Addition Of Ovalbumin Research Articles

Interaction of ovalbumin with lutein dipalmitate and their effects on the color stability of marigold lutein esters extracts

In this study, the interaction of ovalbumin with lutein dipalmitate and the effect of ovalbumin on marigold lutein esters extracts were investigated. Lutein dipalmitate quenched the fluorescence of ovalbumin by static quenching. Binding and thermodynamic parameters proved that lutein dipalmitate bound to ovalbumin spontaneously by van der Waals force and hydrogen bond, and the complex stoichiometry was 1:1. Through three-dimensional fluorescence spectroscopy, Fourier transform infrared spectroscopy and circular dichroism experiments, the conformation of ovalbumin was unfolded, and alteration in the ovalbumin secondary structure induced by lutein dipalmitate was observed. The results of transmission electron microscopy and particle size revealed that there were spherical and nano-sized aggregates in the ovalbumin-lutein dipalmitate system, indicating the lutein dipalmitate not only could bind to ovalbumin at molecular level, but also promote the aggregation of ovalbumin. Additionally, the addition of ovalbumin had a positive effect on the stability of marigold lutein esters extracts.

Selective Hydrolysis of Ovalbumin Promoted by Hf(IV)-Substituted Wells-Dawson-Type Polyoxometalate.

The reactivity and selectivity of Wells-Dawson type polyoxometalate (POM), K16[Hf(α2-P2W17O61)2]·19H2O (Hf1-WD2), have been examined with respect to the hydrolysis of ovalbumin (OVA), a storage protein consisting of 385 amino acids. The exact cleavage sites have been determined by Edman degradation experiments, which indicated that Hf1-WD2 POM selectively cleaved OVA at eight peptide bonds: Phe13-Asp14, Arg85-Asp86, Asn95-Asp96, Ala139-Asp140, Ser148-Trp149, Ala361-Asp362, Asp362-His363, and Pro364-Phe365. A combination of spectroscopic methods including 31P NMR, Circular Dichroism (CD), and Tryptophan (Trp) fluorescence spectroscopy were employed to gain better understanding of the observed selective cleavage and the underlying hydrolytic mechanism. 31P NMR spectra have shown that signals corresponding to Hf1-WD2 gradually broaden upon addition of OVA and completely disappear when the POM-protein molar ratio becomes 1:1, indicating formation of a large POM/protein complex. CD demonstrated that interactions of Hf1-WD2 with OVA in the solution do not result in protein unfolding or denaturation even upon adding an excess of POM. Trp fluorescence spectroscopy measurements revealed that the interaction of Hf1-WD2 with OVA (Kq = 1.1 × 105 M−1) is both quantitatively and qualitatively slightly weaker than the interaction of isostructural Zr-containing Wells-Dawson POM (Zr1-WD2) with human serum albumin (HAS) (Kq = 5.1 × 105 M−1).

The impact of protein characteristics on the protein network in and properties of fresh and cooked wheat-based noodles

The relation between protein characteristics, protein network formation and wheat noodle properties was studied. Bovine serum albumin (BSA), soy glycinin, ovalbumin, S-ovalbumin and lysozyme were included in the recipe of wheat-based noodles. The characteristics of these non-wheat proteins impacted the type, rate and extent of protein network formation during noodle dough preparation and cooking, and thereby the properties of fresh and cooked noodles. None of the added proteins enhanced the properties of fresh noodles. BSA and soy glycinin enhanced Kieffer-rig extensibility parameters of cooked noodles. Addition of ovalbumin or S-ovalbumin led to excessive protein polymerization in cooked noodles and lowered their quality. Inclusion of lysozyme lowered the rate and extent of polymerization during cooking. Experiments in which urea, olive oil or urea were added in the recipe showed that non-covalent interactions dominate the properties of fresh noodles while covalent cross-links and hydrogen bonds mainly impact the properties of cooked noodles.

Highly Selective Fluorescent Turn-On Probe for Protein Thiols in Biotin Receptor-Positive Cancer Cells.

Sulfhydryl-containing proteins play critical roles in various physiological and biological processes, and the activities of those proteins have been reported to be susceptible to thiol oxidation. Therefore, the development of protein thiol target fluorescent probe is highly desirable. In the present work, a biotinylated coumarin fluorescence "off-on" probe SQ for selectively detecting protein thiols in biotin receptor-positive cancer cells was designed with a 2,4-dinitrobenzenesulfony as the thiol receptor. The probe exhibited dramatic fluorescence responses toward sulfhydryl-containing proteins (ovalbumin (OVA), bovine serum albumin (BSA)): up to 170-fold fluorescence enhancement with 70 nm blue-shift was observed with the addition of OVA. However, low molecular weight thiols (Cys, glutathione (GSH), Hcy) caused negligible fluorescence changes of SQ. In addition, biotin receptor-positive Hela cells displayed strong red and green fluorescence after incubation of SQ for 1 h; neither red nor green fluorescence signal could be visualized in biotin-negative normal lung Wi38 cells. These results imply that the probe has potential application in fluorescent imaging protein thiols on the surface of Hela cells.

Application of Magnesium Pyrophosphate–Based Sponge-Like Microparticles to Enhance the Delivery Efficiency and Adjuvant Effects of Polyriboinosinic-Polyribocytidylic Acid in Immune Cells

The magnesium pyrophosphate particle (MgPP) is a unique and safe carrier that is prepared by simply mixing magnesium chloride and sodium pyrophosphate. In this study, we investigated whether MgPP can be used to deliver nucleic acid–based adjuvants to immune cells. Polyriboinosinic-polyribocytidylic acid (polyI:C), a ligand for toll-like receptor 3, was selected as a model nucleic acid–based adjuvant. PolyI:C-loaded MgPP (polyI:C-MgPP) was prepared by adding polyI:C during the MgPP preparation process. Efficient loading of polyI:C into MgPP was confirmed by measuring the absorbance at 260 nm after disruption of polyI:C-MgPP by ethylenediaminetetraacetic acid. Scanning electron microscopy revealed that both MgPP and polyI:C-MgPP had a unique sponge-like shape with a diameter of approximately 1 μm. PolyI:C-MgPP was more efficiently taken up by toll-like receptor 3–positive RAW264.7 cells than naked polyI:C, and its uptake stimulated increased tumor necrosis factor–α production. When the presentation of ovalbumin (OVA), a model antigen, was evaluated after the addition of OVA along with naked polyI:C or polyI:C-MgPP to mouse dendritic DC2.4 cells, polyI:C-MgPP substantially increased OVA presentation. These results indicate that MgPP is a useful delivery vehicle for polyI:C and that polyI:C-MgPP is an effective immune cell adjuvant.

Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning.

The formation of minerals in living organisms occurs in crowded microenvironments generated by the organization of soft matter. Here, we used a biphasic aqueous polymer medium to mimic the macromolecular crowding and compartmentalization of intracellular environments. Mineralization was performed in an aqueous two-phase system (ATPS) containing two nonionic polymers, poly(ethylene glycol) (PEG, 8 kDa) and dextran (Dx, 10 kDa). The enzyme urease was used to catalyze CaCO3 formation by hydrolyzing urea to produce CO3 2-, which reacted with Ca2+ already present in solution. Urease partitioning into the Dx-rich phase provided a mechanism for localizing the hydrolysis reaction, which consequently restricted mineral formation to this phase, despite the initially equal concentration of Ca2+ in both phases. Spatially confined mineralization was quantified by sampling the phases during bulk reactions and also directly observed in microscale systems by optical microscopy. Decreasing the volume of the Dx-rich phase relative to that of the PEG-rich phase significantly enhanced the local urease concentration in the Dx-rich phase, increasing local reaction rates. The PEG and Dx polymers, though present at up to 30 wt% in the ATPS, did not strongly influence the morphology of CaCO3(s) observed. However, addition of ovalbumin (1.5 wt%) caused marked changes in crystal morphology. The PEG/dextran ATPS reaction medium captured several key aspects of the biological environment including macromolecular crowding, localized reagent production via enzymatic activity, and reaction compartmentalization while not precluding the use of structure-directing additives such as proteins.

Nonmyeloablative Chemotherapy Followed by T-cell Adoptive Transfer and Dendritic Cell-based Vaccination Results in Rejection of Established Melanoma

We demonstrated previously that dendritic cell (DC)-based vaccines could mediate a specific and long-lasting antitumor immune response during early lymphoid reconstitution after lethal irradiation and bone marrow transplant. The purpose of this current study was to examine the potential therapeutic efficacy of DC-based vaccines in combination with sublethal lymphodepletion and T-cell transfer. In an aggressive model of melanoma, treatment with the combination of 200 mg/kg cyclophosphamide (Cy) and 100 mg/kg fludarabine (Flu) led to a lymphopenic state lasting approximately 14 days, but had no effect on the growth of an established M05 melanoma. Addition of ovalbumin (OVA) peptide-pulsed DC-based immunization resulted in a delay in tumor growth but did not enhance overall survival in this model. To improve treatment, adoptively transferred naive T cells were added. After induction of lymphopenia with Cy and Flu, transferred T cells demonstrated an activated memory phenotype including high expression of CD44 and low expression of CD62L. Induction of lymphopenia with Cy and Flu in combination with adoptive transfer of naive T cells and OVA peptide-pulsed DCs immunization led to an enhancement in the number of OVA specific, CD8 T cells that demonstrated specific cytotoxic activity, proliferation, and interferon-gamma production in response to the OVA expressing M05 melanoma. This combination therapy also led to tumor regression and enhanced survival in mice bearing M05 melanoma.

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.

Electrodeposition of Cu onto Pt in presence of egg ovalbumin

In the present paper, the effect of egg ovalbumin (OVA) on the electrodeposition of Cu and on the underpotential deposition (UPD) and overpotential deposition (OPD) behaviour of Cu on polycrystalline Pt electrodes is studied. The interfacial behaviour of OVA was studied at a polycrystalline Pt electrode, using cyclic voltammetry (CV) below (25°C) and above (80°C) the OVA denaturation temperature. Ovalbumin was found to adsorb strongly at the Pt surface. As the temperature was increased above the denaturation value, the surface charge density for OVA adsorption also increased, owing to conformational unfolding. Above the denaturation temperature, the surface charge density continued to increase, suggesting multilayer adsorption. Cyclic voltammetry measurements during Cu electroplating from an acidic chloride bath in the presence of OVA at 25°C highlighted a remarkable electrodeposition and stripping inhibition. This effect was not observed at 80°C. Potentiodynamic measurements in the Cu plating bath highlight a strong OVA adsorption in a well defined potential interval. In situ surface enhanced Raman spectra were measured both during electrodeposition and corrosion of Cu in the Cu plating bath. Potential dependent changes of spectral pattern have been found, which can be interpreted in terms of conformational changes of adsorbed OVA. The study was complemented by morphological and optical characterisation of electrodeposited Cu by scanning electron microscopy and spectral reflectivity measurements. Scanning electron microscopy observations reveal notable changes of morphology, possibly related to the assembly of adsorbed protein at the electrode and to the modification of the electrodic charge transfer process. Finally, comparative spectral reflectivity measurements showed that the addition of OVA brings about changes of the electronic structure of the Cu film.

Role of the tachykinin NK 1 receptor in mediating contraction to 5-hydroxytryptamine and antigen in the mouse trachea

Neuroimmune interactions are important in airway diseases such as asthma. We evaluated the role of the tachykinin NK(1) receptor in the contractile response of isolated trachea from tachykinin NK(1) receptor wild type (WT) and knockout (KO) mice, to the antigen ovalbumin and the contractile agonist serotonin (5-hydroxytryptamine). One percent ovalbumin induced contractions of tracheas obtained from ovalbumin-immunized and exposed mice. The tracheas from WT animals showed larger contractions compared to the KO mice. Tracheas from sensitized and ovalbumin-exposed animals released 5-hydroxytyptamine upon addition of ovalbumin. No higher levels of 5-hydroxytryptamine were released from tracheas of WT animals. Tracheas of non-sensitized animals did not release 5-hydroxytryptamine upon ovalbumin challenge. Responses to ovalbumin were abrogated by methysergide, a broad 5-hydroxytryptamine receptor antagonist. Exogenous 5-hydroxytryptamine contracted tracheas but WT tracheas responded significantly more. Atropine and tetrodotoxin (TTX) reduced 5-hydroxytryptamine-induced contractions of the WT tracheas, while they did not affect 5-hydroxytryptamine-induced contractions of KO tracheas. 5-Hydroxytryptamine-induced contractions from atropine- or TTX-treated WT tracheas did not differ significantly from the contractions of the KO tracheas. Single tachykinin NK(1) receptor antagonists SR140,333 and RP67,580 had no effect on 5-hydroxytryptamine-induced contractions. In conclusion, the 5-hydroxytryptamine-induced tracheal contraction includes a cholinergic mechanism that requires the presence of the tachykinin NK(1) receptor.

Rheological Behavior and Organoleptic Effects of Ovalbumin Addition in Yogurt Mousse Production

Yogurt mousse is a novel, high added-value dairy product that has been well received by the market. This paper presents a study of the effect of the addition of ovalbumin to the product on its rheological and organoleptic qualities. The ovalbumin was previously separated from egg white with a high grade of purity using an ion exchange resin synthesized by the authors. Diverse rheological tests at different temperatures and corresponding sensorial assessments were conducted to compare samples without and with added ovalbumin.The obtained results confirm that the product is viscoelastic and combines the properties of foams and emulsions; the elastic component is greater than the viscous component. Moreover, at temperatures ranging from 5 to 15°C, a usual interval of consumption, the product behaves rheologically in a similar way. Conversely, the addition of ovalbumin under the assayed conditions also makes the elastic character of the product increase at a given temperature. Finally, the sensorial assessment tests and determinations of stability and volume yield enabled us to verify that the addition of ovalbumin at an amount of 1.3% hardly alters the stability, resistance to shear stress, or the texture and improves the degree of foaming. Therefore, the product with additive is of good commercial quality.

Simplified Aqueous Gelcasting of Silicon Carbide Ceramics

A new ceramic forming process, protein coagulation casting (PCC), has been applied for consolidation of green silicon carbide bodies. The process utilizes aqueous ceramic slurries based on egg white (ovalbumin). The silicon carbide powder was dispersed in an aqueous medium without any pH adjustment or addition of any dispersing agents. The ovalbumin based slurries could be gelled simply by heating without use of any chemical additives and it provided sufficient strength for handling the bodies. An addition of ovalbumin to aqueous silicon carbide slurries enhanced the viscosity and thus limited the solid loading. The ovalbumin containing slurries were thixotropic in nature. With the use of the present process, defect free near-net shaped silicon carbide bodies were prepared.

Food components affecting the oil absorption and crispness of fried batter

Frying batters are used to add value to a product by improving texture, flavour, weight and volume and reducing water loss during frying which, in turn, lessens oil absorption. The physical characteristics of fried batters were studied using model systems based on rice flours. Crispness was positively correlated with amylose content, while oil absorption was negatively correlated with amylose content. Addition of pregelatinised rice flour improved crispness but resulted in increased oil absorption because of the porous nature of the fried product. Amongst the proteins (egg yolk, gluten, skimmed milk, whey and ovalbumin) studied, ovalbumin was able to reduce oil absorption and improve the crispness of the fried batter. Addition of calcium chloride also reduced oil absorption and improved crispness, while a little oil reduced the oil content of the fried batter and improved its taste. Addition of ovalbumin, oil or calcium chloride beyond the optimal levels reduced crispness and increased oil absorption in a linear manner. Modified tapioca starch and diglyceride emulsifiers improved crispness but increased oil absorption. The amount of water added to the formulation affected the physical characteristics of the fried batter. The optimal formulation for a crisp frying batter with reasonably low oil absorption was an amylose/amylopectin ratio of about 18: 67; with (g kg−1) 850 pregelatinised rice flour; up to 150 modified tapioca starch; 30 ovalbumin; 1 calcium chloride; 20 oil; 20 emulsifier and a water/flour ratio of 2:1. © 1998 Society of Chemical Industry.

Food components affecting the oil absorption and crispness of fried batter

Frying batters are used to add value to a product by improving texture, flavour, weight and volume and reducing water loss during frying which, in turn, lessens oil absorption. The physical characteristics of fried batters were studied using model systems based on rice flours. Crispness was positively correlated with amylose content, while oil absorption was negatively correlated with amylose content. Addition of pregelatinised rice flour improved crispness but resulted in increased oil absorption because of the porous nature of the fried product. Amongst the proteins (egg yolk, gluten, skimmed milk, whey and ovalbumin) studied, ovalbumin was able to reduce oil absorption and improve the crispness of the fried batter. Addition of calcium chloride also reduced oil absorption and improved crispness, while a little oil reduced the oil content of the fried batter and improved its taste. Addition of ovalbumin, oil or calcium chloride beyond the optimal levels reduced crispness and increased oil absorption in a linear manner. Modified tapioca starch and diglyceride emulsifiers improved crispness but increased oil absorption. The amount of water added to the formulation affected the physical characteristics of the fried batter. The optimal formulation for a crisp frying batter with reasonably low oil absorption was an amylose/amylopectin ratio of about 18: 67; with (g kg−1) 850 pregelatinised rice flour; up to 150 modified tapioca starch; 30 ovalbumin; 1 calcium chloride; 20 oil; 20 emulsifier and a water/flour ratio of 2:1. © 1998 Society of Chemical Industry.

Characterization and morphological analysis of a cholecystokinin derivative peptide-loaded poly(lactide-co-glycolide) microspheres prepared by a water-in-oil-in-water emulsion solvent evaporation method

The water soluble peptide, pBC 264, derived from cholecystokinin and composed of seven aminoacids was encapsulated in poly(lactide-co-glycolide) (PLG) microspheres prepared by a multiple emulsion [(W 1/O)W 2] solvent evaporation method. The encapsulation efficiency of pBC 264 was very low when the inner emulsion contained no stabilizing agent. However, the encapsulation rate was improved by the addition of ovalbumin (OVA) used as stabilizer of the inner emulsion. In addition, the presence of a pH gradient between the inner and the outer aqueous phases of the multiple emulsion led to an increase of the retention of the peptide within the microspheres. Nevertheless, the molecular weight of the polymer did not have any influence on the encapsulation efficiency of the peptide. All microspheres batches showed a mean diameter below 10 μm. In vitro release studies were carried out in both phosphate (pH 7.4) and TRIS buffer (pH 8), with microspheres made from several copolymers differing in their molecular weight. Release kinetics were characterized by a dramatic burst effect corresponding to the release of the major part of the entrapped pBC 264. DSC analysis were able to show that there was no interaction between OVA and the polymer suggesting that the protein is not dissolved in the polymer network but mainly present at the interfaces of the inner aqueous phase/polymer and microsphere surface/outer aqueous phase. The OVA that is present on microspheres might create a porous structure that was observed by electron microscopy. These pores would be responsible for the fast release of pBC 264. These results allow better comprehension of the effect of pH gradient and the use of OVA as a stabilizer on the encapsulation and release pattern of PLG microsphere-loaded small peptides.

Enhancement of demyelination in the central nervous system of Semliki Forest virus infected mice after pretreatment with muramyl dipeptide.

Semliki Forest virus infections in mice produce an encephalitis with demyelination. If before giving the virus the mice are treated with muramyl dipeptide in Freund's incomplete adjuvant, there is a significant increase in demyelination. If ovalbumin is added to the above and then followed after an interval by a second dose of ovalbumin and finally by the virus, the demyelination is further, but only marginally increased. The addition of ovalbumin without muramyl dipeptide in the schedule appears to increase the number of infiltrating cells and to a lesser extent the perivascular cuffing, but does not increase the demyelination as compared to that obtained when Semliki Forest virus is given on its own.

766 CYSTINOTIC FIBROBLASTS ACCUMULATE CYSTINE FROM CYSTINE BUT NOT CYSTEINE RESIDUES OF EXTRACELLULAR PROTEINS

Cystine depleted cystinotic fibroblasts reaccumulate cystine when incubated in cystine free medium supplemented with a variety of disulfide (cystine)-containing proteins. The cystine accumulation is linear with the concentration of added protein. Correlation of the amount of degradation of 125I labelled protein with cystine accumulation yields a significant correlation (r=0.97 for bovine serum albumin [BSA], and r=0.81 for bovine insulin). Similar results have been found for human and rat albumin, and bovine y globulin & thyroglobulin. This cystine accumulation is inhibited by inhibitors of proteolysis, and inhibitors of selective endocytosis. Reduced BSA (RBSA) produced by pre-treatment with mercaptoethanol in 6 M urea yields diminished cystine accumulation even though more of the reduced protein is degraded:0.18 nmol/106 cells cystine accumulated and 0.50 mg/106 cells RBSA degraded in 24 hrs; 0.70 nmol/106 cells cystine accumulated and 0.20 mg BSA degraded in 24 hrs. Ovalbumin (OVA), which contains 4 mol of cysteine/mol of protein and 1 mol cystine yields no cystine accumulation after 24 hrs incubation in which 0.86 mg/106 cells OVA was degraded. Addition of OVA or RBSA to non-depleted cystinotic fibroblasts produces cystine depletion (30% in 4 hrs, 55% in 24 hrs). We conclude that cystinotic cells accumulate cystine through the lysosomal retention of cystine, but not cysteine residues following their release from peptide chains during proteolysis. Supported by USPHS AM 25548.

Identification and Isolation of Ovalbumin-synthesizing Polysomes: I. SPECIFIC BINDING OF 125I-ANTI-OVALBUMIN TO POLYSOMES

Abstract Polysomes involved in ovalbumin synthesis were identified by the binding of 125I-anti-ovalbumin to hen oviduct polysomes. Techniques were developed for the isolation of undegraded hen oviduct polysomes and for the preparation of 125I-γ-globulin free of ribonuclease activity. The distribution of 125I-anti-ovalbumin in the polysome profile is in accordance with the size of the polysomes that are expected to be synthesizing ovalbumin. Anti-ovalbumin does not react with polysomes extracted from tissues that do not synthesize ovalbumin, and antibodies directed against a protein that is not synthesized in the oviduct do not bind to hen oviduct polysomes. These criteria indicate that the reaction is specific for polysomes that synthesize ovalbumin. Polysomes that bind 125I-anti-ovalbumin can be precipitated by the addition of ovalbumin and an excess of specific antibody.