alkaloids; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Models, Molecular; Molecular Structure; Neoplasm Invasiveness; Olea; Ovomucin; Penicillium chrysogenum; Proto-Oncogene Proteins c-met; Structure-Activity Relationship
PubMed: 26692349
DOI: 10.1016/j.bmc.2015.11.038 -
Journal of Nutritional Science and... 2015Oral immunotherapy (OIT) is a promising therapeutic approach for treating food allergy. Past studies have shown that OIT reduces allergic response only in severe allergy...
Oral immunotherapy (OIT) is a promising therapeutic approach for treating food allergy. Past studies have shown that OIT reduces allergic response only in severe allergy model mice. We worked to establish mild allergy model mice, and investigated whether 'rush' OIT for 10 d improved the allergic response and biomarkers in these mice. Balb/c mice were sensitized to ovomucoid (OM) in alum. The rush OIT was done for 10 d. Oral OM challenge was used to determine the impact of OIT on the allergic response. We measured allergic biomarkers, such as vascular permeability in the skin, plasma levels of total IgE, OM-specific IgE, IgG1 and IgG2a and cytokines in splenocyte culture supernatant. OIT for 10 d did not improve allergy symptoms and increased vascular permeability. Total IgE in the plasma of OIT-treated mice was significantly higher than in that of non-treated mice. OM-specific IgG1 and IgG2a plasma levels were not significantly different between OIT-treated and non-treated mice. Among the cytokine secretion of splenocyte from OIT-treated mice, IFN-γ and IL-10 were significantly lower than in non-treated mice, and IL-4 and IL-5 were significantly higher. Total TGF-β in the OIT-treated group was not detected. The IFN-γ/IL-4 ratio of the OIT-treated group was about 1/8 that of the non-treated group. OIT for 10 d was not effective and some biomarkers showed negative responses in the mild allergy model mice. We suggest OIT should be used very carefully as this treatment carries a risk of worsening allergy symptoms for mice with mild allergy.
Topics: Animals; Biomarkers; Cells, Cultured; Disease Models, Animal; Egg Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Immunotherapy; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukin-5; Mice; Mice, Inbred BALB C; Ovomucin; Spleen; Transforming Growth Factor beta
PubMed: 26639848
DOI: 10.3177/jnsv.61.400 -
Poultry Science Sep 2015Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if...
Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point.
Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antioxidants; Avian Proteins; Chelating Agents; Chickens; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Hydrolysis; Metals; Ovomucin; Ovum; Peptides; Tandem Mass Spectrometry
PubMed: 26195809
DOI: 10.3382/ps/pev196 -
PloS One 2015We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with...
BACKGROUND
We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins.
OBJECTIVE
The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.
DESIGN
BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.
RESULTS
A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.
CONCLUSION
Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.
Topics: Allergens; Anaphylaxis; Animals; Body Temperature; Chemokine CCL2; Circular Dichroism; Disease Models, Animal; Egg Hypersensitivity; Immunization; Injections, Intraperitoneal; Lactoglobulins; Mice; Milk Hypersensitivity; Models, Molecular; Nitrogen; Ovomucin; Protein Structure, Secondary
PubMed: 25955653
DOI: 10.1371/journal.pone.0126279