Effect of guar gum conjugation on functional, antioxidant and antimicrobial activity of egg white lysozyme.

PubMed

Hamdani, Afshan Mumtaz; Wani, Idrees Ahmed; Bhat, Naseer Ahmad; Siddiqi, Raushid Ahmad

2018-02-01

This study was undertaken to analyze the effect of conjugation of egg-white lysozyme with guar gum. Lysozyme is an antimicrobial polypeptide that can be used for food preservation. Its antibacterial activity is limited to gram positive bacteria. Conjugation with polysaccharides like guar gum may broaden its activity against gram negatives. Conjugate was developed through Maillard reaction. Assays carried out included sugar estimation, SDS-PAGE, GPC, color, FT-IR, DSC, thermal stability, solubility, emulsifying, foaming and antioxidant activity. In addition, antimicrobial activity of the conjugate was determined against two gram positive (Staphyllococcus aureus and Enterococcus) and two gram negative pathogens (E. coli and Salmonella). Results showed higher functional properties of lysozyme-guar gum conjugate. The antioxidant properties increased from 2.02-35.80% (Inhibition of DPPH) and 1.65-4.93AAE/g (reducing power) upon guar gum conjugation. Conjugate significantly inhibited gram negative bacteria and the antibacterial activity also increased significantly against gram positive pathogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structure of a shark single-domain antibody V region in complex with lysozyme.

PubMed

Stanfield, Robyn L; Dooley, Helen; Flajnik, Martin F; Wilson, Ian A

2004-09-17

Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies.

High-pressure protein crystallography of hen egg-white lysozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Hiroyuki; Nagae, Takayuki; Watanabe, Nobuhisa, E-mail: nobuhisa@nagoya-u.jp

The crystal structure of hen egg-white lysozyme (HEWL) was analyzed under pressures of up to 950 MPa. The high pressure modified the conformation of the molecule and induced a novel phase transition in the tetragonal crystal of HEWL. Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phasemore » transition from P4{sub 3}2{sub 1}2 to P4{sub 3}. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.« less

Water molecules in the antibody-antigen interface of the structure of the Fab HyHEL-5-lysozyme complex at 1.7 A resolution: comparison with results from isothermal titration calorimetry.

PubMed

Cohen, Gerson H; Silverton, Enid W; Padlan, Eduardo A; Dyda, Fred; Wibbenmeyer, Jamie A; Willson, Richard C; Davies, David R

2005-05-01

The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315-326]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A resolution. The refined structure has yielded a detailed picture of the Fab-lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an R(work) of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed.

Fate of egg proteins during the development of Columba livia domestica embryo.

PubMed

Shbailat, Seba Jamal; Aslan, Ibtisam Omar

2018-01-01

The transfer of egg white into the yolk and consumption of yolk proteins by the embryo are largely unexplored in the pigeon Columba livia domestica. Here, we investigated the route of egg white transfer as well as the degradation and uptake of yolk proteins by the pigeon embryo. Initially, we tested the electrophoretic patterns of proteins in different egg compartments throughout development. Then, we used lysozyme as a reference protein to follow the egg white transfer, and we measured its activity using Micrococcus lysodeikticus as a substrate. Moreover, we determined the general protease activity during different developmental stages in the yolk using casein. Finally, we examined the expression of aminopeptidase-N (APN) and oligopeptide transporter PepT1 genes in the yolk sac membrane (YSM) from incubation day 8 until day 17. Several electrophoretic bands of presumptive egg white proteins appeared in different egg compartments. Also, lysozyme activity was detected chronologically in the egg compartments. It appeared on day 12 in the amniotic and intestinal fluids and on day 14 in the yolk. Moreover, protease activity in the yolk increased significantly on day 14 and thereafter. APN expression was largest on day 8 and reduced generally afterward, whereas PepT1 expression peaked between days 13 and 15 but then reduced substantially. Our results suggest that the egg white proteins move through the amnion and intestine into the yolk where they undergo degradation by the activated proteases. Furthermore, the YSM appears to have a role in protein consumption, and this role decreases toward hatch. © 2018 Wiley Periodicals, Inc.

Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications.

PubMed

Perevedentseva, E; Cai, P-J; Chiu, Y-C; Cheng, C-L

2011-02-01

Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.

Local pH at the surface of hen egg white lysozyme

NASA Astrophysics Data System (ADS)

Otosu, Takuhiro; Kobayashi, Kaito; Yamaguchi, Shoichi

2018-02-01

The microenvironment at the surface of hen-egg-white lysozyme (HEWL) was examined by analyzing the change in pKa of fluorescein isothiocyanate (FITC) upon binding to the N-terminus of HEWL. The result showed that the local pH at the HEWL surface is higher than the bulk pH. Furthermore, the data showed that the difference between the local and bulk pH becomes larger with decreasing pH, suggesting HEWL repels more protons at lower pH. Because the local pH affects the protonation states of functional amino-acids at the protein surface, the results provide the fundamental insight into the microenvironment at the protein surface.

Control of effect on the nucleation rate for hen egg white lysozyme crystals under application of an external ac electric field.

PubMed

Koizumi, H; Uda, S; Fujiwara, K; Nozawa, J

2011-07-05

The effect of an external ac electric field on the nucleation rate of hen egg white lysozyme crystals increased with an increase in the concentration of the precipitant used, which enabled the design of an electric double layer (EDL) formed at the inner surface of the drop in the oil. This is attributed to the thickness of the EDL controlled by the ionic strength of the precipitant used. Control of the EDL formed at the interface between the two phases is important to establishing this novel technique for the crystallization of proteins under the application of an external ac electric field. © 2011 American Chemical Society

Science Study Aids 6: Lysozyme - The Cooperative Enzyme.

ERIC Educational Resources Information Center

Boeschen, John; Alderton, Gordon

This publication is the sixth of a series of seven supplementary investigative materials for use in secondary science classes providing up-to-date research-related investigations. This unit is structured for grade levels 10 through 12. It is concerned with the crystallization of an enzyme, lysozyme, from egg white. The first part of this guide…

Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

ERIC Educational Resources Information Center

Schwinefus, Jeffrey J.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L.; Clark, Christopher A.

2008-01-01

As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to…

The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI{sub 6} octahedral complex to a PtI{sub 3} moiety bound to His15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanley, Simon W. M.; Starkey, Laurina-Victoria; Lamplough, Lucinda

The platinum hexahalides have an octahedral arrangement of six halogen atoms bound to a Pt centre, thus having an octahedral shape that could prove to be useful in interpreting poor electron-density maps. In a detailed characterization, PtI{sub 6} chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15 of HEWL was also observed, which was not observed for PtBr{sub 6} or PtCl{sub 6}. This study examines the binding and chemical stability of the platinum hexahalides K{sub 2}PtCl{sub 6}, K{sub 2}PtBr{sub 6} and K{sub 2}PtI{sub 6} when soaked into pre-grown hen egg-white lysozyme (HEWL) crystalsmore » as the protein host. Direct comparison of the iodo complex with the chloro and bromo complexes shows that the iodo complex is partly chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15, a chemical behaviour that is not exhibited by the chloro or bromo complexes. Each complex does, however, bind to HEWL in its octahedral form either at one site (PtI{sub 6}) or at two sites (PtBr{sub 6} and PtCl{sub 6}). As heavy-atom derivatives of a protein, the octahedral shape of the hexahalides could be helpful in cases of difficult-to-interpret electron-density maps as they would be recognisable ‘objects’.« less

Determination of protein phase diagrams by microbatch experiments: exploring the influence of precipitants and pH.

PubMed

Baumgartner, Kai; Galm, Lara; Nötzold, Juliane; Sigloch, Heike; Morgenstern, Josefine; Schleining, Kristina; Suhm, Susanna; Oelmeier, Stefan A; Hubbuch, Jürgen

2015-02-01

Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution. Copyright © 2014 Elsevier B.V. All rights reserved.

The Question of Impurities in Macromolecule Crystal Quality Improvement in Microgravity

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, Edward H.; Pusey, Marc L.; Sportiello, Michael G.; Todd, Paul; Bellamy, Henry; Borgstahl, Gloria E.; Pokros, Matthew; Cassanto, John M.

2000-01-01

While macromolecule impurities may affect crystal size and morphology the over-riding question is how do macromolecule impurities effect crystal X-ray quality and diffraction resolution. In the case of chicken egg white lysozyme previous researchers have reported that crystals grown in the presence of ovalbumin, ovotransferrin, and turkey egg white lysozyme show no difference in diffraction resolution compared to those grown in pure solutions. One impurity however, a naturally occurring lysozyme dimer, does negatively impact the X-ray crystal properties. For this impurity it has been reported that crystal quality improvement in microgravity may be due to improved impurity partitioning during crystallization. In this study we have examined the incorporation of the dimer into lysozyme crystals, both on the ground and in microgravity experiments, and have performed detailed X-ray analysis of the crystals using a new technique for finely probing the mosaicity of the crystal at the Stanford Synchrotron Radiation Laboratory. Dimer partitioning was not significantly different in microgravity compared to the ground based experiments, although it is significantly better than that previously reported in microgravity. Mosaicity analysis of pure crystals, 1422 indexed reflections (microgravity) and 752 indexed reflections (ground), gave average results of 0.0066 and 0.0092 degrees (FWHM) respectively. The microgravity crystals also provided an increased signal to noise. Dimer incorporation increased the average mosaicity in microgravity but not on the ground. However, dimer incorporation did greatly reduce the resolution limit in both ground and microgravity grown crystals. The data is being treated anisotropically to explore these effects. These results indicate that impurity effects in microgravity are complex and that the conditions or techniques employed may greatly affect the role of impurities.

Improved detection limits for electrospray ionization on a magnetic sector mass spectrometer by using an array detector.

PubMed

Cody, R B; Tamura, J; Finch, J W; Musselman, B D

1994-03-01

Array detection was compared with point detection for solutions of hen egg-white lysozyme, equine myoglobin, and ubiquitin analyzed by electrospray ionization with a magnetic sector mass spectrometer. The detection limits for samples analyzed by using the array detector system were at least 10 times lower than could be achieved by using a point detector on the same mass spectrometer. The minimum detectable quantity of protein corresponded to a signal-to-background ratio of approximately 2∶1 for a 500 amol/μL solution of hen egg-white lysozyme. However, the ultimate practical sample concentrations appeared to be in the 10-100 fmol/μL range for the analysis of dilute solutions of relatively pure proteins or simple mixtures.

Crystallization of proteins by dynamic control of supersaturation. Ph.D. Thesis Semiannual Status Report, 21 Mar. - 20 Sep. 1990

NASA Technical Reports Server (NTRS)

Wilson, Lori June

1990-01-01

The growth of protein crystals is known to be the limiting factor in the determination of the three-dimensional structures of most proteins. It is expected that the kinetics of supersaturation, which is directly related to solvent evaporation, will affect protein crystal growth and nucleation and accordingly determine the quality, number, size, and morphology of the crystals. With a technique that controls the evaporation of solvent from a protein solution with N2(g) it is possible to determine the effect of different evaporation profiles on hen egg white lysozyme crystals. Hen egg white lysozyme was chosen as the model protein because it crystallizes easily and has solubility data available for most salt, pH, and temperature ranges. Commercially available lysozyme was further purified by a number of methods. Crystals grown with the purified lysozyme and with the unpurified lysozyme in citrate buffer were different shapes but were found to be of the same symmetry space group by precession photos. Differences were seen in the lysozyme crystals grown using different evaporation rates. At three of the four initial conditions for lysozyme crystal growth, longer evaporation times yielded better crystals. The evaporation times required to see a change in the appearance of the crystals was much longer than expected. The number of rates studied so far represent only a small fraction of the ones now available with the gas evaporation device. The technique also provides for control of both solution pH and temperature which are related to the solubilities of proteins.

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

NASA Astrophysics Data System (ADS)

Das, Sourav; Ghosh, Pooja; Koley, Sudipta; Singha Roy, Atanu

2018-03-01

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV-vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300 K, the value of binding constant (Kb) of HEWL-NG complex (5.596 ± 0.063 × 104 M- 1) was found to be greater than that of HEWL-NR complex (3.404 ± 0.407 × 104 M- 1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2 +, Cu2 + and Fe2 +) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D.

PubMed

Strynadka, N C; James, M N

1991-07-20

A structure of the trisaccharide 2-acetamido-2-deoxy-D-muramic acid-beta (1----4)-2-acetamido-2-deoxy-D-glucose-beta (1----4)-2-acetamido-2-deoxy-D-muramic acid (NAM-NAG-NAM), bound to subsites B, C and D in the active-site cleft of hen egg-white lysozyme has been determined and refined at 1.5 A resolution. The resulting atomic co-ordinates indicate that the NAM residue in site D is distorted from the full 4C1 chair conformation to one in which the ring atoms C-1, C-2, O-5 and C-5 are approximately coplanar, and the hydroxymethyl group is positioned axially (a conformation best described as a sofa). This finding supports the original proposals that suggested the ground-state conformation of the sugar bound in site D is strained to one that more closely resembles the geometry required for the oxocarbonium-ion transition state, the next step along the reaction pathway. Additionally, detailed analysis at 1.5 A resolution of the environments of the catalytic residues Glu35 and Asp52 provides new information on the properties that may allow lysozyme to promote the stabilization of an unusually long-lived oxocarbonium-ion transition state. Intermolecular interactions between the N-acetylmuramic acid residue in site D and the lysozyme molecule that contribute to the saccharide ring distortion include: close packing of the O-3' lactyl group with a hydrogen-bonded "platform" of enzyme residues (Asp52, Asn46, Asn59, Ser50 and Asp48), a close contact between the hydroxymethyl group of ring D and the 2'-acetamido group of ring C and a strong hydrogen-bonded interaction between the NH group of Val109 and O-6 of ring D that stabilizes the observed quasi-axial orientation of the -CH2OH group. Additionally, the structure of this complex shows a strong hydrogen bond between the carboxyl group of Glu35 and the beta-anomeric hydroxyl group of the NAM residue in site D. The hydrogen-bonded environment of Asp52 in the native enzyme and in the complex coupled with the very unfavorable direction of approach of the potential carboxylate nucleophile makes it most unlikely that there is a covalent glycosylenzyme intermediate on the hydrolysis pathway of hen egg-white lysozyme.

Copper(II) directs formation of toxic amorphous aggregates resulting in inhibition of hen egg white lysozyme fibrillation under alkaline salt-mediated conditions.

PubMed

Ghosh, Sudeshna; Pandey, Nitin K; Banerjee, Priyanka; Chaudhury, Koel; Nagy, Nóra Veronika; Dasgupta, Swagata

2015-01-01

Hen egg white lysozyme (HEWL) adopts a molten globule-like state at high pH (~12.75) and is found to form amyloid fibrils at alkaline pH. Here, we report that Cu(II) inhibits self-association of HEWL at pH 12.75 both at 37 and 65 °C. A significant reduction in Thioflavin T fluorescence intensity, attenuation in β-sheet content and reduction in hydrophobic exposure were observed with increasing Cu(II) stoichiometry. Electron paramagnetic resonance spectroscopy suggests a 4N type of coordination pattern around Cu(II) during fibrillation. Cu(II) is also capable of altering the cytotoxicity of the proteinaceous aggregates. Fibrillar species of diverse morphology were found in the absence of Cu(II) with the generation of amorphous aggregates in the presence of Cu(II), which are more toxic compared to the fibrils alone.

Scientist prepare Lysozyme Protein Crystal

NASA Technical Reports Server (NTRS)

1996-01-01

Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida).

PubMed

Fiołka, Marta J; Zagaja, Mirosław P; Hułas-Stasiak, Monika; Wielbo, Jerzy

2012-01-01

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantifying Main Trends in Lysozyme Nucleation: The Effects of Precipitant Concentration, Supersaturation and Impurities

NASA Technical Reports Server (NTRS)

Burke, Michael W.; Leardi, Riccardo; Judge, Russell A.; Pusey, Marc L.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Full factorial experimental design incorporating multi-linear regression analysis of the experimental data allows quick identification of main trends and effects using a limited number of experiments. In this study these techniques were employed to identify the effect of precipitant concentration, supersaturation, and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal forin of chicken egg white lysozyme. Decreasing precipitant concentration, increasing supers aturation, and increasing impurity, were found to increase crystal numbers. The crystal axial ratio decreased with increasing precipitant concentration, independent of impurity.

Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

2014-11-01

Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

Do females invest more into eggs when males sing more attractively? Postmating sexual selection strategies in a monogamous reed passerine.

PubMed

Krištofík, Ján; Darolová, Alžbeta; Majtan, Juraj; Okuliarová, Monika; Zeman, Michal; Hoi, Herbert

2014-04-01

Maternal investment can play an important role for offspring fitness, especially in birds, as females have to provide their eggs with all the necessary nutrients for the development of the embryo. It is known that this type of maternal investment can be influenced by the quality of the male partner. In this study, we first verify that male song is important in the mate choice of female Eurasian reed warblers, as males mate faster when their singing is more complex. Furthermore, female egg investment varies in relation to male song characteristics. Interestingly, clutch size, egg weight, or size, which can be considered as an high-cost investment, is not influenced by male song characteristics, whereas comparably low-cost investment types like investment into diverse egg components are adjusted to male song characteristics. In line with this, our results suggest that female allocation rules depend on investment type as well as song characteristics. For example, egg white lysozyme is positively correlated with male song complexity. In contrast, a negative correlation exists between-song speed and syllable repetitiveness and egg yolk weight as well as egg yolk testosterone concentration. Thus, our results suggest that female egg investment is related to male song performance in several aspects, but female investment patterns regarding various egg compounds are not simply correlated.

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters.

PubMed

Borana, Mohanish S; Mishra, Pushpa; Pissurlenkar, Raghuvir R S; Hosur, Ramakrishna V; Ahmad, Basir

2014-03-01

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

The antibacterial protein lysozyme identified as the termite egg recognition pheromone.

PubMed

Matsuura, Kenji; Tamura, Takashi; Kobayashi, Norimasa; Yashiro, Toshihisa; Tatsumi, Shingo

2007-08-29

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

PubMed Central

Matsuura, Kenji; Tamura, Takashi; Kobayashi, Norimasa; Yashiro, Toshihisa; Tatsumi, Shingo

2007-01-01

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus ‘termite-ball’ and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites. PMID:17726543

In Vitro Determination of the Allergenic Potential of Egg White in Processed Meat

PubMed Central

Hildebrandt, Sabine; Schütte, Larsen; Stoyanov, Stefan; Hammer, Günther; Steinhart, Hans; Paschke, Angelika

2010-01-01

Hen's egg white has been reported as a causative agent of allergic reactions, with ovalbumin, conalbumin, ovomucoid, and lysozyme being the major allergens. However, little is known about the effects of processing with heat and high pressure on the allergenicity of egg white proteins as ingredients in meat. For this purpose, the allergenic characteristics of such treated preparations were studied. The IgE-binding capacity was analyzed by EAST inhibition in raw and processed meat preparations using sera from patients with hen's egg specific IgE. Increasing heat treatment as well as the application of high pressure decreased IgE binding, which is a measure of allergenic potential. The combined application of heat (70°C) and high pressure had synergistic effects in reducing the allergenic potential nearly twice as the sum of the single treatments conducted separately. PMID:20948881

Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme

NASA Astrophysics Data System (ADS)

Arteca, Gustavo A.; Tapia, O.

Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded β-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.

Macromolecule Crystal Quality Improvement in Microgravity: The Role of Impurities

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, Edward H.; Pusey, Marc L.; Sportiello, Michael G.; Todd, Paul; Bellamy, Henry; Borgstahl, Gloria E.; Pokros, Matt; Cassanto, John M.

2000-01-01

While macromolecule impurities may affect crystal size and morphology the over-riding question is; "How do macromolecule impurities effect crystal X-ray quality and diffraction resolution?" In the case of chicken egg white lysozyme, crystals can be grown in the presence of a number of impurities without affecting diffraction resolution. One impurity however, the lysozyme dimer, does negatively impact the X-ray crystal properties. Crystal quality improvement as a result of better partitioning of this impurity during crystallization in microgravity has been reported'. In our recent experimental work dimer partitioning was found to be not significantly different between the two environments. Mosaicity analysis of pure crystals showed a reduced mosaicity and increased signal to noise for the microgravity grown crystals. Dimer incorporation however, did greatly reduce the resolution limit in both ground and microgravity grown crystals. These results indicate that impurity effects in microgravity are complex and may rely on the conditions or techniques employed.

Crystallization of Chicken Egg-White Lysozyme from Ammonium Sulfate

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Snell, Edward H.; Pusey, Marc L.

1997-01-01

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg/ml. Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(sub 3)2(sub 1)2 space group with crystals grown at ph 4.4 having unit-cell dimensions of a = b = 78.7 1, c=38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(sub 1)2(sub 1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Heterogeneity Determination and Purification of Commercial Hen Egg-White Lysozyme

NASA Technical Reports Server (NTRS)

Thomas, B. R.; Vekilov, P. G.; Rosenberger, F.

1998-01-01

Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6%(w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(sub r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9%(w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.

Counter ion induced irreversible denaturation of hen egg white lysozyme upon electrostatic interaction with iron oxide nanoparticles: a predicted model.

PubMed

Ghosh, Goutam; Panicker, Lata; Ningthoujam, R S; Barick, K C; Tewari, R

2013-03-01

The effects of electrostatic interaction between the hen egg white lysozyme (HEWL) and the functionalized iron oxide nanoparticles (IONPs) have been investigated using several techniques, e.g., CD, DSC, ζ-potential, UV-visible spectroscopy, DLS, TEM. Nanoparticles (IONPs) were functionalized with three hydrophilic ligands, viz., poly(ethylene glycol) (PEG), trisodium citrate (TSC) and sodium triphosphate (STP); where both TSC and STP contain Na(+) counter ions. It has been observed that the secondary structure of HEWL was not affected by PEG functionalized IONPs, but was partially and almost completely perturbed by TSC and STP functionalized IONPs, respectively. The perturbation of the secondary structure was irreversible. We have predicted an interaction model to explain the origin of perturbation of HEWL structure. We have also investigated the stability of nanoparticles dispersions after interaction with HEWL and used the DLVO theory to explain results. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantifying Main Trends in Lysozyme Nucleation: The Effect of Precipitant Concentration and Impurities

NASA Technical Reports Server (NTRS)

Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.; Rose, M. Franklin (Technical Monitor)

2000-01-01

Full factorial experiment design incorporating multi-linear regression analysis of the experimental data allows the main trends and effects to be quickly identified while using only a limited number of experiments. These techniques were used to identify the effect of precipitant concentration and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal form of chicken egg white lysozyme. Increasing precipitant concentration was found to decrease crystal numbers, the magnitude of this effect also depending on the supersaturation. The presence of the dimer generally increased nucleation. The crystal axial ratio decreased with increasing precipitant concentration independent of impurity.

Protecting Gram-negative bacterial cell envelopes from human lysozyme: Interactions with Ivy inhibitor proteins from Escherichia coli and Pseudomonas aeruginosa.

PubMed

Liu, Zhihong; García-Díaz, Beatriz; Catacchio, Bruno; Chiancone, Emilia; Vogel, Hans J

2015-11-01

Lysozymes play an important role in host defense by degrading peptidoglycan in the cell envelopes of pathogenic bacteria. Several Gram-negative bacteria can evade this mechanism by producing periplasmic proteins that inhibit the enzymatic activity of lysozyme. The Escherichia coli inhibitor of vertebrate lysozyme, Ivyc and its Pseudomonas aeruginosa homolog, Ivyp1 have been shown to be potent inhibitors of hen egg white lysozyme (HEWL). Since human lysozyme (HL) plays an important role in the innate immune response, we have examined the binding of HL to Ivyc and Ivyp1. Our results show that Ivyp1 is a weaker inhibitor of HL than Ivyc even though they inhibit HEWL with similar potency. Calorimetry experiments confirm that Ivyp1 interacts more weakly with HL than HEWL. Analytical ultracentrifugation studies revealed that Ivyp1 in solution is a monomer and forms a 30kDa heterodimer with both HL and HEWL, while Ivyc is a homodimer that forms a tetramer with both enzymes. The interaction of Ivyp1 with HL was further characterized by NMR chemical shift perturbation experiments. In addition to the characteristic His-containing Ivy inhibitory loop that binds into the active site of lysozyme, an extended loop (P2) between the final two beta-strands also participates in forming protein-protein interactions. The P2 loop is not conserved in Ivyc and it constitutes a flexible region in Ivyp1 that becomes more rigid in the complex with HL. We conclude that differences in the electrostatic interactions at the binding interface between Ivy inhibitors and distinct lysozymes determine the strength of this interaction. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides. Copyright © 2015 Elsevier B.V. All rights reserved.

Lysozyme

NASA Technical Reports Server (NTRS)

2004-01-01

Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity experiments with data from laboratory experiments to study the equilibrium rate of hanging drop experiments in microgravity.

Incorporation of impurity to a tetragonal lysozyme crystal

NASA Astrophysics Data System (ADS)

Kurihara, Kazuo; Miyashita, Satoru; Sazaki, Gen; Nakada, Toshitaka; Durbin, Stephen D.; Komatsu, Hiroshi; Ohba, Tetsuhiko; Ohki, Kazuo

1999-01-01

Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton-Prim-Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.

The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helixmore » secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.« less

Sequential /sup 1/H NMR assignments and secondary structure of hen egg white lysozyme in solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redfield, C.; Dobson, C.M.

Assignments of /sup 1/H NMR resonances of 121 of the 129 residues of hen egg white lysozyme have been obtained by sequence-specific methods. Spin systems were identified with phase-sensitive two-dimensional (2-D) correlated spectroscopy and single and double relayed coherence transfer spectroscopy. For key types of amino acid residues, particularly alanine, threonine, valine, and glycine, complete spin systems were identified. For other residues a less complete definition of the spin system was found to be adequate for the purpose of sequential assignment. Sequence-specific assignments were achieved by phase-sensitive 2-D nuclear Overhauser enhancement spectroscopy (NOESY). Exploitation of the wide range of hydrogenmore » exchange rates found in lysozyme was a useful approach to overcoming the problem of spectral overlap. The sequential assignment was built up from 21 peptide segments ranging in length from 2 to 13 residues. The NOESY spectra were also used to provide information about the secondary structure of the protein in solution. Three helical regions and two regions of ..beta..-sheet were identified from the NOESY data; these regions are identical with those found in the X-ray structure of hen lysozyme. Slowly exchanging amides are generally correlated with hydrogen bonding identified in the X-ray structure; a number of exceptions to this general trend were, however, found. The results presented in this paper indicate that highly detailed information can be obtained from 2-D NMR spectra of a protein that is significantly larger than those studies previously.« less

HPLC of the Polypeptides in a Hydrolyzate of Egg-White Lysozyme. An Experiment for the Undergraduate Biochemistry Laboratory.

ERIC Educational Resources Information Center

Richardson, W. S., III; Burns, L.

1988-01-01

Describes a simple high-performance liquid chromatography experiment for undergraduate biochemistry laboratories. The experiment illustrates the separation of polypeptides by a step gradient elution using a single pump instrument with no gradient attachments. Discusses instrumentation, analysis, a sample preparation, and results. (CW)

Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

1998-01-01

Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Four different crystal morphologies have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed, Crystals grown at 15 C were generally tetragonal, with space group P43212. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P21212 1. The tetragonal much less than orthorhombic morphology transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 0.8 -1.2M magnesium sulfate at pH 7.6 - 8.0 gave a hexagonal (trigonal) crystal form, space group P3121, which diffracted to 2.8 A. Ammonium sulfate was also found to result in a monoclinic form, space group C2. Small twinned monoclinic crystals of approx. 0.2 mm on edge were grown by dialysis followed by seeded sitting drop crystallization.

Trehalose and Magnesium Chloride Exert a Common Anti-amyloidogenic Effect Towards Hen Egg White Lysozyme.

PubMed

Chatterjee, Rupsa; Kolli, Vidyalatha; Sarkar, Nandini

2017-04-01

Many degenerative disorder such as Parkinsons, Alzheimers, Huntingtons disease, etc are caused due to the deposition of amyloid fibrils, formed due to the ordered aggregation of misfolded/unfolded proteins. Misfolded or unfolded proteins aggregate mostly through hydrophobic interactions which are unexposed in native state, but become exposed upon unfolding. To counteract amyloid related diseases, inhibition of the protein self assembly into fibril is a potential therapeutic strategy. The study aims at investigating the effect of selected compounds, namely trehalose and magnesium chloride hexahydrate towards inhibition and disaggregation of amyloid fibrils using Hen Egg White Lysozyme as a model. We further attempted to understand the mechanism of action with the help of various biophysical, microscopic as well as computational studies. A common mechanism of action was identified where the selected compounds exert their anti-amyloidogenic effects by altering HEWL conformations characterized by reduction in the beta sheet content and decrease in exposed hydrophobic surfaces. The altered conformation seems to have lesser amyloidogenic propensity leading to inhibition as well as disaggregation of amyloids.

Volume properties and spectroscopy: A terahertz Raman investigation of hen egg white lysozyme

NASA Astrophysics Data System (ADS)

Sassi, Paola; Perticaroli, Stefania; Comez, Lucia; Giugliarelli, Alessandra; Paolantoni, Marco; Fioretto, Daniele; Morresi, Assunta

2013-12-01

The low frequency depolarized Raman spectra of 100 mg/ml aqueous solutions of hen egg white lysozyme (HEWL) have been collected in the 25-85 °C range. Short and long exposures to high temperatures have been used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. A peculiar temperature evolution of spectra is evidenced under prolonged exposure of the protein solution at temperatures higher than 65 °C. This result is connected to the self-assembling of polypeptide chains and testifies the sensitivity of the technique to the properties of both protein molecule and its surrounding. Solvent free spectra have been obtained after subtraction of elastic and solvent components and assigned to a genuine vibrational contribution of hydrated HEWL. A straight similarity is observed between the solvent-free THz Raman feature and the vibrational density of states as obtained by molecular dynamics simulations; according to this, we verify the relation between this spectroscopic observable and the effective protein volume, and distinguish the properties of this latter respect to those of the hydration shell in the pre-melting region.

Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study.

PubMed

Cappannella, Elena; Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Bavaro, Teodora; Esti, Marco

2016-11-01

Lysozyme from hen egg white (HEWL) was covalently immobilized on spherical supports based on microbial chitosan in order to develop a system for the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. The objective is to limit the sulfur dioxide dosage required to control malolactic fermentation, via a cell concentration typical during this process. The immobilization procedure was optimized in batch mode, evaluating the enzyme loading, the specific activity, and the kinetic parameters in model wine. Subsequently, a bench-scale fluidized-bed reactor was developed, applying the optimized process conditions. HEWL appeared more effective in the immobilized form than in the free one, when the reactor was applied in real white and red wine. This preliminary study suggests that covalent immobilization renders the enzyme less sensitive to the inhibitory effect of wine flavans. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.

2001-01-01

Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study, we show that possible conformational changes induced by heating are stable and apparently non-reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for four weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 hours at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Burke, Michael W.; Judge, Russell A.; Pusey, Marc L.; Rose, M. Franklin (Technical Monitor)

2000-01-01

Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study we show that possible conformational changes induced by heating are stable and apparently non- reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for 4 weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 h at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

Characterization of amyloidogenesis of hen egg lysozyme in concentrated ethanol solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holley, Mikel; Eginton, Chris; Schaefer, David

We show that hen egg white lysozyme [HEWL] reproducibly forms amyloid fibrils in 80% ethanol at 22 deg. C with constant agitation. Fibril formation occurs over a time course of approximately 30 days, displays polymerization nucleation kinetics, and demonstrates a marked decrease in {alpha}-helical structure. Seeding with as little as 0.05% v/v of fibrils cleaved into smaller seed fragments by sonication abolishes the lag phase. Thioflavin T assays confirm the amyloid nature of the fibrils. Atomic force microscopy reveals unbranched amyloid fibrils with lengths varying between 1 and 3 {mu}m and heights ranging from 6-12 nm. The formation of amyloidmore » fibrils in predominantly organic solvents demonstrates that the basic principles guiding fibril formation arise from interactions of the peptide backbone rather than from interactions between the amino acid side chains.« less

Location of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

1998-01-01

Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

Locations of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

1998-01-01

Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

Cracking the egg: An insight into egg hypersensitivity.

PubMed

Dhanapala, Pathum; De Silva, Chamika; Doran, Tim; Suphioglu, Cenk

2015-08-01

Hypersensitivity to the chicken egg is a widespread disorder mainly affecting 1-2% of children worldwide. It is the second most common food allergy in children, next to cow's milk allergy. Egg allergy is mainly caused by hypersensitivity to four allergens found in the egg white; ovomucoid, ovalbumin, ovotransferrin and lysozyme. However, some research suggests the involvement of allergens exclusively found in the egg yolk such as chicken serum albumin and YGP42, which may play a crucial role in the overall reaction. In egg allergic individuals, these allergens cause conditions such as itching, atopic dermatitis, bronchial asthma, vomiting, rhinitis, conjunctivitis, laryngeal oedema and chronic urticaria, and anaphylaxis. Currently there is no permanent cure for egg allergy. Upon positive diagnosis for egg allergy, strict dietary avoidance of eggs and products containing traces of eggs is the most effective way of avoiding future hypersensitivity reactions. However, it is difficult to fully avoid eggs since they are found in a range of processed food products. An understanding of the mechanisms of allergic reactions, egg allergens and their prevalence, egg allergy diagnosis and current treatment strategies are important for future studies. This review addresses these topics and discusses both egg white and egg yolk allergy as a whole. Copyright © 2015 Elsevier Ltd. All rights reserved.

Control of solvent evaporation in hen egg white lysozyme crystallization

NASA Technical Reports Server (NTRS)

Wilson, L. J.; Suddath, F. L.

1992-01-01

An investigation of the role of solvent evaporation in tetragonal lysozyme crystallization was preformed with a device that employs N2(g) to control the evaporation of solvent from a micro-volume crystallization hanging drop. The number of crystals was found to vary with the rate at which the final supersaturation level was achieved. It was found that the more rapid the approach to supersaturation the larger the number of crystals. Accordingly, the crystals reached a smaller terminal size. Elongation of the (110) face parallel to the four-fold axis was observed with the slower evaporation rates.

Protein-Lipid Interactions and Mechanisms of Antioxidant Activity of Proteins.

DTIC Science & Technology

1984-06-25

lysozyme and peroxidizing methyl fatty acid esters 3 F.~~~ ~ ~ -: 7 7-7777- 77.:1 71 7: .and decreases lipid hydroperoxide and malonadelhyde...freeze-dried emulsions of methyl linoleate with chicken egg lysozyme and other proteins, and in solution with amino acids and several aldehydes. Generation...4 Z,. ..- : , , , , . .:: . . ,.. -. , . . . .b - ? . Egg lecithin liposomes with hen- egg lysozyme localized either inside or outside the vesicles

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.

PubMed

Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus

2012-01-01

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme. Copyright © 2012 S. Karger AG, Basel.

Influences of animal mucins on lysozyme activity in solution and on hydroxyapatite surfaces.

PubMed

Park, Won-Kyu; Chung, Jin-Woo; Kim, Young-Ku; Chung, Sung-Chang; Kho, Hong-Seop

2006-10-01

The purpose of this study was to investigate the influence of animal mucins on lysozyme activity in solution and on the surface of hydroxyapatite (HA) beads. The effects of animal mucins on lysozyme activity in solution were examined by incubating porcine gastric mucin (PGM) or bovine submaxillary mucin (BSM) with hen egg-white lysozyme (HEWL) or salivary samples. HA-immobilised animal mucins or lysozyme were used to determine the influence of animal mucins on lysozyme activity on HA surfaces. Lysozyme activity was determined by turbidity measurement of a Micrococcus lysodeikticus substrate suspension. Protein concentration was determined by ninhydrin assay. PGM inhibited the activity of HEWL and salivary lysozyme in solution. The amount of inhibition was dependent on mucin concentration, incubation time and temperature, and the structural integrity of the mucin. The inhibition of salivary lysozyme activity by PGM was greater in submandibular/sublingual saliva than in parotid saliva. The inhibition of lysozyme activity by PGM was markedly dependent on pH. However, BSM did not inhibit the in-solution lysozyme activities of HEWL and clarified saliva. Both PGM and BSM bound to HA surfaces, and HA-adsorbed animal mucins increased the subsequent adsorption of lysozyme. When HA beads were exposed to a mixture of HEWL and PGM or BSM, lysozyme activity on the HA surfaces was significantly increased. The results suggest that animal mucins affect lysozyme activity, and the effects are different on HA surfaces compared with in solution. Further research is needed to determine the effect of animal mucins on lysozyme activity in vivo.

Crystallization of chicken egg white lysozyme from assorted sulfate salts

NASA Astrophysics Data System (ADS)

Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

1999-01-01

Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4°C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed. Crystals grown at 15°C were generally tetragonal, with space group P4 32 12. Crystallization at 20°C typically resulted in the formation of orthorhombic crystals, space group P2 12 12 1. The tetragonal ↔ orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20°C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium sulfate at pH 7.8 gave a trigonal crystal, space group P3 12 1, a= b=87.4, c=73.7, γ=120°, which diffracted to 2.8 Å. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form, space group C2, a=65.6, b=95.0, c=41.2, β=119.2°. A crystal of ˜0.2×0.2×0.5 mm grown from bulk solution diffracted to ˜3.5 Å.

Composite cryogels for lysozyme purification.

PubMed

Baydemir, Gözde; Türkoğlu, Emir Alper; Andaç, Müge; Perçin, Işık; Denizli, Adil

2015-01-01

Beads-embedded novel composite cryogel was synthesized to purify lysozyme (Lyz) from chicken egg white. The poly(hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) (PHEMAPA) beads of smaller than 5 µm size were synthesized by suspension polymerization and then embedded into a poly(hydroxyethyl methacrylate) (PHEMA)-based cryogel column. The PHEMAPA bead-embedded cryogel (BEC) column was characterized by swelling tests, scanning electron microscopy (SEM), surface area measurements by the Brunauer-Emmett-Teller (BET) method, elemental analysis, and flow dynamics. The specific surface area of the PHEMAPA BEC was found as 41.2 m(2) /g using BET measurements. Lyz-binding experiments were performed using aqueous solutions in different conditions such as initial Lyz concentration, pH, flow rate, temperature, and NaCl concentration of an aqueous medium. The PHEMAPA BEC column could be used after 10 adsorption-desorption studies without any significant loss in adsorption capacity of Lyz. The PHEMAPA BEC column was used to purify Lyz from chicken egg white, and gel electrophoresis was used to estimate the purity of Lyz. The chromatographic application of the PHEMAPA BEC column was also performed using fast protein liquid chromatography. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

NASA Astrophysics Data System (ADS)

Siepi, Marialuisa; Politi, Jane; Dardano, Principia; Amoresano, Angela; De Stefano, Luca; Monti, Daria Maria; Notomista, Eugenio

2017-08-01

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes.

The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria.

PubMed

Guzzo, F; Cappello, M S; Azzolini, M; Tosi, E; Zapparoli, G

2011-08-15

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating. Copyright © 2011 Elsevier B.V. All rights reserved.

Microwave-radiation-induced molecular structural rearrangement of hen egg-white lysozyme

NASA Astrophysics Data System (ADS)

Singh, Anang K.; Burada, P. S.; Bhattacharya, Susmita; Bag, Sudipta; Bhattacharya, Amitabha; Dasgupta, Swagata; Roy, Anushree

2018-05-01

We have investigated the nonthermal effect of 10 GHz/22 dBm microwave radiation on hen egg-white lysozyme (HEWL) over different irradiation times, ranging from 2 min to 1 h. To ensure a control over the radiation parameters, a pair of microwave rectangular waveguides is used to irradiate the samples. Optical spectroscopic measurements, which include UV-visible absorption spectroscopy, Raman spectroscopy, and far UV CD spectroscopy, reveal the exposure of the buried tryptophan (Trp) residues of the native molecule between 15 and 30 min of radiation. The higher duration of the perturbation leads to a compact structure of the protein and Trp residues are buried again. Interestingly, we do not find any change in the secondary structure of the protein even for 1 h duration of radiation. The relaxation dynamics of the irradiated molecules also has been discussed. We have shown that the molecules relax to their native configuration in 7-8 h after the radiation field is turned off. The structural rearrangement over the above timescale has further been probed by a model calculation, based on a modified Langevin equation. Our coarse-grained simulation approach utilizes the mean of atomic positions and net atomic charge of each amino acid of native HEWL to mimic the initial conformation of the molecule. The modified positions of the residues are then calculated for the given force fields. The simulation results reveal the nonmonotonous change in overall size of the molecule, as observed experimentally. The radiation parameters used in our experiments are very similar to those of some of the electronic devices we often come across. Thus, we believe that the results of our studies on a simple protein structure may help us in understanding the effect of radiation on complex biological systems as well.

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

PubMed

Hadfield, A T; Harvey, D J; Archer, D B; MacKenzie, D A; Jeenes, D J; Radford, S E; Lowe, G; Dobson, C M; Johnson, L N

1994-11-11

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein thermal stabilization in aqueous solutions of osmolytes.

PubMed

Bruździak, Piotr; Panuszko, Aneta; Jourdan, Muriel; Stangret, Janusz

2016-01-01

Proteins' thermal stabilization is a significant problem in various biomedical, biotechnological, and technological applications. We investigated thermal stability of hen egg white lysozyme in aqueous solutions of the following stabilizing osmolytes: Glycine (GLY), N-methylglycine (NMG), N,N-dimethylglycine (DMG), N,N,N-trimethylglycine (TMG), and trimethyl-N-oxide (TMAO). Results of CD-UV spectroscopic investigation were compared with FTIR hydration studies' results. Selected osmolytes increased lysozyme's thermal stability in the following order: Gly>NMG>TMAO≈DMG>TMG. Theoretical calculations (DFT) showed clearly that osmolytes' amino group protons and water molecules interacting with them played a distinctive role in protein thermal stabilization. The results brought us a step closer to the exact mechanism of protein stabilization by osmolytes.

Protein crystallization in a 100 nl solution with new stirring equipment

PubMed Central

Maki, S.; Murai, R.; Yoshikawa, H. Y.; Kitatani, T.; Nakata, S.; Kawahara, H.; Hasenaka, H.; Kobayashi, A.; Okada, S.; Sugiyama, S.; Adachi, H.; Matsumura, H.; Takano, K.; Murakami, S.; Inoue, T.; Sasaki, T.; Mori, Y.

2008-01-01

To investigate quantitatively the effects of stirring on protein crystallization, a new stirring system which can agitate a protein solution, ∼100 nl, by providing Hagen–Poiseuille flow has been successfully developed. In addition, this new stirring system provides flow with a well defined pattern and velocity. Using this system, hen egg-white lysozyme was crystallized in 100–200 nl solutions while being stirred. The optimum stirring conditions for lysozyme crystals have been explored by evaluating the Reynolds (Re) number and the crystals obtained. Intermittent flow, as well as a low Re number, was found to contribute significantly to the growth of a smaller number of larger crystals. PMID:18421156

Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Snell, Edward H.; Malone, Christine C.; Pusey, Marc L.

1999-01-01

Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4 C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed. Crystals grown at 15 C were generally tetragonal, with space group P4(sub 3)2(sub 1)2. Crystallization at 20 C typically resulted in the formation of orthorhombic crystals, space group P2(sub 1)2(sub 1)2(sub 1). The tetragonal reversible reaction orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20 C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium sulfate at pH 7.8 gave a trigonal crystal, space group P3(sub 1)2(sub 1), a = b = 87.4, c = 73.7, gamma = 120 deg, which diffracted to 2.8 A. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form. space group C2, a = 65.6, b = 95.0, c = 41.2, beta = 119.2 deg. A crystal of approximately 0.2 x 0.2 x 0.5 mm grown from bulk solution diffracted to approximately 3.5 A.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.

PubMed

Vogtt, K; Winter, R

2005-08-01

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

An attractive way of egg white protein by-product use for producing of novel anti-hypertensive peptides.

PubMed

Pokora, M; Zambrowicz, A; Dąbrowska, A; Eckert, E; Setner, B; Szołtysik, M; Szewczuk, Z; Zabłocka, A; Polanowski, A; Trziszka, T; Chrzanowska, J

2014-05-15

The aim of this study was to (i) examine how enzymatic hydrolysis with a non-commercially available proteinase of fig-leaf gourd fruit (Cucurbita ficifolia) increased the use value of egg white protein preparations, generated as byproducts in the industrial process of lysozyme and cystatin isolation from egg white, and (ii) evaluate the inhibition of angiotensin I-converting enzyme (ACE) by the obtained hydrolysates. Purification procedures including membrane filtration, gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) led to the production of several peptide fractions. Two novel ovalbumin-derived tetrapeptides: SWVE (f 148-151) and DILN (f 86-89) with ACE inhibitory activity were obtained. Study of their inhibitory kinetics revealed a non-competitive binding mode, with an IC50 value against ACE of 33.88 and 73.44 μg for SWVE and DILN, respectively. Synthetic peptides which were designed on the basis of peptide SWVE were examined. A tripeptide sequence of SWV revealed the strongest ACE-inhibitory activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Size Exclusion Chromatography Studies of the Initial Self-Association Steps of Chicken Egg White Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Ewing, Felecia; Donovan, David; Pusey, Marc

2000-01-01

Nucleation is one of the least understood aspects of crystallogenesis. In the case of macromolecule nucleation, this understanding is further hampered by uncertainty over what precisely is being discussed. We define the process of solute self-association (aggregation, oligomerization, interaction, clustering, etc.) whereby n-mers (n > or = 2) having a crystallographic or nascent crystallographic arrangement leading to the critical nucleus reversibly form in the solution, to be part of the nucleation process. This reversible self-association process is a fundamental part of the nucleation process, and occurs as a function of the solute concentration. In the case of chicken egg white lysozyme, a considerable body of experimental evidence leads us to the conclusion that it also forms the crystal growth units. Size exclusion chromatography is a simple and direct method for determining the equilibrium constants for the self-association process. A Pharmacia FPLC system was used to provide accurate solution flow rates. The column, injection valve, and sample loop were all mounted within a temperature-controlled chamber. Chromatographically re-purified lysozyme was first dialyzed against the column equilibration buffer, with injection onto the column after several hours pre-incubation at the running temperature. Preliminary experiments, were carried out using a Toyopearl HW-50F column (1 x 50cm), equilibrated with 0.1 M sodium acetate, 5% sodium chloride, pH 4.6, at 15C. Protein concentrations from 0.1 to 4 mg/ml were employed (C(sub sat) = 1.2 mg/ml). The data from several different protein preparations consistently shows a progressively decreasing elution volume with increasing protein concentration, indicating that reversible self-association is occurring. The dotted line indicates the monomeric lysozyme elution volume. However, lysozyme interacts with the column matrix in these experiments, which complicates data analysis.Accordingly, we are testing silica-based HPLC columns in an effort to eliminate this problem and substantially reduce the column volume and experimental run time. The results and data analysis from these and subsequent experiments will be presented.

Microgravity

NASA Image and Video Library

1996-01-25

Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

Macromolecular powder diffraction : structure solution via molecular.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doebbler, J.; Von Dreele, R.; X-Ray Science Division

Macromolecular powder diffraction is a burgeoning technique for protein structure solution - ideally suited for cases where no suitable single crystals are available. Over the past seven years, pioneering work by Von Dreele et al. [1,2] and Margiolaki et al. [3,4] has demonstrated the viability of this approach for several protein structures. Among these initial powder studies, molecular replacement solutions of insulin and turkey lysozyme into alternate space groups were accomplished. Pressing the technique further, Margiolaki et al. [5] executed the first molecular replacement of an unknown protein structure: the SH3 domain of ponsin, using data from a multianalyzer diffractometer.more » To demonstrate that cross-species molecular replacement using image plate data is also possible, we present the solution of hen egg white lysozyme using the 60% identical human lysozyme (PDB code: 1LZ1) as the search model. Due to the high incidence of overlaps in powder patterns, especially in more complex structures, we have used extracted intensities from five data sets taken at different salt concentrations in a multi-pattern Pawley refinement. The use of image plates severely increases the overlap problem due to lower detector resolution, but radiation damage effects are minimized with shorter exposure times and the fact that the entire pattern is obtained in a single exposure. This image plate solution establishes the robustness of powder molecular replacement resulting from different data collection techniques.« less

A comparative study on the aggregating effects of guanidine thiocyanate, guanidine hydrochloride and urea on lysozyme aggregation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emadi, Saeed, E-mail: emadi@iasbs.ac.ir; Behzadi, Maliheh

Highlights: • Lysozyme aggregated in guanidine thiocyanate (1.0 and 2.0 M). • Lysozyme aggregated in guanidine hydrochloride (4 and 5 M). • Lysozyme did not aggregated at any concentration (0.5–5 M) of urea. • Unfolding pathway is more important than unfolding per se in aggregation. - Abstract: Protein aggregation and its subsequent deposition in different tissues culminate in a diverse range of diseases collectively known as amyloidoses. Aggregation of hen or human lysozyme depends on certain conditions, namely acidic pH or the presence of additives. In the present study, the effects on the aggregation of hen egg-white lysozyme via incubationmore » in concentrated solutions of three different chaotropic agents namely guanidine thiocyanate, guanidine hydrochloride and urea were investigated. Here we used three different methods for the detection of the aggregates, thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy. Our results showed that upon incubation with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 5.0 M) of the chemical denaturants, lysozyme was aggregated at low concentrations of guanidine thiocyanate (1.0 and 2.0 M) and at high concentrations of guanidine hydrochloride (4 and 5 M), although no fibril formation was detected. In the case of urea, no aggregation was observed at any concentration.« less

Bacteriolytic Activity Of Human Interleukin-2, Chicken Egg Lysozyme In The Presence Of Potential Effectors

PubMed Central

Levashov, P. A.; Matolygina, D. A.; Ovchinnikova, E. D.; Atroshenko, D. L.; Savin, S. S.; Belogurova, N. G.; Smirnov, S. A.; Tishkov, V. I.; Levashov, A. V.

2017-01-01

The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various substances has been studied. Glycine and lysine do not affect the activity of interleukin-2 but increase that of lysozyme, showing a bell-shape concentration dependence peaking at 1.5 mM glycine and 18 mM lysine. Arginine and glutamate activate both interleukin-2 and lysozyme with a concentration dependence of the saturation type. Aromatic amino acids have almost no effect on the activity of both interleukin-2 and lysozyme. Aromatic amines, tryptamine, and tyramine activate interleukin-2 but inhibit lysozyme. Peptide antibiotics affect interleukin and lysozyme similarly and exhibit maximum activity in the micromolar range of antibiotics. Taurine has no effect on the activity of interleukin-2 and lysozyme. Mildronate showed no influence on lysozyme, but it activated interleukin-2 with the activity maximum at 3 mM. EDTA activates both interleukin-2 and lysozyme at concentrations above 0.15 mM. PMID:28740730

Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

PubMed

Chaturvedi, Sumit K; Khan, Javed M; Siddiqi, Mohammad K; Alam, Parvez; Khan, Rizwan H

2016-02-01

Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

NASA Technical Reports Server (NTRS)

Snell, Edward H.; Judge, Russell A.; Crawford, Lisa; Forsythe, Elizabeth L.; Pusey, Marc L.; Sportiello, Michael; Todd, Paul; Bellamy, Henry; Lovelace, Jeff; Cassanto, John M.;

A microcrystal selection technique in protein crystallization

NASA Astrophysics Data System (ADS)

Han, Qing; Lin, Sheng-Xiang

1996-10-01

The goal of protein crystallization is to obtain high quality single crystals for X-ray diffraction analysis. A new and easy technique was employed to control the number and quality of crystals by eliminating poor microcrystals after the spontaneous nucleation. The process was carried out with two samples: human 17β-hydroxysteroid dehydrogenase (17β-HSD) and hen egg white lysozyme. The present study suggests a useful method for the successful crystal growth of biomacromolecules.

Are antimicrobial defences in bird eggs related to climatic conditions associated with risk of trans-shell microbial infection?

PubMed Central

2014-01-01

Introduction All bird eggs are exposed to microbes in the environment, which if transmitted to the developing embryo, could cause hatching failure. However, the risk of trans-shell infection varies with environmental conditions and is higher for eggs laid in wetter environments. This might relate to generally higher microbial abundances and diversity in more humid environments, including on the surface of eggshells, as well as the need for moisture to facilitate microbial penetration of the eggshell. To protect against microbial infection, the albumen of avian eggs contains antimicrobial proteins, including lysozyme and ovotransferrin. We tested whether lysozyme and ovotransferrin activities varied in eggs of larks (Alaudidae) living along an arid-mesic gradient of environmental aridity, which we used as a proxy for risk of trans-shell infection. Results Contrary to expectations, lysozyme activity was highest in eggs from hotter, more arid locations, where we predicted the risk of trans-shell infection would be lower. Ovotransferrin concentrations did not vary with climatic factors. Temperature was a much better predictor of antimicrobial protein activity than precipitation, a result inconsistent with studies stressing the importance of moisture for trans-shell infection. Conclusions Our study raises interesting questions about the links between temperature and lysozyme activity in eggs, but we find no support for the hypothesis that antimicrobial protein deposition is higher in eggs laid in wetter environments. PMID:25057281

Are antimicrobial defences in bird eggs related to climatic conditions associated with risk of trans-shell microbial infection?

PubMed

Horrocks, Nicholas Pc; Hine, Kathryn; Hegemann, Arne; Ndithia, Henry K; Shobrak, Mohammed; Ostrowski, Stéphane; Williams, Joseph B; Matson, Kevin D; Tieleman, B Irene

2014-01-01

All bird eggs are exposed to microbes in the environment, which if transmitted to the developing embryo, could cause hatching failure. However, the risk of trans-shell infection varies with environmental conditions and is higher for eggs laid in wetter environments. This might relate to generally higher microbial abundances and diversity in more humid environments, including on the surface of eggshells, as well as the need for moisture to facilitate microbial penetration of the eggshell. To protect against microbial infection, the albumen of avian eggs contains antimicrobial proteins, including lysozyme and ovotransferrin. We tested whether lysozyme and ovotransferrin activities varied in eggs of larks (Alaudidae) living along an arid-mesic gradient of environmental aridity, which we used as a proxy for risk of trans-shell infection. Contrary to expectations, lysozyme activity was highest in eggs from hotter, more arid locations, where we predicted the risk of trans-shell infection would be lower. Ovotransferrin concentrations did not vary with climatic factors. Temperature was a much better predictor of antimicrobial protein activity than precipitation, a result inconsistent with studies stressing the importance of moisture for trans-shell infection. Our study raises interesting questions about the links between temperature and lysozyme activity in eggs, but we find no support for the hypothesis that antimicrobial protein deposition is higher in eggs laid in wetter environments.

[Influence of Different Type of Surfactant on Bacteriolytic Activity of Lysozyme].

PubMed

Ivanov, R A; Soboleva, O A; Smirnov, S A; Levashov, P A

2015-01-01

The influence ofvarious surfactants (anionic sodium dodecyl sulfate, SDS, cationic dodecyltrimethylarnmonium bromide, DTAB, and zwitterionic cocoamidopropylbetaine, CAPB) on the activity of the chicken egg lysozyme is investigated. Lysis of Gram-positive bacteria by the enzyme was carried out at pH 7.2 and ionic strength of 0.15 M. It was found that at low SDS and DTAB concentrations (less than 1 x 10(-5) M) the bacteriolytic activity increases by 30-140%. At higher concentrations (1 x 10(-5) - 1 x 10(4) M) the activity returns to the level observed in the absence of the surfactants. The elevated activity correlated with the formation of hydrophobic lysozyme-surfactant complexes. Introduction of CAPB at concentrations above 1 x 10(-5) M sig, nificantly diminished the bacteriolytic activity due to CAPB induced aggregation of lysozyme.

Hen Egg as an Antioxidant Food Commodity: A Review

PubMed Central

Nimalaratne, Chamila; Wu, Jianping

2015-01-01

Intake of antioxidants through diet is known to be important in reducing oxidative damage in cells and improving human health. Although eggs are known for their exceptional, nutritional quality, they are not generally considered as antioxidant foods. This review aims to establish the importance of eggs as an antioxidant food by summarizing the current knowledge on egg-derived antioxidants. Eggs have various natural occurring compounds including the proteins ovalbumin, ovotransferrin and lysozyme in egg white, as well as phosvitin, carotenoids and free aromatic amino acids in egg yolk. Some lipophilic antioxidants such as vitamin E, carotenoids, selenium, iodine and others can be transferred from feed into egg yolk to produce antioxidant-enriched eggs. The bioactivity of egg antioxidants can be affected by food processing, storage and gastrointestinal digestion. Generally thermal processing methods can promote loss of antioxidant properties in eggs due to oxidation and degradation, whereas gastrointestinal digestion enhances the antioxidant properties, due to the formation of new antioxidants (free amino acids and peptides). In summary, in addition to its well-known nutritional contribution to our diet, this review emphasizes the role of eggs as an important antioxidant food. PMID:26404361

Polyethyleneimine assisted-two-step polymerization to develop surface imprinted cryogels for lysozyme purification.

PubMed

Erol, Kadir; Köse, Kazım; Uzun, Lokman; Say, Rıdvan; Denizli, Adil

2016-10-01

Surface imprinting strategy is one of the promising approaches to synthesize plastic antibodies while overcoming the problems in the protein imprinting research. In this study, we focused our attentions on developing two-step polymerization to imprint on the bare surface employing polyethyleneimine (PEI) assisted-coordination of template molecules, lysozyme. For this aim, we firstly synthesized poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA) cryogels as a bare structure. Then, we immobilized PEI onto the cryogels through the addition reaction between GMA and PEI molecules. After that, we determined the amount of free amine (NH2) groups of PEI molecules, subsequently immobilized methacrylate functionalities onto the half of them and another half was used to chelate Cu(II) ions as a mediator between template, lysozyme and PEI groups. After the characterization of the materials developed by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and the micro-computed tomography (μCT), we optimized the lysozyme adsorption conditions from aqueous solution. Before performing lysozyme purification from chicken egg white, we evaluated the effects of pH, interaction time, the initial lysozyme concentration, temperature and ionic strength on the lysozyme adsorption. Moreover, the selectivity of surface imprinted cryogels was examined against cytochrome c and bovine serum albumin (BSA) as the competitors. Finally, the mathematical modeling, which was applied to describe the adsorption process, showed that the experimental data is very well-fitted to the Langmuir adsorption isotherm. Copyright © 2016 Elsevier B.V. All rights reserved.

Influence of osmolytes on protein and water structure: a step to understanding the mechanism of protein stabilization.

PubMed

Bruździak, Piotr; Panuszko, Aneta; Stangret, Janusz

2013-10-03

Results concerning the thermostability of hen egg white lysozyme in aqueous solutions with stabilizing osmolytes, trimethylamine-N-oxide (TMAO), glycine (Gly), and its N-methyl derivatives, N-methylglycine (NMG), N,N-dimethylglycine (DMG), and N,N,N-trimethylglycine (betaine, TMG), have been presented. The combination of spectroscopic (IR) and calorimetric (DSC) data allowed us to establish a link between osmolytes' influence on water structure and their ability to thermally stabilize protein molecule. Structural and energetic characteristics of stabilizing osmolytes' and lysozyme's hydration water appear to be very similar. The osmolytes increase lysozyme stabilization in the order bulk water < TMAO < TMG < Gly < DMG < NMG, which is consistent with the order corresponding to the value of the most probable oxygen-oxygen distance of water molecules affected by osmolytes in their surrounding. Obtained results verified the hypothesis concerning the role of water molecules in protein stabilization, explained the osmophobic effect, and finally helped to bring us nearer to the exact mechanism of protein stabilization by osmolytes.

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody.

PubMed

Ito, W; Nishimura, M; Sakato, N; Fujio, H; Arata, Y

1987-09-01

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.

Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements.

PubMed Central

Kuehner, D E; Heyer, C; Rämsch, C; Fornefeld, U M; Blanch, H W; Prausnitz, J M

1997-01-01

The diffusion of hen egg-white lysozyme has been studied by dynamic light scattering in aqueous solutions of ammonium sulfate as a function of protein concentration to 30 g/liter. Experiments were conducted under the following conditions: pH 4-7 and ionic strength 0.05-5.0 M. Diffusivity data for ionic strengths up to 0.5 M were interpreted in the context of a two-body interaction model for monomers. From this analysis, two potential-of-mean-force parameters, the effective monomer charge, and the Hamaker constant were obtained. At higher ionic strength, the data were analyzed using a model that describes the diffusion coefficient of a polydisperse system of interacting protein aggregates in terms of an isodesmic, indefinite aggregation equilibrium constant. Data analysis incorporated multicomponent virial and hydrodynamic effects. The resulting equilibrium constants indicate that lysozyme does not aggregate significantly as ionic strength increases, even at salt concentrations near the point of salting-out precipitation. PMID:9414232

Different analytical approaches in assessing antibacterial activity and the purity of commercial lysozyme preparations for dairy application.

PubMed

Brasca, Milena; Morandi, Stefano; Silvetti, Tiziana; Rosi, Veronica; Cattaneo, Stefano; Pellegrino, Luisa

2013-05-21

Hen egg-white lysozyme (LSZ) is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing) caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.

Preparation and preliminary characterization of crystallizing fluorescent derivatives of chicken egg white lysozyme

NASA Astrophysics Data System (ADS)

Sumida, John P.; Forsythe, Elizabeth L.; Pusey, Marc L.

2001-11-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp 101 using a carbodiimide coupling procedure. Asp 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactive groups have been bound to His 15, located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp 101-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His 15 have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.

Hen Egg-White Lysozyme Crystallisation: Protein Stacking and Structure Stability Enhanced by a Tellurium(VI)-Centred Polyoxotungstate

PubMed Central

Bijelic, Aleksandar; Molitor, Christian; Mauracher, Stephan G; Al-Oweini, Rami; Kortz, Ulrich; Rompel, Annette

2015-01-01

As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson–Evans-type polyoxometalate (POM), specifically Na6[TeW6O24]⋅22 H2O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid–liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein–protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation. PMID:25521080

Isolation and quantitation of a minor determinant of hen egg white lysozyme bound to I-Ak by using peptide-specific immunoaffinity.

PubMed

Gugasyan, R; Vidavsky, I; Nelson, C A; Gross, M L; Unanue, E R

1998-12-01

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-Ak of APCs. We isolated and concentrated the peptides from the I-Ak extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34-45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33-45), and the longest peptide was a 19-mer (residues 31-49). The two most abundant were 31-47 (1.3 pmol) and 31-46 (1 pmol), while the least abundant were 31-45 (40 fmol) and 32-45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31-47 peptide, the predominant member of this series, was 22 times lower than that of 48-62, the major epitope of HEL. The 31-47 peptide bound about 20-fold weaker to I-Ak compared with the dominant 48-62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.

Evaluation of Proteins' Rotational Diffusion Coefficients from Simulations of Their Free Brownian Motion in Volume-Occupied Environments.

PubMed

Długosz, Maciej; Antosiewicz, Jan M

2014-01-14

We have investigated the rotational dynamics of hen egg white lysozyme in monodisperse aqueous solutions of concentrations up to 250 mg/mL, using a rigid-body Brownian dynamics method that accurately accounts for anisotropies of diffusing objects. We have examined the validity of the free diffusion concept in the analysis of computer simulations of volume-occupied molecular solutions. We have found that, when as the only intermolecular interaction, the excluded volume effect is considered, rotational diffusion of molecules adheres to the free diffusion model. Further, we present a method based on the exact (in the case of the free diffusion) analytic forms of autocorrelation functions of particular vectors rigidly attached to diffusing objects, which allows one to obtain from results of molecular simulations the three principal rotational diffusion coefficients characterizing rotational Brownian motion of an arbitrarily shaped rigid particle for an arbitrary concentration of crowders. We have applied this approach to trajectories resulting from Brownian dynamics simulations of hen egg white lysozyme solutions. We show that the apparent anisotropy of proteins' rotational motions increases with an increasing degree of crowding. Finally, we demonstrate that even if the hydrodynamic anisotropy of molecules is neglected and molecules are simulated using their average translational and rotational diffusion coefficients, excluded volume effects still lead to their anisotropic rotational dynamics.

Modified lysozymes as novel broad spectrum natural antimicrobial agents in foods.

PubMed

Aminlari, Ladan; Hashemi, Marjan Mohammadi; Aminlari, Mahmoud

2014-06-01

In recent years much attention and interest have been directed toward application of natural antimicrobial agents in foods. Some naturally occurring proteins such as lactoperoxidase, lactoferrin, and lysozyme have received considerable attention and are being considered as potential antimicrobial agents in foods. Lysozyme kills bacteria by hydrolyzing the peptidoglycan layer of the cell wall of certain bacterial species, hence its application as a natural antimicrobial agent has been suggested. However, limitations in the action of lysozyme against only Gram-positive bacteria have prompted scientists to extend the antimicrobial effects of lysozyme by several types of chemical modifications. During the last 2 decades extensive research has been directed toward modification of lysozyme in order to improve its antimicrobial properties. This review will report on the latest information available on lysozyme modifications and examine the applicability of the modified lysozymes in controlling growth of Gram-positive and Gram-negative bacteria in foods. The results of modifications of lysozyme using its conjugation with different small molecule, polysaccharides, as well as modifications using proteolytic enzymes will be reviewed. These types of modifications have not only increased the functional properties of lysozyme (such as solubility and heat stability) but also extended the antimicrobial activity of lysozyme. Many examples will be given to show that modification can decrease the count of Gram-negative bacteria in bacterial culture and in foods by as much as 5 log CFU/mL and in some cases essentially eliminated Escherichia coli. In conclusion this review demonstrates that modified lysozymes are excellent natural food preservatives, which can be used in food industry. The subject described in this review article can lead to the development of methods to produce new broad-spectrum natural antimicrobial agents, based on modification of chicken egg white lysozyme, which might potentially replace the currently used synthetic food preservatives. © 2014 Institute of Food Technologists®

Transport phenomena in the crystallization of lysozyme by osmotic dewatering and liquid-liquid diffusion in low gravity

NASA Technical Reports Server (NTRS)

Todd, Paul; Sportiello, Michael G.; Gregory, Derek; Cassanto, John M.; Alvarado, Ulises A.; Ostroff, Robert; Korszun, Z. R.

1993-01-01

Two methods of protein crystallization, osmotic dewatering and liquid-liquid diffusion, like the vapor diffusion (hanging-drop and sessile-drop) methods allow a gradual approach to supersaturation conditions. The crystallization of hen egg-white lysozyme, an extensively characterized protein crystal, in the presence of sodium chloride was used as an experimental model with which to compare these two methods in low gravity and in the laboratory. Comparisons of crystal growth rates by the two methods under the two conditions have, to date, indicated that the rate of crystal growth by osmotic dewatering is nearly the same in low gravity and on the ground, while much faster crystal growth rates can be achieved by the liquid-liquid diffusion method in low gravity.

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

PubMed

Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

2005-08-01

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

On the dynamics of water molecules at the protein solute interfaces.

PubMed

Bernini, A; Spiga, O; Ciutti, A; Chiellini, S; Menciassi, N; Venditti, V; Niccolai, N

2004-10-01

Proteins, with the large variety of chemical groups they present at their molecular surface, are a class of molecules which can be very informative on most of the possible solute-solvent interactions. Hen egg white lysozyme has been used as a probe to investigate the complex solvent dynamics occurring at the protein surface, by analysing the results obtained from Nuclear Magnetic Resonance, X-ray diffractometry and Molecular Dynamics simulations. A consistent overall picture for the dynamics of water molecules close to the protein is obtained, suggesting that a rapid exchange occurs, in a picosecond timescale, among all the possible hydration surface sites both in solution and the solid state, excluding the possibility that solvent molecules can form liquid-crystal-like supramolecular adducts, which have been proposed as a molecular basis of 'memory of water'.

Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods

PubMed Central

Chaari, Ali; Fahy, Christine; Chevillot-Biraud, Alexandre; Rholam, Mohamed

2015-01-01

Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer’s and Parkinson’s diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils. PMID:26571264

A simple apparatus for controlling nucleation and size in protein crystal growth

NASA Technical Reports Server (NTRS)

Gernert, Kim M.; Smith, Robert; Carter, Daniel C.

1988-01-01

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the 'hanging drop' method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.

Nucleation of insulin crystals in a wide continuous supersaturation gradient.

PubMed

Penkova, Anita; Dimitrov, Ivaylo; Nanev, Christo

2004-11-01

Modifying the classical double pulse technique, by using a supersaturation gradient along an insulin solution contained in a glass capillary tube, we found conditions appropriate for the direct measurement of nucleation parameters. The nucleation time lag has been measured. Data for the number of crystal nuclei versus the nucleation time were obtained for this hormone. Insulin was chosen as a model protein because of the availability of solubility data in the literature. A comparison with the results for hen-egg-white lysozyme, HEWL was performed.

Fluorescence Studies of Protein Crystal Nucleation

NASA Technical Reports Server (NTRS)

Pusey, Marc L.

1999-01-01

Fluorescence can be used to study protein crystal nucleation through methods such as anisotropy, quenching, and resonance energy transfer (FRET), to follow pH and ionic strength changes, and follow events occurring at the growth interface. We have postulated, based upon a range of experimental evidence that the growth unit of tetragonal hen egg white lysozyme is an octamer. Several fluorescent derivatives of chicken egg white lysozyme have been prepared. The fluorescent probes lucifer yellow (LY), cascade blue, and 5-((2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS), have been covalently attached to ASP 101. All crystallize in the characteristic tetragonal form, indicating that the bound probes are likely laying within the active site cleft. Crystals of the LY and EDANS derivatives have been found to diffract to at least 1.7 A. A second group of derivatives is to the N-terminal amine group, and these do not crystallize as this site is part of the contact region between the adjacent 43 helix chains. However derivatives at these sites would not interfere with formation of the 43 helices in solution. Preliminary FRET studies have been carried out using N-terminal bound pyrene acetic acid (Ex 340 nm, Em 376 nm) lysozyme as a donor and LY (Ex -425 nm, Em 525 nm) labeled lysozyme as an acceptor. FRET data have been obtained at pH 4.6, 0.1 M NaAc buffer, at 5 and 7% NaCl, 4 C. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10(exp -5) M respectively). The data at both salt concentrations show a consistent trend of decreasing fluorescence intensity of the donor species (PAA) with increasing total protein concentration. This decrease is more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations reflected in the lower solubility. The calculated average distance between any two protein molecules at 5 x 10(exp -6) M is approximately 70nm, well beyond the range where any FRET can be expected. Results from these and ongoing studies will be presented.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

PubMed

Madrigal-Carballo, Sergio; Haas, Linda; Vestling, Martha; Krueger, Christian G; Reed, Jess D

2016-12-01

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Expression of lysozyme in the life history of the house fly (Musca domestica l.).

PubMed

Nayduch, Dana; Joyner, Chester

2013-07-01

From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments.

The Effects of Thermal History on Nucleation of Tetragonal Lysozyme Crystals, or Hot Protein and Cold Nucleation

NASA Technical Reports Server (NTRS)

Burke, Michael; Judge, Russell; Pusey, Marc

2000-01-01

Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.2 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubilities are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to addition of precipitant solution. Warming a lysozyme solution above the phase transition point, then cooling it back below this point, has been shown to affect the subsequent nucleation rate, as determined by the numbers and size of crystals formed, but not the growth rate for the tetragonal crystal form . We have now measured the kinetics of this process and investigated its reversibility. The transition effects are progressive with temperature, having a half time of about 1 hour at 37C at pH 4.8. After holding a lysozyme solution at 37C (prior to addition of precipitant) for 16 hours, then cooling it back to 4C no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. Putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. This differential behaviour may be exploited to elucidate how and where flow affects the lysozyme crystal growth process. The presentation will focus on the results of these and ongoing studies in this area.

Exploration of electrostatic interaction in the hydrophobic pocket of lysozyme: Importance of ligand-induced perturbation of the secondary structure on the mode of binding of exogenous ligand and possible consequences.

PubMed

Panja, Sudipta; Halder, Mintu

2016-08-01

Exogenous ligand binding can be adequate to alter the secondary structure of biomolecules besides other external stimuli. In such cases, structural alterations can complicate on the nature of interaction with the exogenous molecules. In order to accommodate the exogenous ligand, the biomolecule has to unfold resulting in a considerable change to its properties. If the bound ligand can be unbound, the biomolecule gets the opportunity to refold back and return to its native state. Keeping this in mind, we have purposely investigated the interaction of tartrazine (TZ), a well abundant azo food colorant, with two homologous lysozymes, namely, human lysozyme (HLZ) and chicken egg white lysozyme (CEWLZ) in physiological pH condition. The binding of TZ with lysozymes has been identified to accompany a ligand-induced secondary structure alteration as indicated by the circular dichroism spectra, and the reduction of α-helical content is more with HLZ than CEWLZ. Interestingly, the binding is identified to occur in the electronic ground state of TZ with lysozyme in its hydrophobic cavity, containing excess of positive charge, predominantly via electrostatic interaction. With increase of salinity of the medium the protein tends to refold back due to wakening of electrostatic forces and consequent reduction of strength of ligand interaction and unbinding. The entropy enthalpy compensation (EEC) has been probed to understand the binding features and it is found that CEWLZ-TZ shows better compensation than HLZ-TZ complex. This is presumably due to the fact that with CEWLZ the binding does not accompany substantial change in the protein secondary structure and hence ineffective to scramble the EEC. The present study initiates the importance of ligand-perturbed structural alteration of biomolecule in controlling the thermodynamics of binding. If there is a considerable alteration of the protein secondary structure due to binding, it is indicative that such changes should bring in the overall loss of activity of protein. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities.

PubMed

Oh, Hye Young; Kim, Chan-Hee; Go, Hye-Jin; Park, Nam Gyu

2018-05-09

Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5' untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3' UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys. Copyright © 2018. Published by Elsevier Ltd.

On the possibility of using polycrystalline material in the development of structure-based generic assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allaire, Marc, E-mail: allaire@bnl.gov; Moiseeva, Natalia; Botez, Cristian E.

The correlation coefficients calculated between raw powder diffraction profiles can be used to identify ligand-bound/unbound states of lysozyme. The discovery of ligands that bind specifically to a targeted protein benefits from the development of generic assays for high-throughput screening of a library of chemicals. Protein powder diffraction (PPD) has been proposed as a potential method for use as a structure-based assay for high-throughput screening applications. Building on this effort, powder samples of bound/unbound states of soluble hen-egg white lysozyme precipitated with sodium chloride were compared. The correlation coefficients calculated between the raw diffraction profiles were consistent with the known bindingmore » properties of the ligands and suggested that the PPD approach can be used even prior to a full description using stereochemically restrained Rietveld refinement.« less

Rapid localized crystallization of lysozyme by laser trapping.

PubMed

Yuyama, Ken-Ichi; Chang, Kai-Di; Tu, Jing-Ru; Masuhara, Hiroshi; Sugiyama, Teruki

2018-02-28

Confining protein crystallization to a millimetre size was achieved within 0.5 h after stopping 1 h intense trapping laser irradiation, which shows excellent performance in spatial and temporal controllability compared to spontaneous nucleation. A continuous-wave near-infrared laser beam is tightly focused into a glass/solution interfacial layer of a supersaturated buffer solution of hen egg-white lysozyme (HEWL). The crystallization is not observed during laser trapping, but initiated by stopping the laser irradiation. The generated crystals are localized densely in a circular area with a diameter of a few millimetres around the focal spot and show specific directions of the optical axes of the HEWL crystals. To interpret this unique crystallization, we propose a mechanism that nucleation and the subsequent growth take place in a highly concentrated domain consisting of HEWL liquid-like clusters after turning off laser trapping.

Biophysical insights into the interaction of hen egg white lysozyme with therapeutic dye clofazimine: modulation of activity and SDS induced aggregation of model protein.

PubMed

Ajmal, Mohammad Rehan; Chaturvedi, Sumit Kumar; Zaidi, Nida; Alam, Parvez; Zaman, Masihuz; Siddiqi, Mohammad Khursheed; Nusrat, Saima; Jamal, Mohammad Sarwar; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

2017-08-01

The present study details the binding process of clofazimine to hen egg white lysozyme (HEWL) using spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and molecular docking techniques. Clofazimine binds to the protein with binding constant (K b ) in the order of 1.57 × 10 4 at 298 K. Binding process is spontaneous and exothermic. Molecular docking results suggested the involvement of hydrogen bonding and hydrophobic interactions in the binding process. Bacterial cell lytic activity in the presence of clofazimine increased to more than 40% of the value obtained with HEWL only. Interaction of the drug with HEWL induced ordered secondary structure in the protein and molecular compaction. Clofazimine also effectively inhibited the sodium dodecyl sulfate (SDS) induced amyloid formation in HEWL and caused disaggregation of preformed fibrils, reinforcing the notion that there is involvement of hydrophobic interactions and hydrogen bonding in the binding process of clofazimine with HEWL and clofazimine destabilizes the mature fibrils. Further, TEM images confirmed that fibrillar species were absent in the samples where amyloid induction was performed in the presence of clofazimine. As clofazimine is a drug less explored for the inhibition of fibril formation of the proteins, this study reports the inhibition of SDS-induced amyloid formation of HEWL by clofazimine, which will help in the development of clofazimine-related molecules for the treatment of amyloidosis.

Buffer Effects in the Solubility, Nucleation and Growth of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Gibson, Ursula J.

1999-01-01

The growth of protein crystals is important for determination of their three-dimensional structure, which relates to their biochemical functions and to the practical goal of designing pharmaceuticals to modify that function. While many proteins have been successfully crystallized by a variety of methods, there is still limited understanding of the process of nucleation and growth of even the simplest proteins. Chicken egg-white lysozyme (CEWL) is readily crystallized under a variety of conditions, and studies underway at MSFC are designed to elucidate the mechanisms by which the crystals nucleate and grow. We have investigated the effect of buffer choice on the solubility, nucleation and growth of CEWL. CEWL was purified by dialysis against a .05M phosphate buffer and chromatographic separation from contaminants in a sepharose column. Solubility studies were made as a function of buffer concentration for phosphate and formate buffers, and the nucleation and growth of crystals at 10 C was studied as a function of pH for oxalate, succinate, formate, butyrate, carbonate, phosphate and acetate buffer solutions. The solubility data support the conclusion that there is a solubility minimum as a function of buffer concentration for amphiphilic molecules, while no minimum is observed for a phosphate buffer. Nucleation is suppressed at pH greater than pKa for all buffers except phosphate. The aspect ratio of the (110) faces is shown to be a function of crystal size, rather than pH.

Preparation and Preliminary Characterization of Crystallizing Fluorescent Derivatives of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Sumida, John; Forsythe, Elizabeth L.; Pusey, Marc L.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp(sup 101) using a carbodiimide coupling procedure. Asp(sup 101) lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to His(sup 15), located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp(sup 101)-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His(sup 15) have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.

Preparation and Preliminary Characterization of Crystallizing Fluorescent Derivatives of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Sumida, John P.; Forsythe, Elizabeth L.; Pusey, Marc L.

2001-01-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-i-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp(sup 101) using a carbodiimide coupling procedure. Asp(sup 101) lies within the active site cleft, and it is believed that the probes are 'buried' within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive five groups have been bound to His(sup 15), located on the 'back side' of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp(sup 101)-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His(sup 15) have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.

The Effect of Complex Solvents on the Structure and Dynamics of Protein Solutions: the case of Lysozyme in Trehalose/Water Mixtures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghattyvenkatakrishna, Pavan K; Carri, Gustavo A.

We present a Molecular Dynamics simulation study of the effect of trehalose concentration on the structure and dynamics of individual proteins immersed in trehalose/water mixtures. Hen Egg White Lysozyme is used in this study and trehalose concentrations of 0%, 10%, 20%, 30% and 100% by weight are explored. Surprisingly, we have found that changes in trehalose concentration do not change the global structural characteristics of the protein as measured by standard quantities like the mean square deviation, radius of gyration, solvent accessible surface area, inertia tensor and asphericity. Only in the limit of pure trehalose these metrics change significantly. Specifically,more » we found that the protein is compressed by 2% when immersed in pure trehalose. At the amino acid level there is noticeable rearrangement of the surface residues due to the change in polarity of the surrounding environment with the addition of trehalose. From a dynamic perspective, our computation of the Incoherent Intermediate Scattering Function shows that the protein slows down with increasing trehalose concentration; however, this slowdown is not monotonic. Finally, we also report in-depth results for the hydration layer around the protein including its structure, hydrogen- bonding characteristics and dynamic behavior at different length scales.« less

Coarse-graining of proteins based on elastic network models

NASA Astrophysics Data System (ADS)

Sinitskiy, Anton V.; Voth, Gregory A.

2013-08-01

To simulate molecular processes on biologically relevant length- and timescales, coarse-grained (CG) models of biomolecular systems with tens to even hundreds of residues per CG site are required. One possible way to build such models is explored in this article: an elastic network model (ENM) is employed to define the CG variables. Free energy surfaces are approximated by Taylor series, with the coefficients found by force-matching. CG potentials are shown to undergo renormalization due to roughness of the energy landscape and smoothing of it under coarse-graining. In the case study of hen egg-white lysozyme, the entropy factor is shown to be of critical importance for maintaining the native structure, and a relationship between the proposed ENM-mode-based CG models and traditional CG-bead-based models is discussed. The proposed approach uncovers the renormalizable character of CG models and offers new opportunities for automated and computationally efficient studies of complex free energy surfaces.

Crystallization of Chicken Egg White Lysozyme from Sulfate Salts

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth; Pusey, Marc

1998-01-01

It has been "known" that chicken egg white lysozyme does not crystallize from sulfate, particularly ammonium sulfate, salts, but instead gives amorphous precipitates. This has been the basis of several studies using lysozyme comparing macromolecule crystal nucleation and amorphous precipitation. Recently Ries-Kautt et al (Acta Cryst D50, (1994) 366) have shown that purified isoionic CEWL could be crystallized from low concentrations of sulfate at basic pH, and we subsequently showed that in fact CEWL could be purified in both the tetragonal and orthorhombic forms using ammonium sulfate over the pH range 4.0 to 7.8 (Acta Cryst D53, (1997) 795). We have now extended these observations to include a range of common sulfate salts, specifically sodium, potassium, rubidium, magnesium, and manganese sulfates. In all cases but the manganese sulfates both the familiar tetragonal and orthorhombic forms were obtained, with unit cell dimensions close to those known for the "classic" sodium chloride crystallized forms. Manganese sulfate has only yielded orthorhombic crystals to date. All crystallizations were carried out using low (typically less than or equal to 6 M) salt and high (greater than approximately 90 mg/ml) protein concentrations. As with ammonium sulfate, the tetragonal - orthorhombic phase shift appears to be a function of both the temperature and the protein concentration, with higher temperatures and concentrations favoring the orthorhombic and lower the tetragonal form. The phase change range is somewhat reduced for the sulfate salts, depending upon conditions being typically between approximately 15 - 20 C. Both the magnesium and manganese sulfates gave crystals at salt concentrations over 0.6 M as well, with magnesium sulfate giving a very slowly nucleating and growing hexagonal form. A triclinic crystal form, characterized by aggressively small crystals (typically 0.1 mm in size) has been occasionally obtained from ammonium sulfate. Finally, preliminary spot solubility determinations have suggested that in some cases the solubility increases with increasing salt concentrations.

Inhibition of Growth of Candida albicans by a Lysozyme-chitosan Conjugate, LYZOX and its Combination with Decanoic Acid.

PubMed

Kageshima, Hiroki; Hayama, Kazumi; Takahashi, Miki; Abe, Miho; Yamada, Tsuyoshi; Saito, Akira; Hirano, Shoichiro; Murakami, Yoichi; Abe, Shigeru

2017-01-01

A lysozyme-chitosan conjugate preparation (LYZOX), produced from egg white lysozyme and chitosan by Maillard reaction, is a commercial product developed as a cosmetic ingredient or food additive. Effects of LYZOX on in vitro growth of Candida albicans were examined. C. albicans cells were treated with LYZOX for 3 hrs, and then washed and cultured for an additional 16 hrs in modified RPMI1640 medium. Mycelial growth of C. albicans was clearly inhibited by more than 100 μg/ml of LYZOX in a concentration-dependent manner. On the other hand, corresponding concentration of chitosan or lysozyme or their mixture only scarcely showed clear inhibitory effect. Similarly, anti-Candida activity of the combination of LYZOX and decanoic acid, a middle-chain fatty acid, was also examined. Inhibitory activity of this combination against mycelial growth of C. albicans was very potent and appeared synergistic, since fractionated inhibitory concentration (FIC) index for 70% growth inhibition was calculated to be 0.20. Oral application of this combination improved the symptoms of Candida-infected-tongue in an experimental murine candidiasis model. On the basis of these results, the possible application of LYZOX as a new functional product with anti-candida activity was discussed.

Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

NASA Astrophysics Data System (ADS)

Mariño, Laura; Pauwels, Kris; Casasnovas, Rodrigo; Sanchis, Pilar; Vilanova, Bartolomé; Muñoz, Francisco; Donoso, Josefa; Adrover, Miquel

2015-07-01

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

Optimization of protein solution by a novel experimental design method using thermodynamic properties.

PubMed

Kim, Nam Ah; An, In Bok; Lee, Sang Yeol; Park, Eun-Seok; Jeong, Seong Hoon

2012-09-01

In this study, the structural stability of hen egg white lysozyme in solution at various pH levels and in different types of buffers, including acetate, phosphate, histidine, and Tris, was investigated by means of differential scanning calorimetry (DSC). Reasonable pH values were selected from the buffer ranges and were analyzed statistically through design of experiment (DoE). Four factors were used to characterize the thermograms: calorimetric enthalpy (ΔH), temperature at maximum heat flux (T( m )), van't Hoff enthalpy (ΔH( V )), and apparent activation energy of protein solution (E(app)). It was possible to calculate E(app) through mathematical elaboration from the Lumry-Eyring model by changing the scan rate. The transition temperature of protein solution, T( m ), increased when the scan rate was faster. When comparing the T( m ), ΔH( V ), ΔH, and E(app) of lysozyme in various pH ranges and buffers with different priorities, lysozyme in acetate buffer at pH 4.767 (scenario 9) to pH 4.969 (scenario 11) exhibited the highest thermodynamic stability. Through this experiment, we found a significant difference in the thermal stability of lysozyme in various pH ranges and buffers and also a new approach to investigate the physical stability of protein by DoE.

Determination of allergenic egg proteins in food by protein-, mass spectrometry-, and DNA-based methods.

PubMed

Lee, Ji-Yun; Kim, Chang Jong

2010-01-01

Egg allergy is one of the most common food allergies in both adults and children, and foods including eggs and their byproducts should be declared under food allergen labeling policies in industrial countries. Therefore, to develop and validate a sensitive and specific method to detect hidden egg allergens in foods, we compared immunochemical, DNA-based, and proteomic methods for detecting egg allergens in foods using egg allergen standards such as egg whole protein, egg white protein, egg yolk protein, ovomucoid, ovalbumin, ovotransferrin, lysozyme, and alpha-livetin. Protein-based immunochemical methods, including ELISA as an initial screening quantitative analysis and immunoblotting as a final confirmatory qualitative analysis, were very sensitive and specific in detecting potentially allergenic egg residues in processed foods in trace amounts. In contrast, the proteomics-based, matrix-assisted laser desorption/ionization time-of-flight MS and LC-tandem quadrupole time-of-flight MS methods were not able to detect some egg allergens, such as ovomucoid, because of its nondenaturing property under urea and trypsin. The DNA-based PCR method could not distinguish between egg and chicken meat because it is tissue-nonspecific. In further studies for the feasibility of these immunochemical methods on 100 real raw dietary samples, four food samples without listed egg ingredients produced a positive response by ELISA, but exhibited negative results by immunoblotting.

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

PubMed Central

Oliveira Silva, Catarina; Petersen, Steffen B.; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation. PMID:26656259

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability.

PubMed

Oliveira Silva, Catarina; Petersen, Steffen B; Pinto Reis, Catarina; Rijo, Patrícia; Molpeceres, Jesús; Vorum, Henrik; Neves-Petersen, Maria Teresa

2015-01-01

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.

Nucleation and Convection Effects in Protein Crystal Growth

NASA Technical Reports Server (NTRS)

Rosenberger, Franz

1997-01-01

Work during the second year under this grant (NAG8-1161) resulted in several major achievements. We have characterized protein impurities as well as microheterogeneities in the proteins hen egg white lysozyme and horse spleen apoferritin, and demonstrated the effects of these impurities on nucleation and crystallization. In particular, the purification of apoferritin resulted in crystals with an X-ray diffraction resolution of better than 1.8 A, i.e. a 1 A improvement over earlier work on the cubic form. Furthermore, we have shown, in association with studies of liquid-liquid phase separation, that depending on the growth conditions, lysozyme can produce all growth morphologies that have been observed with other proteins. Finally, in connection with our experimental and simulation work on growth step bunching, we have developed a system-dependent criterion for advantages and disadvantages of crystallization from solution under reduced gravity. In the following, these efforts are described in some detail.

Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation.

PubMed

Goldberg, M E; Expert-Bezançon, N; Vuillard, L; Rabilloud, T

1996-01-01

Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase. The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

Preferential solvation of lysozyme in dimethyl sulfoxide/water binary mixture probed by terahertz spectroscopy.

PubMed

Das, Dipak Kumar; Patra, Animesh; Mitra, Rajib Kumar

2016-09-01

We report the changes in the hydration dynamics around a model protein hen egg white lysozyme (HEWL) in water-dimethyl sulfoxide (DMSO) binary mixture using THz time domain spectroscopy (TTDS) technique. DMSO molecules get preferentially solvated at the protein surface, as indicated by circular dichroism (CD) and Fourier transform infrared (FTIR) study in the mid-infrared region, resulting in a conformational change in the protein, which consequently modifies the associated hydration dynamics. As a control we also study the collective hydration dynamics of water-DMSO binary mixture and it is found that it follows a non-ideal behavior owing to the formation of DMSO-water clusters. It is observed that the cooperative dynamics of water at the protein surface does follow the DMSO-mediated conformational modulation of the protein. Copyright © 2016 Elsevier B.V. All rights reserved.

A genome-wide screen identifies Salmonella Enteritidis lipopolysaccharide biosynthesis and the HtrA heat shock protein as crucial factors involved in egg white persistence at chicken body temperature.

PubMed

Raspoet, R; Shearer, N; Appia-Ayme, C; Haesebrouck, F; Ducatelle, R; Thompson, A; Van Immerseel, F

2014-05-01

Eggs contaminated with Salmonella Enteritidis are an important source of human foodborne Salmonella infections. Salmonella Enteritidis is able to contaminate egg white during formation of the egg within the chicken oviduct, and it has developed strategies to withstand the antimicrobial properties of egg white to survive in this hostile environment. The mechanisms involved in the persistence of Salmonella Enteritidis in egg white are likely to be complex. To address this issue, a microarray-based transposon library screen was performed to identify genes necessary for survival of Salmonella Enteritidis in egg white at chicken body temperature. The majority of identified genes belonged to the lipopolysaccharide biosynthesis pathway. Additionally, we provide evidence that the serine protease/heat shock protein (HtrA) appears essential for the survival of Salmonella Enteritidis in egg white at chicken body temperature.

Characterization of protein hydration by solution NMR spectroscopy

NASA Astrophysics Data System (ADS)

Wand, Joshua

A comprehensive understanding of the interactions between protein molecules and hydration water remains elusive. Solution nuclear magnetic resonance (NMR) spectroscopy has been proposed as a means to characterize these interactions but is plagued with artifacts when employed in bulk aqueous solution. Encapsulation of proteins in reverse micelles prepared in short chain alkane solvents can overcome these technical limitations. Application of this approach has revealed that the interaction of water with the surface of protein molecules is quite heterogeneous with some regions of the protein having long-lived interactions while other regions show relatively transient hydration. Results from several proteins will be presented including ubiquitin, staphylococcal nuclease, interleukin 1beta, hen egg white lysozyme (HEWL) and T4 lysozyme. Ubiquitin and interleukin 1beta are signaling proteins and interact with other proteins through formation of dry protein-protein interfaces. Interestingly, the protein surfaces of the free proteins show relatively slowed (restricted) motion at the surface, which is indicative of low residual entropy. Other regions of the protein surface have relatively high mobility water. These results are consistent with the idea that proteins have evolved to maximize the hydrophobic effect in optimization of binding with protein partners. As predicted by simulation and theory, we find that hydration of internal hydrophobic cavities of interleukin 1beta and T4 lysozyme is highly disfavored. In contrast, the hydrophilic polar cavity of HEWL is occupied by water. Initial structural correlations suggest that hydration of alpha helical structure is characterized by relatively mobile water while those of beta strands and loops are more ordered and slowed. These and other results from this set of proteins reveals that the dynamical and structural character of hydration of proteins is heterogeneous and complex. Supported by the National Science Foundation.

The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth; Pusey, Marc Lee

1994-01-01

Measurements were made of the (110) and (101) face growth rates of the tetragonal form of hen egg white lysozyme at 0.1M sodium acetate buffer, pH 4.0, from 4 to 22 C and with 3.0%, 5.0%, and 7.0% NaCl used as the precipitating salt. The data were collected at supersaturation ratios ranging from approximately 4 to approximately 63. Both decreasing temperature and increasing salt concentrations shifted plots of the growth rate versus C/C(sat) to the right, i.e. higher supersaturations were required for comparable growth rates. The observed trends in the growth data are counter to those expected from the solubility data. If tetragonal lysozyme crystal growth is by addition of ordered aggregates from the solution, then the observed growth data could be explained as a result of the effects of lowered temperature and increased salt concentration on the kinetics and equilibrium processes governing protein-protein interactions in solution. The data indicate that temperature would be a more tractable means of controlling the growth rate for tetragonal lysozyme crystals contrary to the usual practice in, e.g., vapor diffusion protein crystal growth, where both the precipitant and protein concentrations are simultaneously increased. However, the available range for control is dependent upon the protein concentration, with the greatest growth rate control being at the lower concentration.

Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions

NASA Technical Reports Server (NTRS)

Nadarajah, Arunan

1996-01-01

Molecular forces, such as electrostatic, hydrophobic, van der Waals and steric forces, are known to be important in determining protein interactions. These forces are affected by the solution conditions and changing the pH, temperature or the ionic strength of the solution can sharply affect protein interactions. Several investigations of protein crystallization have shown that this process is also strongly dependent on solution conditions. As the ionic strength of the solution is increased, the initially soluble protein may either crystallize or form an amorphous precipitate at high ionic strengths. Studies done on the model protein hen egg white lysozyme have shown that different crystal forms can be easily and reproducibly obtained, depending primarily on the anion used to desolubilize the protein. In this study we employ pyranine to probe the effect of various anions on the water structure. Additionally, lysozyme crystallization was carried out at these conditions and the crystal form was determined by X-ray crystallography. The goal of the study was to understand the physico-chemical basis for the effect of changing the anion concentration on the equilibrium form of lysozyme crystals. It will also verify the hypothesis that the anions, by altering the bulk water structure in the crystallizing solutions, alter the surface energy of the between the crystal faces and the solution and, consequently, the equilibrium form of the crystals.

The effect of protein–precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reis, Nuno M.; Chirgadze, Dimitri Y.; Blundell, Tom L.

The nucleation of lysozyme in microbatch experiments was linked to the formation of protein–precipitant interfaces. The use of oscillatory shear allowed decreasing the nucleation rate and extending the growth period for lysozyme crystals, presumably through the control of the number of interfaces and removal of impurities or defects. This paper is concerned with the effect of protein–precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a proteinmore » drop and the optical development of the protein–precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable ‘fingers’ that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography.« less

Passive maternal exposure to environmental microbes selectively modulates the innate defences of chicken egg white by increasing some of its antibacterial activities

PubMed Central

2013-01-01

Background Egg defence against bacterial contamination relies on immunoglobulins (IgY) concentrated in the yolk and antimicrobial peptides/proteins predominantly localized in the egg white (EW). Hens contaminated with pathogenic microorganisms export specific IgYs to the egg (adaptative immunity). No evidence of such regulation has been reported for the antimicrobial peptides/proteins (innate immunity) which are preventively secreted by the hen oviduct and are active against a large range of microbes. We investigated whether the egg innate defences can be stimulated by the environmental microbial contamination by comparing the antimicrobial activity of EW of hens raised in three extreme breeding conditions: Germ-free (GF), Specific Pathogen Free (SPF) and Conventional (C) hens. Results The difference in the immunological status of GF, SPF and C hens was confirmed by the high stimulation of IL-1β, IL-8 and TLR4 genes in the intestine of C and SPF groups. EW from C and SPF groups demonstrated higher inhibitory effect against Staphylococcus aureus (13 to 18%) and against Streptococcus uberis (31 to 35%) as compared to GF but showed similar activity against Salmonella Enteritidis, Salmonella Gallinarum, Escherichia coli and Listeria monocytogenes. To further investigate these results, we explored putative changes amongst the three main mechanisms of egg antimicrobial defence: the sequestration of bacterial nutrients, the inactivation of exogenous proteases and the direct lytic action on microorganisms. Lysozyme activity, chymotrypsin-, trypsin- and papain-inhibiting potential of EW and the expression of numerous antimicrobial genes were not stimulated suggesting that these are not responsible for the change in anti-S. aureus and anti-S. uberis activity. Moreover, whereas the expression levels of IL-1β, IL-8 and TLR4 genes were modified by the breeding conditions in the intestine of C and SPF groups they were not modified in the magnum where egg white is formed. Conclusions Altogether, these data revealed that the degree of environmental microbial exposure of the hen moderately stimulated the egg innate defence, by reinforcing some specific antimicrobial activities to protect the embryo and to insure hygienic quality of table eggs. PMID:23758641

Structure-based analysis reveals hydration changes induced by arginine hydrochloride.

PubMed

Nakakido, Makoto; Tanaka, Yoshikazu; Mitsuhori, Mariko; Kudou, Motonori; Ejima, Daisuke; Arakawa, Tsutomu; Tsumoto, Kouhei

2008-10-01

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

PubMed

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens

PubMed Central

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies. PMID:26713728

Maternally derived egg hormones, antibodies and antimicrobial proteins: common and different pathways of maternal effects in Japanese quail.

PubMed

Okuliarova, Monika; Kankova, Zuzana; Bertin, Aline; Leterrier, Christine; Mostl, Erich; Zeman, Michal

2014-01-01

Avian eggs contain a variety of maternally-derived substances that can influence the development and performance of offspring. The levels of these egg compounds vary in relation to environmental and genetic factors, but little is known about whether there are correlative links between maternal substances in the egg underlying common and different pathways of maternal effects. In the present study, we investigated genetically determined variability and mutually adjusted deposition of sex hormones (testosterone-T, androstenedione-A4 and progesterone-P4), antibodies (IgY) and antimicrobial proteins (lysozyme) in eggs of Japanese quail (Coturnix japonica). We used different genetic lines that were independently selected for yolk T concentrations, duration of tonic immobility and social reinstatement behaviour, since both selections for behavioural traits (fearfulness and social motivation, respectively) produced considerable correlative responses in yolk androgen levels. A higher selection potential was found for increased rather than decreased yolk T concentrations, suggesting that there is a physiological minimum in egg T levels. Line differences in yolk IgY concentrations were manifested within each selection experiment, but no consistent inter-line pattern between yolk IgY and T was revealed. On the other hand, a consistent inverse inter-line pattern was recorded between yolk IgY and P4 in both selections for behavioural traits. In addition, selections for contrasting fearfulness and social motivation were associated with changes in albumen lysozyme concentrations and an inverse inter-line pattern between the deposition of yolk IgY and albumen lysozyme was found in lines selected for the level of social motivation. Thus, our results demonstrate genetically-driven changes in deposition of yolk T, P4, antibodies and albumen lysozyme in the egg. This genetic variability can partially explain mutually adjusted maternal deposition of sex hormones and immune-competent molecules but the inconsistent pattern of inter-line differences across all selections indicates that there are other underlying mechanisms, which require further studies.

Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanfield, R.L.; Dooley, H.; Verdino, P.

2007-07-13

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts withmore » antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.« less

A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Lu; Velikovsky, C. Alejandro; Xu, Gang

Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called 'variable lymphocyte receptors' (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like {gamma}{delta} T-cell receptors (TCRs) but unlike {alpha}{beta} TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus,more » these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and {gamma}{delta} TCRs, and the exclusive expression on the lymphocyte surface that is unique to {alpha}{beta} and {gamma}{delta} TCRs.« less

Effects of pH, temperature and pulsed electric fields on the turbidity and protein aggregation of ovomucin-depleted egg white.

PubMed

Liu, Ya-Fei; Oey, Indrawati; Bremer, Phil; Carne, Alan; Silcock, Pat

2017-01-01

The effect of either pulsed electric fields (PEF) or thermal processing on protein aggregation of ovomucin-depleted egg white (OdEW) solutions at different pH was assessed by solution turbidity and SDS-PAGE. Heating to 60°C for 10min caused marked protein aggregation of OdEW at pH5, 7, and 9. At constant electric field strength (E=1.4-1.8kV/cm), PEF processing under high specific energy input (W spec =260-700kJ/kg) induced some protein aggregation at pH5 and 7, but not at either pH4 or 9. Similar effects of pH on protein aggregation were observed upon PEF processing at varied E (from 0.7 to 1.7kV/cm) but with constant W spec (713kJ/kg). Analysis by SDS-PAGE revealed that proteins in the OdEW solution at pH5 were most susceptible to both PEF- and heat-induced protein aggregation and lysozyme was only involved in the formation of insoluble aggregates under PEF. The present study shows that PEF treatment has considerable potential for minimizing protein aggregation in the processing of heat-labile egg white proteins. Retaining the OdEW proteins in solution during processing has potential industry application, for example, protein fortification of drinks with OdEW, where minimizing solution turbidity would be advantageous. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase.

PubMed

Kim, Jihoon; Chang, Ji-Youn; Kim, Yoon-Young; Kim, Moon-Jong; Kho, Hong-Seop

2018-05-01

To investigate the effects of the molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase in solution and on the hydroxyapatite surface. Hyaluronic acids of four different molecular weights (10 kDa, 100 kDa, 1 MDa, and 2 MDa), hen egg-white lysozyme, bovine lactoperoxidase, and human whole saliva were used. Viscosity values of hyaluronic acids were measured using a cone-and-plate viscometer at six different concentrations (0.1-5.0 mg/mL). Enzymatic activities of lysozyme and peroxidase were examined by hydrolysis of fluorescein-labeled Micrococcus lysodeikticus and oxidation of fluorogenic 2',7'-dichlorofluorescein to fluorescing 2',7'-dichlorofluorescein, respectively. In solution assays, only 2 MDa-hyaluronic acid significantly inhibited lysozyme activities in saliva. In surface assays, hyaluronic acids inhibited lysozyme and peroxidase activities; the inhibitory activities were more apparent with high-molecular-weight ones in saliva than in purified enzymes. The 100 kDa-hyaluronic acid at 5.0 mg/mL, 1 MDa-one at 0.5 mg/mL, and 2 MDa-one at 0.2 mg/mL showed viscosity values similar to those of human whole saliva at a shear rate range required for normal oral functions. The differences among the influences of the three conditions on the enzymatic activities were not statistically significant. High-molecular-weight hyaluronic acids at low concentration and low-molecular-weight ones at high concentration showed viscosity values similar to those of human whole saliva. Inhibitory effects of hyaluronic acids on lysozyme and peroxidase activities were more significant with high-molecular-weight ones on the surface and in saliva compared with in solution and on purified enzymes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

1999-01-01

A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

Crawford, Lisa; Karr, Laurel; Pusey, Marc

1998-01-01

A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

Protein nanoparticle electrostatic interaction: size dependent counterions induced conformational change of hen egg white lysozyme.

PubMed

Ghosh, Goutam; Panicker, Lata; Barick, K C

2014-06-01

In our earlier paper (Ghosh et al., 2013), we have shown that (i) the positively charged hen egg white lysozyme (HEWL), dispersed in water, binds electrostatically with the negatively functionalized iron oxide nanoparticles (IONPs), and (ii) the Na(+) counterions, associated with functionalized IONPs, diffuse into bound proteins and irreversibly unfold them. Having this information, we have extended our investigation and report here the effect of the size and the charge of alkaline metal counterions on the conformational modification of HEWL. In order to obtain a negative functional 'shell' on IONPs and the counterions of different size and charge we have functionalized IONPs with different derivatives of citrate, namely, tri-lithium citrate (TLC, Li3C6H5O7), tri-sodium citrate (TSC, Na3C6H5O7), tri-potassium citrate (TKC, K3C6H5O7) and tri-magnesium citrate (TMC, Mg3C12H10O14). The size of counterions varies as Mg(2+)

Hen egg-white lysozyme crystallisation: protein stacking and structure stability enhanced by a Tellurium(VI)-centred polyoxotungstate.

PubMed

Bijelic, Aleksandar; Molitor, Christian; Mauracher, Stephan G; Al-Oweini, Rami; Kortz, Ulrich; Rompel, Annette

2015-01-19

As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson-Evans-type polyoxometalate (POM), specifically Na6 [TeW6 O24 ]⋅22 H2 O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid-liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein-protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Superactivity of MOF-808 toward Peptide Bond Hydrolysis.

PubMed

Ly, Hong Giang T; Fu, Guangxia; Kondinski, Aleksandar; Bueken, Bart; De Vos, Dirk; Parac-Vogt, Tatjana N

2018-05-03

MOF-808, a Zr(IV)-based metal-organic framework, has been proven to be a very effective heterogeneous catalyst for the hydrolysis of the peptide bond in a wide range of peptides and in hen egg white lysozyme protein. The kinetic experiments with a series of Gly-X dipeptides with varying nature of amino acid side chain have shown that MOF-808 exhibits selectivity depending on the size and chemical nature of the X side chain. Dipeptides with smaller or hydrophilic residues were hydrolyzed faster than those with bulky and hydrophobic residues that lack electron rich functionalities which could engage in favorable intermolecular interactions with the btc linkers. Detailed kinetic studies performed by 1 H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10 -4 s -1 ( t 1/2 = 0.72 h), which is more than 4 orders of magnitude faster compared to the uncatalyzed reaction. Importantly, MOF-808 can be recycled several times without significantly compromising the catalytic activity. A detailed quantum-chemical study combined with experimental data allowed to unravel the role of the {Zr 6 O 8 } core of MOF-808 in accelerating Gly-Gly hydrolysis. A mechanism for the hydrolysis of Gly-Gly by MOF-808 is proposed in which Gly-Gly binds to two Zr(IV) centers of the {Zr 6 O 8 } core via the oxygen atom of the amide group and the N-terminus. The activity of MOF-808 was also demonstrated toward the hydrolysis of hen egg white lysozyme, a protein consisting of 129 amino acids. Selective fragmentation of the protein was observed with 55% yield after 25 h under physiological pH.

Preparation and application of hollow molecularly imprinted polymers with a super-high selectivity to the template protein.

PubMed

Chen, Yang; He, Xi-Wen; Mao, Jie; Li, Wen-You; Zhang, Yu-Kui

2013-10-01

Protein-imprinted polymers with hollow cores that have a super-high imprinting factor were prepared by etching the core of the surface-imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single-protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super-high imprinting factor was obtained. The as-prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP-induced noncooperative thermal unfolding of hen lysozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Honglin; Yin, Peidong; He, Shengnan

To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperativemore » unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.« less

Membrane crystallization of lysozyme: kinetic aspects

NASA Astrophysics Data System (ADS)

Profio, Gianluca Di; Curcio, Efrem; Cassetta, Alberto; Lamba, Doriano; Drioli, Enrico

2003-10-01

The relevant kinetic aspects related to an innovative method of biological macromolecules crystallization based on microporous hydrophobic membranes, used both as active surfaces to promote heterogeneous nucleation and as a mass-transfer apparatus to concentrate macromolecular solutions by solvent removal in vapour phase, have been evaluated. Polypropylene membranes, supplied in the form of hollow fibres, have been aligned in a versatile system, designed for an on-line spectrophotometric monitoring of hen egg white lysozyme crystallizing solutions (experimental conditions: 0.1 M NaAc/HAc Buffer pH 4.6, 0.5-5.8% wt/vol NaCl, 20°C). The turbidity measurements have been exploited in order to follow: (i) the induction time of crystallization, (ii) the early stage nucleation kinetics based on the Rayleigh scattering theory, and (iii) the crystal growth rate (coupled with data evaluated from image-analysis carried out by optical microscopy) under a model hypothesis of exponential growth of clusters. The crystals have been qualitatively assessed by an X-ray crystallographic analysis carried out at the synchrotron light laboratory ELETTRA.

Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme

PubMed Central

León-Sicairos, Nidia; López-Soto, Fernando; Reyes-López, Magda; Godínez-Vargas, Delfino; Ordaz-Pichardo, Cynthia; de la Garza, Mireya

2006-01-01

Objectives: To identify amoebicidal components in human milk and the effect of iron on the amoebicidal activity. Design: Investigation in axenic cultures of Entamoeba histolytica trophozoites. Methods: Amoebas were treated with 5%–20% of human, bovine and swine milk, with 10% of human milk fractions (i.e., casein, proteins except casein and fat) or with 1 mg/ml of human milk apo-lactoferrin, human secretory immunoglobulin type A (sIgA) and chicken egg-white lysozyme (i.e., purified proteins). Milk proteins were detected using immunoblot. Confocal microscopy was used to define the interaction of milk proteins (100 μM each) and amoebas. Experiments were done at least three times in triplicate, and mean and standard deviations were calculated. Results: Human and bovine milk were amoebicidal showing a concentration-dependent effect. The amoebicidal effect was increased in the absence of iron. Milk protein fractions, with the exception of casein, were the components responsible for the amoebicidal activity found. Apo-lactoferrin, sIgA and lysozyme were identified in the amoebicidal milk protein fraction. Apo-lactoferrin showed the major amoebicidal effect. These proteins, either alone or in combination, showed a killing effect on the trophozoites. They bound to the amoebic membrane causing cell rounding, lipid disruption and damage. Conclusions: Milk proteins such as apo-lactoferrin, sIgA and lysozyme are able to kill Entamoeba histolytica trophozoites. This study confirms the importance of feeding breast milk to newborns. PMID:16809402

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhancement of crystal homogeneity of protein crystals under application of an external alternating current electric field

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koizumi, H.; Uda, S.; Fujiwara, K.

X-ray diffraction rocking-curve measurements were performed on tetragonal hen egg white (HEW) lysozyme crystals grown with and without the application of an external alternating current (AC) electric field. The crystal quality was assessed by the full width at half maximum (FWHM) value for each rocking curve. For two-dimensional maps of the FWHMs measured on the 440 and the 12 12 0 reflection, the crystal homogeneity was improved under application of an external electric field at 1 MHz, compared with that without. In particular, the significant improvement of the crystal homogeneity was observed for the 12 12 0 reflection.

High Resolution X-Ray Diffraction of Macromolecules with Synchrotron Radiation

NASA Technical Reports Server (NTRS)

Stojanoff, Vivian; Boggon, Titus; Helliwell, John R.; Judge, Russell; Olczak, Alex; Snell, Edward H.; Siddons, D. Peter; Rose, M. Franklin (Technical Monitor)

2000-01-01

We recently combined synchrotron-based monochromatic X-ray diffraction topography methods with triple axis diffractometry and rocking curve measurements: high resolution X-ray diffraction imaging techniques, to better understand the quality of protein crystals. We discuss these methods in the light of results obtained on crystals grown under different conditions. These non destructive techniques are powerful tools in the characterization of the protein crystals and ultimately will allow to improve, develop, and understand protein crystal growth. High resolution X-ray diffraction imaging methods will be discussed in detail in light of recent results obtained on Hen Egg White Lysozyme crystals and other proteins.

Does Warming a Lysozyme Solution Cook Ones Data?

NASA Technical Reports Server (NTRS)

Pusey, Marc; Burke, Michael; Judge, Russell

2000-01-01

Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.0 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubility are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to the initiation of the crystallization process. We have now measured the kinetics of this process and investigated its reversibility. An aliquot of a stock protein solution is held at a given temperature, and at periodic intervals used to set up batch crystallization experiments. The batch solutions were incubated at 20 C until macroscopic crystals were obtained, at which point the number of crystals in each well were counted. The transition effects increased with temperature, slowly falling off at 30 C with a half time (time to approx. 1/2 the t = 0 number of crystals) of approx. 5 hours, and an estimated half time of approx. 0.5 hours at 43 C. Further, the process was not reversible by simple cooling. After holding a lysozyme solution at 37 C (prior to addition of precipitant) for 16 hours, then cooling and holding it at 4 C, no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Thus every thermal excursion above the phase transition point results in a further decrease in the nucleation rate of that solution, the extent being a function of the time and temperature. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. We have previously shown that putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. We may be able to use this differential behavior to elucidate how flow affects tile lysozyme crystal growth process.

Protein profiles of hatchery egg shell membrane.

PubMed

Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

2016-01-01

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae.

PubMed

de-la-Re-Vega, Enrique; García-Galaz, Alfonso; Díaz-Cinco, Martha E; Sotelo-Mundo, Rogerio R

2006-03-01

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.

Cutting edge: the relative distribution of T cells responding to chemically dominant or minor epitopes of lysozyme is not affected by CD40-CD40 ligand and B7-CD28-CTLA-4 costimulatory pathways.

PubMed

DiPaolo, Richard J; Unanue, Emil R

2002-09-15

We examined the frequencies and specificities of the CD4+ T cell responses to the protein hen egg white lysozyme in mice deficient in the CD40-CD40 ligand or B7-CD28 costimulatory pathways. The frequency of T cells was decreased by between 3- and 4-fold in CD40-/- mice, and 12-fold in B7-1/B7-2-/- mice, but surprisingly, the relative distribution of T cells responding to peptides that were presented at levels that differed by >250-fold was similar. We also examined the CD4 response after blocking the regulatory molecule CTLA-4 during immunization. We observed no difference in either the frequency or specificity of the CD4+ T cell response if CTLA-4 was blocking during priming. Thus, the T cell response was generated toward the constellation of chemically dominant and subdominant epitopes as a whole, and did not discriminate among them based on their relative abundance.

Free-falling Crystals: Biological Macromolecular Crystal Growth Studies in Low Earth Orbit

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, E. H.; Pusey, M. L.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Spacecraft orbiting the earth experience a reduced acceleration environment due to being in a state of continuous free-fall. This state colloquially termed microgravity, has produced improved X-ray diffraction quality crystals of biological macromolecules. Improvements in X-ray diffraction resolution (detail) or signal to noise, provide greater detail in the three-dimensional molecular structure providing information about the molecule, how it works, how to improve its function or how to impede it. Greater molecular detail obtained by crystallization in microgravity, has important implications for structural biology. In this article we examine the theories behind macromolecule crystal quality improvement in microgravity using results obtained from studies with the model protein, chicken egg white lysozyme.

Hot Spots in a Network of Functional Sites

PubMed Central

Ozbek, Pemra; Soner, Seren; Haliloglu, Turkan

2013-01-01

It is of significant interest to understand how proteins interact, which holds the key phenomenon in biological functions. Using dynamic fluctuations in high frequency modes, we show that the Gaussian Network Model (GNM) predicts hot spot residues with success rates ranging between S 8–58%, C 84–95%, P 5–19% and A 81–92% on unbound structures and S 8–51%, C 97–99%, P 14–50%, A 94–97% on complex structures for sensitivity, specificity, precision and accuracy, respectively. High specificity and accuracy rates with a single property on unbound protein structures suggest that hot spots are predefined in the dynamics of unbound structures and forming the binding core of interfaces, whereas the prediction of other functional residues with similar dynamic behavior explains the lower precision values. The latter is demonstrated with the case studies; ubiquitin, hen egg-white lysozyme and M2 proton channel. The dynamic fluctuations suggest a pseudo network of residues with high frequency fluctuations, which could be plausible for the mechanism of biological interactions and allosteric regulation. PMID:24023934

The active site of hen egg-white lysozyme: flexibility and chemical bonding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Held, Jeanette, E-mail: jeanette.netzel@uni-bayreuth.de; Smaalen, Sander van

Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters. Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) ofmore » HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C—H⋯O hydrogen bonds are identified by means of the existence of bond critical points (BCPs) in the multipole electron density. It is proposed that these weak interactions might be important for defining the tertiary structure and activity of HEWL. The deprotonated state of Glu35 prevents a distinction between the Phillips and Koshland mechanisms.« less

Metal-assisted and microwave accelerated-evaporative crystallization: Application to lysozyme protein

NASA Astrophysics Data System (ADS)

Mauge-Lewis, Kevin

In response to the growing need for new crystallization techniques that afford for rapid processing times along with control over crystal size and distribution, the Aslan Research Group has recently demonstrated the use of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization MA-MAEC technique in conjunction with metal nanoparticles and nanostructures for the crystallization of amino acids and organic small molecules. In this study, we have employed the newly developed MA-MAEC technique to the accelerated crystallization of chicken egg-white lysozyme on circular crystallization platforms in order to demonstrate the proof-of-principle application of the method for protein crystallization. The circular crystallization platforms are constructed in-house from poly (methyl methacrylate) (PMMA) and silver nanoparticle films (SNFs), indium tin oxide (ITO) and iron nano-columns. In this study, we prove the MA-MAEC method to be a more effective technique in the rapid crystallization of macromolecules in comparison to other conventional methods. Furthermore, we demonstrate the use of the novel iCrystal system, which incorporates the use of continuous, low wattage heating to facilitate the rapid crystallization of the lysozyme while still retaining excellent crystal quality. With the incorporation of the iCrystal system, we observe crystallization times that are even shorter than those produced by the MA-MAEC technique using a conventional microwave oven in addition to significantly improved crystal quality.

The Effect of Solution Conditions on the Nucleation Kinetics of Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Baird, James K.; Pusey, Marc L.

1998-01-01

An understanding of protein crystal nucleation rates and the effect of solution conditions upon them, is fundamental to the preparation of protein crystals of the desired size and shape for X-ray diffraction analysis. The ability to predict the effect of supersaturation, temperature, pH and precipitant concentration on the number and size of crystals formed is of great benefit in the pursuit of protein structure analysis. In this study we experimentally examine the effect of supersaturation, temperature, pH and sodium chloride concentration on the nucleation rate of tetragonal chicken egg white lysozyme crystals. In order to do this batch crystallization plates were prepared at given solution concentrations and incubated at three different temperatures over the period of one week. The number of crystals per well with their size and dimensions were recorded and correlated against solution conditions. Duplicate experiments indicate the reproducibility of the technique. Although it is well known that crystal numbers increase with increasing supersaturation, large changes in crystal number were also correlated against solution conditions of temperature, pH and salt concentration over the same supersaturation ranges. Analysis of these results enhance our understanding of the effect of solution conditions such as the dramatic effect that small changes in charge and ionic strength can have on the number of tetragonal lysozyme crystals that form and grow in solution.

Developmental toxicity in white leghorn chickens following in ovo exposure to perfluorooctane sulfonate (PFOS)

USGS Publications Warehouse

Peden-Adams, M. M.; Stuckey, Joyce E.; Gaworecki, K.M.; Berger-Ritchie, J.; Bryant, K.; Jodice, P.G.; Scott, T.R.; Ferrario, J.B.; Guan, B.; Vigo, C.; Boone, J.S.; McGuinn, W.D.; DeWitt, J.C.; Keil, D.E.

2009-01-01

Studies show that perfluorinated compounds cause various toxicological effects; nevertheless, effects on immune function and developmental endpoints have not been addressed at length. This study examined the effects of perfluorooctane sulfonate (PFOS) in white leghorn hatchlings on various developmental, immunological, and clinical health parameters. In addition, serum PFOS concentrations were determined by LC/MS/MS. Embryonic day (ED) 0 eggs were injected with either safflower oil/10% DMSO (control, 0 mg/kg egg wt) or PFOS in safflower oil/10% DMSO at 1, 2.5, or 5 mg/kg egg wt, and the chicks were grown to post-hatch day (PHD) 14. Treatment with PFOS did not affect hatch rate. Following in ovo exposure chicks exhibited increases in spleen mass at all treatment levels, in liver mass at 2.5 and 5 mg/kg egg wt, and in body length (crown-rump length) at the 5 mg/kg treatment. Right wings were shorter in all treatments compared to control. Increases in the frequency of brain asymmetry were evident in all treatment groups. SRBC-specific immunoglobulin (IgM and IgY combined) titers were decreased significantly at all treatment levels, while plasma lysozyme activity was increased at all treatment levels. The PHA skin test response decreased in relation to increasing PFOS dose. Serum concentrations where significant immunological, morphological, and neurological effects were observed at the lowest dose (1 mg/kg egg wt) averaged 154 ng PFOS/g serum. These concentrations fall within environmental ranges reported in blood samples from wild caught avian species; thereby, verifying that the environmental egg concentrations used for the injections do indeed relate to serum levels in hatchlings that are also environmentally relevant. These data indicate that immune alterations and brain asymmetry can occur in birds following in ovo exposure to environmentally relevant concentrations of PFOS and demonstrates the need for further research on the developmental effects of perfluorinated compounds in various species. ?? 2009 Elsevier Inc.

Elucidation of metal-ion accumulation induced by hydrogen bonds on protein surfaces by using porous lysozyme crystals containing Rh(III) ions as the model surfaces.

PubMed

Ueno, Takafumi; Abe, Satoshi; Koshiyama, Tomomi; Ohki, Takahiro; Hikage, Tatsuo; Watanabe, Yoshihito

2010-03-01

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Fabrication of diverse pH-sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples.

PubMed

Wang, Jiaojiao; Lan, Jingfeng; Li, Huihui; Liu, Xiaoyan; Zhang, Haixia

2017-01-01

In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH-sensitive functional mesoporous silica materials composed of 2-(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH-sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five-protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum albumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms. Copyright © 2016 Elsevier B.V. All rights reserved.

An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

PubMed

Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

2017-11-08

The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

PubMed

Planque, M; Arnould, T; Dieu, M; Delahaut, P; Renard, P; Gillard, N

2016-09-16

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity. Copyright © 2016 Elsevier B.V. All rights reserved.

Consumption of whole eggs promotes greater stimulation of postexercise muscle protein synthesis than consumption of isonitrogenous amounts of egg whites in young men.

PubMed

van Vliet, Stephan; Shy, Evan L; Abou Sawan, Sidney; Beals, Joseph W; West, Daniel Wd; Skinner, Sarah K; Ulanov, Alexander V; Li, Zhong; Paluska, Scott A; Parsons, Carl M; Moore, Daniel R; Burd, Nicholas A

2017-12-01

Background: Protein in the diet is commonly ingested from whole foods that contain various macro- and micronutrients. However, the effect of consuming protein within its natural whole-food matrix on postprandial protein metabolism remains understudied in humans. Objective: We aimed to compare the whole-body and muscle protein metabolic responses after the consumption of whole eggs with egg whites during exercise recovery in young men. Design: In crossover trials, 10 resistance-trained men [aged 21 ± 1 y; 88 ± 3 kg; body fat: 16% ± 1% (means ± SEMs)] received primed continuous l-[ ring - 2 H 5 ]phenylalanine and l-[1- 13 C]leucine infusions and performed a single bout of resistance exercise. After exercise, participants consumed intrinsically l-[5,5,5- 2 H 3 ]leucine-labeled whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat). Repeated blood and muscle biopsy samples were collected to assess whole-body leucine kinetics, intramuscular signaling, and myofibrillar protein synthesis. Results: Plasma appearance rates of protein-derived leucine were more rapid after the consumption of egg whites than after whole eggs ( P = 0.01). Total plasma availability of leucine over the 300-min postprandial period was similar ( P = 0.75) between the ingestion of whole eggs (68% ± 1%) and egg whites (66% ± 2%), with no difference in whole-body net leucine balance ( P = 0.27). Both whole-egg and egg white conditions increased the phosphorylation of mammalian target of rapamycin complex 1, ribosomal protein S6 kinase 1, and eukaryotic translation initiation factor 4E-binding protein 1 during postexercise recovery (all P < 0.05). However, whole-egg ingestion increased the postexercise myofibrillar protein synthetic response to a greater extent than did the ingestion of egg whites ( P = 0.04). Conclusions: We show that the ingestion of whole eggs immediately after resistance exercise resulted in greater stimulation of myofibrillar protein synthesis than did the ingestion of egg whites, despite being matched for protein content in young men. Our data indicate that the ingestion of nutrient- and protein-dense foods differentially stimulates muscle anabolism compared with protein-dense foods. This trial was registered at clinicaltrials.gov as NCT03117127. © 2017 American Society for Nutrition.

Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

PubMed

Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

2016-08-21

Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection.

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

PubMed Central

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

2012-01-01

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

Molecular dynamics study of unfolding of lysozyme in water and its mixtures with dimethyl sulfoxide.

PubMed

Sedov, Igor A; Magsumov, Timur I

2017-09-01

All-atom explicit solvent molecular dynamics was used to study the process of unfolding of hen egg white lysozyme in water and mixtures of water with dimethyl sulfoxide at different compositions. We have determined the kinetic parameters of unfolding at a constant temperature 450K. For each run, the time of disruption of the tertiary structure of lysozyme t u was defined as the moment when a certain structural criterion computed from the trajectory reaches its critical value. A good agreement is observed between the results obtained using several different criteria. The secondary structure according to DSSP calculations is found to be partially unfolded to the moment of disruption of tertiary structure, but some of its elements keep for a long time after that. The values of t u averaged over ten 30ns-long trajectories for each solvent composition are shown to decrease very rapidly with addition of dimethyl sulfoxide, and rather small amounts of dimethyl sulfoxide are found to change the pathway of unfolding. In pure water, despite the loss of tertiary contacts and disruption of secondary structure elements, the protein preserves its compact globular state at least over 130ns of simulation, while even at 5mol percents of dimethyl sulfoxide it loses its compactness within 30ns. The proposed methodology is a generally applicable tool to quantify the rate of protein unfolding in simulation studies. Copyright © 2017 Elsevier Inc. All rights reserved.

AFM Studies of Salt Concentration Effects on the (110) Surface Structure of Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Pusey, Marc Lee; Gorti, Sridhar; Forsythe, Elizabeth; Konnert, John

2002-01-01

Previous high resolution AFM studies of the (110) surface of tetragonal chicken egg white lysozyme crystals had shown that only one of two possible molecular surfaces is present, those constituting the completed 43 helices. These suggested that the crystal growth process was by the solution-phase assembly of the growth units, which then attach to the surface. However, the best fit for the imaged surfaces, vs. those predicted based upon the bulk crystallographic coordinates, were obtained when the packing about the 43 helices was "tightened up", while maintaining the underlying crystallographic unit cell spacing. This results in a widening of the gap between adjacent helices, and the top- most layer(s) may no longer be in contact. We postulated that the tightened packing about the helices is a result of the high salt concentrations in the bulk solution, used to crystallize the protein, driving hydrophobic interactions. Once the crystal surface is sufficiently buried by subsequent growth layers the ratio of salt to protein molecules decreases and the helices relax to their bulk crystallographic coordinates. The crystal surface helix structure is thus a reflection of the solution structure, and the tightness of the packing about the 43 helices would be a function of the bulk salt concentration. AFM images of the (110) surface of tetragonal lysozyme crystals grown under low (2%) and high (5%) NaCl concentrations reveal differences in the packing about the 43 helices consistent with the above proposal.

An Integrated Biochemistry Laboratory, Including Molecular Modeling

NASA Astrophysics Data System (ADS)

Hall, Adele J. Wolfson Mona L.; Branham, Thomas R.

1996-11-01

The dilemma of designing an advanced undergraduate laboratory lies in the desire to teach and reinforce basic principles and techniques while at the same time exposing students to the excitement of research. We report here on a one-semester, project-based biochemistry laboratory that combines the best features of a cookbook approach (high success rate, achievement of defined goals) with those of an investigative, discovery-based approach (student involvement in the experimental design, excitement of real research). Individual modules may be selected and combined to meet the needs of different courses and different institutions. The central theme of this lab is protein purification and design. This laboratory accompanies the first semester of biochemistry (Structure and Function of Macromolecules, a course taken mainly by junior and senior chemistry and biological chemistry majors). The protein chosen as the object of study is the enzyme lysozyme, which is utilized in all projects. It is suitable for a student lab because it is easily and inexpensively obtained from egg white and is extremely stable, and its high isoelectric point (pI = 11) allows for efficient separation from other proteins by ion-exchange chromatography. Furthermore, a literature search conducted by the resourceful student reveals a wealth of information, since lysozyme has been the subject of numerous studies. It was the first enzyme whose structure was determined by crystallography (1). Hendrickson et al. (2) have previously described an intensive one-month laboratory course centered around lysozyme, although their emphasis is on protein stability rather than purification and engineering. Lysozyme continues to be the focus of much exciting new work on protein folding and dynamics, structure and activity (3 - 5). This lab course includes the following features: (i) reinforcement of basic techniques, such as preparation of buffers, simple enzyme kinetics, and absorption spectroscopy; (ii) experience with methods of protein purification; (iii) incorporation of appropriate controls into experiments; (iv) use of basic statistics in data analysis; (v) writing papers and grant proposals in accepted scientific style; (vi) peer review; (vii) oral presentation of results and proposals; and (viii) introduction to molecular modeling. Figure 1 illustrates the modular nature of the lab curriculum. Elements from each of the exercises can be separated and treated as stand-alone exercises, or combined into short or long projects. We have been able to offer the opportunity to use sophisticated molecular modeling in the final module through funding from an NSF-ILI grant. However, many of the benefits of the research proposal can be achieved with other computer programs, or even by literature survey alone. Figure 1.Design of project-based biochemistry laboratory. Modules (projects, or portions of projects) are indicated as boxes. Each of these can be treated independently, or used as part of a larger project. Solid lines indicate some suggested paths from one module to the next. The skills and knowledge required for protein purification and design are developed in three units: (i) an introduction to critical assays needed to monitor degree of purification, including an evaluation of assay parameters; (ii) partial purification by ion-exchange techniques; and (iii) preparation of a grant proposal on protein design by mutagenesis. Brief descriptions of each of these units follow, with experimental details of each project at the end of this paper. Assays for Lysozyme Activity and Protein Concentration (4 weeks) The assays mastered during the first unit are a necessary tool for determining the purity of the enzyme during the second unit on purification by ion exchange. These assays allow an introduction to the concept of specific activity (units of enzyme activity per milligram of total protein) as a measure of purity. In this first sequence, students learn a turbidimetric assay for lysozyme activity and a colorimetric one for protein concentration. Familiarity with the assays is reinforced by an independently designed project to modify a variable in one of these assays. The assay for lysozyme activity is that of Shugar (6), based on hydrolysis of a cell-wall suspension from the bacterium Micrococcus lysodeikticus, a substrate that is particularly sensitive to lysozyme. As the cell walls are broken down by the enzyme, the turbidity of the sample decreases. This decrease can be conveniently measured by following the decrease in absorbance at a wavelength of 450 nm, using a spectrophotometer or other device for measuring light scattering. The Bradford method (7), a standard assay, is used to determine protein concentration. Using the data from both lysozyme activity assays and protein concentration assays, students can calculate the specific activity for commercial lysozyme and an egg- white solution. These calculations clearly demonstrate the increase in specific activity with increasing purity, since the purified (commercial) preparation has a specific activity approximately 20-fold higher than that of the crude egg-white solution. Lysozyme Purification by Ion-Exchange Chromatography (5 weeks) As suggested by Strang (8), students can design a rational purification of lysozyme using ion-exchange chromatography when presented with information on the isoelectric point of the enzyme and the properties of ion- exchange resins. One week is spent discussing protein purification and the relative advantages and disadvantages of different resins. Each group has a choice of anion-exchange (DEAE) or cation-exchange (CM) resins. Because lysozyme is positively charged below a pH of 11, it will not be adsorbed to an anion-exchange resin, but will be adsorbed to the cation-exchange resin. Therefore, for the cation-exchange protocols, there are further options for methods of collecting and eluting the desired protein. A purification table, including information on yield, specific activity, and degree of purification, is constructed. As described by both Strang (8) and Hurst et al. (9), excellent purifications can be obtained by cation-exchange chromatography, especially when elution is carried out with a salt gradient. Even a stepwise elution yields a highly purified fraction. Students' results for degrees of purification using cation exchange range from 15- to 50-fold. Anion-exchange has been less successful, with purification factors less than 5, although the principle that the positively charged lysozyme will not be adsorbed to the resin is still noted. During the last lab for this project, the crude and purified fractions are analyzed by SDS - polyacrylamide gel electrophoresis. The results of electrophoresis correlate well with calculated purification results. It is very clear that for preparations with high degrees of purification, a single band corresponding to standard lysozyme is observed, whereas for the anion-exchange procedures, multiple contaminating bands are still present. Allowing the students to choose and plan their own purification scheme in consultation with the instructor not only gives them experience in experimental design, but also introduces some flexibility in the level of work expected from each student. Those students who have had much previous lab experience in other courses or who have done independent research can choose to carry out a complex purification scheme, giving them exposure to techniques they may not have used before, whereas students with less experience can be guided to a less demanding purification procedure. Grant Proposal on Protein Design (4 weeks) The culminating exercise is a library/computer project on protein design and engineering. The students propose mutational studies of lysozyme to study stability or mechanism, based on the enzyme's known structure. Perhaps the most exciting area in protein biochemistry today is protein design and engineering. This is an appropriate final project, since by then the students have an appreciation for the complexities of dealing with proteins and should be capable of planning an extension of the project. The students by this point are familiar with the structure of hen egg-white lysozyme (1). The structure of the corresponding enzyme from bacteriophage T4 has also been established (10) and rational site-directed mutagenesis has been carried out to answer specific questions about the factors that affect protein stability (11). The relationship between the two lysozymes can provide a starting point for the students' manipulations of the enzyme from hen egg white, especially since there are many amino acids conserved in the two structures (12). Many mutations for the hen enzyme have also been described (13), and students are expected to carry out literature searches beyond the references supplied. The students examine structure - function relationships and decide on changes they would like to make in the lysozyme molecule, based on energy calculations and the scientific literature describing previous mutations in this and similar proteins. Several potential mutation sites are illustrated in Figure 2. Figure 2.The active site of hen egg-white lysozyme. The back-bone structure of lysozyme is illustrated as a ribbon model, with side-chains for tryptophan 62, 63, and 108 as stick models. Atomic coordinates for lysozyme were obtained from the Protein Data Bank. The molecule was displayed and manipulated using Quanta (MSI) on an Iris Indigo Workstation (Silicon Graphics). The active site of the enzyme is depicted, with side-chains of three tryptophan residues indicated. These residues have been implicated in lysozyme's mechanism of action, although not in actual catalysis. Tryptophan residues 62 and 63 participate in hydrogen bonding to substrate, and steric hindrance with Trp 108 has been thought to induce conformational strain in the substrate, favoring the transition state (reviewed in ref 13). In order to evaluate the importance of these interactions, students have proposed conservative or radical mutations at these sites. Once they have changed the amino acid residue and allowed the model to reach a new minimum energy, they can measure interatomic distances to determine whether or not a substrate can fit into the active site and/or form hydrogen bonds. A subtle variation on mutation of Trp 108 was a proposal to mutate an adjacent alanine residue, such as to displace the Trp further toward the helix and open up the active site. Other mutations, outside of the active site, have been proposed to change the overall stability of the protein (e.g., Gly to Ala within an a-helix, or loss of a disulfide bond). This exercise illustrates the possibilities of modeling proteins. Because it requires that the students set certain parameters and defaults for structural prediction, it helps them to understand the limitations of modeling. The final paper takes the form of a grant proposal. It presents the purification results, rationale for the desired mutation, relevance to previous studies, an overview of techniques applicable to the study of the mutant, predictions as to the characteristics of the engineered protein, and a plan for its purification. These proposals pass through at least one stage of peer review, and each student gives an oral presentation. This project serves to tie together many concepts learned during both the lecture and laboratory portions of the course. As noted above, we have been able to acquire workstations and software for modeling through funding from an NSF-ILI grant. However, the research proposal exercise can be modified, depending on facilities available and the goals of a particular course. A literature survey alone yields a wealth of information on this enzyme, and students can design experiments in biochemistry or molecular biology to follow up on the papers they have read. Some recent textbooks are accompanied by diskettes that allow visualization of proteins on computer monitors, although not manipulation of the structures. Computer programs such as Hyperchem (Autodesk, Sausalito CA) and Nanovision (ACS Software) allow display of proteins using PDB coordinates, and students can make decisions about which residues to mutate based on their position in the natural structure or in analogous proteins. Peer review of student work is incorporated into many writing courses (14), as well other courses that require multiple drafts of papers. We have found it to be a useful exercise for students in this laboratory course, both for the short paper and the grant proposal, although some care must be taken that students do not reinforce one another's misconceptions about scientific writing. There are many advantages to the students to this two-stage writing process. Most superficial, but not unimportant, is that it forces them to have their ideas in some written form one week before the final assignment is due. More significantly, it is a part of their professional training. The peer reviews are, in general, full of thoughtful and useful advice to their colleagues. They also learn a good deal from reading other drafts - about styles of writing, effective use of figures and tables, and references that might be useful to their own projects. Feedback from students indicates that they particularly enjoy devising their own experimental plans and that they appreciate the chance to work under the different conditions of small groups and as individuals. Not all of the exercises described here will be appropriate for every undergraduate biochemistry laboratory. However, the principle of projects that take several weeks to develop, beginning with basic concepts and working up to independently designed experiments, can be adapted to many settings. Experimental Details for Each Project Enzyme Assay The substrate, Micrococcus lysodeikticus, is obtained from Sigma (St. Louis). A suspension (0.3 mg/mL) is prepared fresh in 0.1 M phosphate buffer, pH 6.24, each day. Commercial lysozyme (also from Sigma), 2 mg/mL, as well as a 1:5 dilution of egg white, both in the same phosphate buffer, are used as a source of enzyme. From the change in absorbance over the time of assay, students calculate the activity of each enzyme preparation in terms of units per milliliter. A unit of lysozyme activity is defined as the amount of enzyme that will produce a DeltaA450 of 0.001 per min at pH 6.24 at 25 oC. Linearity of the enzyme-catalyzed reaction over the course of 5 min is the criterion used to choose an appropriate dilution of each enzyme preparation. Although the activity of the enzyme is sensitive to effects of pH and ionic strength (15), if these variables are held constant, the assay is quite reproducible and is, in general, very suitable for monitoring purification procedures (16). Protein Assay Students are supplied with a standard solution of lysozyme at a concentration of 2 mg/mL and the Coomassie blue reagent, obtained from BioRad (Hercules, CA), from which they prepare standards over the range 75 to 1500 mg/mL. Unknowns (in this case, the egg-white solution) are diluted appropriately. A standard curve is prepared by plotting the average absorbance at 595 nm for each set of duplicate readings. Using this standard curve, students determine the protein concentration for each unknown sample. Independent Assay Project Working in pairs or groups of three, the students design an experiment in which they vary one condition in the enzyme assay for lysozyme or in the Bradford assay for total protein and determine the effects on the assay. Examples of conditions to be varied for the enzyme assay are enzyme concentration, time of incubation, substrate concentration, addition of salts, change in pH or ionic strength of buffer, and addition of sulfhydryl reagent. Examples of conditions to be varied for the protein assay are identity of the protein used as standard (e.g., bovine serum albumin rather than lysozyme), time of incubation, wavelength of measurement, addition of salts, and addition of detergent. In these independent projects, students sometimes discover ways to improve the methodology for the assays, and these improvements are incorporated into the suggested procedures for the rest of the semester. The results of this project are written up as a short paper in a style appropriate for a rapid communication in a scientific journal. The papers go through a round of peer review and revision. Ion-Exchange Chromatography Once each group has decided on a project and discussed the details with the instructor, they prepare their own buffers and equilibrate the resins. DEAE-Sephacel and CM-Sepharose, both from Pharmacia/LKB (Piscataway, NJ) are supplied, about 20 mL of packed resin for each egg-white preparation. (These resins can be regenerated and reused many times.) Each group uses one egg white for the entire purification project. The egg white is filtered through one layer of cheesecloth and diluted 5-fold with the starting buffer. The first step is done batchwise because of the viscosity of the sample. The diluted egg white is mixed with the resin for approximately 15 min, then centrifuged at 1500 μ g for 15 min. The supernatant, containing those proteins not adsorbed to the resin, is decanted. Subsequent washing and elution can then be carried out batchwise or by transferring to a column [1.5 - 20 cm Econocolumns (BioRad)]. The options for recovery of the enzyme from the CM resin include a batch or column method, elution by change in pH or ionic strength, and gradient or stepwise elution. Those carrying out elutions from a column use an automatic fraction collector; gradient formers are available for those who choose to elute with a linear salt gradient. When elution is carried out by a change in pH, it should be noted that lysozyme may precipitate near its isoelectric point, especially at low ionic strength. SDS - polyacrylamide gel electrophoresis is used to monitor purity of the final preparation. Using mini-gels (BioRad Mini-Protean II apparatus), each group can pour, run, and stain their own 8 7.3-cm gel within the lab period; destaining can be carried out at any time afterward. The main contaminating band observed is ovalbumin, at a molecular weight of 46,000. Computer Modeling Using the program Quanta (MSI, Burlington, MA) on Indigo workstations (Silicon Graphics, Hudson, MA), the students retrieve coordinates from an MSI version of the Protein Data Bank, display the structure, and rationalize what changes would occur with a mutated form of the protein. Even for those who do not have Quanta or analogous programs, structural coordinates are available through the Internet. Students are prepared for their independent use of the molecular modeling workstations through a series of tutorials during the course of the semester. These exercises require that the students become familiar with specific applications of Quanta, including setting secondary conformation and hydrogen bonds, energy calculations, selectively displaying parts of molecules, measuring interatomic distances, and editing existing proteins. This introduction to macromolecular modeling is comparable to that suggested by Harvey and Tan (17) as a brief introduction to the field. Peer Review For each writing assignment (short paper and grant proposal), one week of lab is devoted to the peer review process. Students are to come to lab with a draft of their paper and a cover letter to their reviewers, which states how far they believe they are in the writing process; what they like and don't like about their work at this stage; and in what specific areas they need help (e.g., audience level, organization, use of references). They exchange papers, reading two or three during the course of the lab period. For each paper, they fill out a peer review form, which requires that they summarize the paper; look for clarity of presentation, appropriate citations, and use of others' work; point out any problems in organization or grammar; and suggest the steps necessary to complete the paper. They then go over the peer review form with the writer, referring any questions or disagreements to the instructor. It is most helpful if the instructor can also read and comment on the draft. General comments on the level of detail included and any problems with the reasoning come more appropriately from the instructor than from peers. The paper is then revised and handed in on the following week. The final draft is accompanied by a summary of revisions and copies of all peer reviews. Students are graded on peer reviews and summaries of revisions and cover letters, as well as on the final paper. Acknowledgments The molecular modeling workstations and associated software were purchased with funding from NSF, Instrumentation and Laboratory Improvement program, grant DUE-9350843 to AJW, with matching funds from Wellesley College. Some of the equipment for protein purification was funded by a grant from the Howard Hughes Medical Institute to Wellesley College. The authors are grateful to Paul Reisberg for helpful discussions in the early stages of this project and to Margaret Merritt for a critical review of the manuscript. Literature Cited 1. Blake, C. C. F.; Jonhson, L. N.; Mair, G. A.; North, A. C. T.; Phillips. D. C.; Sarma, V. R. Proc. Roy. Soc. London Ser. B 1967, 167, 378 - 388. 2. Hendrickson, H. S.; Giannini, J. L.; Bergstrom, J. P.; Johnson, S. N.; Leland, P. A. Biochem. Educ. 1995, 23, 14 - 17. 3. Miranker, A., Robinson, C. V.; Radford, S. E.; Aplin, R. T.; Dobson, C. M. Science 1993, 262, 896 - 900. 4. Turner, M. A.; Howell, P. L. Prot. Sci. 1995, 4, 442 - 449. 5. Radmacher, M.; Fritz, M.; Hansma, H. G.; Hansma, P. Science 1994, 265, 1577 - 1579. 6. Shugar, D. Biochim. Biophys. Acta 1952, 8, 302 - 309. 7. Bradford, M. M. Anal. Biochem. 1976, 72, 248 - 254. 8. Strang, R. H. C. In Practical Biochemistry for Colleges; Wood, E. J., Ed.; Pergamon: Oxford, 1989; pp 43 - 44. 9. Hurst, M. O.; Keenan, M. V.; Son, C. C. J. Chem. Educ. 1992, 69, 850 - 851. 10. Matthews, B. W.; Remington, S. J. Proc. Natl. Acad. Sci. USA 1974, 71, 4178 - 4182. 11. Matthews, B. W.; Nicholson, H.; Becktel, W. J. Proc. Natl. Acad. Sci. USA 1987, 84, 6663 - 6667. 12. Matthews, B. W.; Remington, S. J.; Grutter, M. G.; Anderson, W. F. J. Mol. Biol. 1981, 147, 545 - 558. 13. Imoto, T.; Johnson, L; North, A.; Phillips, D.; Rupley, J. In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic: London, 1972; Vol. VII, Chapter 21. 14. Walvoord, B. Helping Students Write Well; MLA: New York, 1986; Chapter 4. 15. Davies, R. C.; Neuberger, A.; Wilson, B. M. Biochim. Biophys. Acta 1969, 178, 294 - 305. 16. Jolles, P. Meth. Enzymol 1962, 5, 137 - 140. 17. Harvey, S. C.; Tan, R. K.-Z. Biophys. J. 1992, 63, 1683 - 1688.

Triclinic lysozyme at 0.65 angstrom resolution.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, J.; Dauter, M.; Alkire, R.

The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 {angstrom} resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R{sub free} of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules weremore » located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 {angstrom} (at the low temperature of 120 K) and at 0.95 {angstrom} resolution (at room temperature).« less

21 CFR 160.140 - Egg whites.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Egg whites. 160.140 Section 160.140 Food and Drugs... CONSUMPTION EGGS AND EGG PRODUCTS Requirements for Specific Standardized Eggs and Egg Products § 160.140 Egg whites. (a) Egg whites, liquid egg whites, liquid egg albumen is the food obtained from eggs of the...

21 CFR 160.140 - Egg whites.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 2 2011-04-01 2011-04-01 false Egg whites. 160.140 Section 160.140 Food and Drugs... CONSUMPTION EGGS AND EGG PRODUCTS Requirements for Specific Standardized Eggs and Egg Products § 160.140 Egg whites. (a) Egg whites, liquid egg whites, liquid egg albumen is the food obtained from eggs of the...

Application Of Empirical Phase Diagrams For Multidimensional Data Visualization Of High Throughput Microbatch Crystallization Experiments.

PubMed

Klijn, Marieke E; Hubbuch, Jürgen

2018-04-27

Protein phase diagrams are a tool to investigate cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphological features, such as crystal size, as well as kinetic features, such as crystal growth time. Common used data visualization techniques include individual line graphs or symbols-based phase diagrams. These techniques have limitations in terms of handling large datasets, comprehensiveness or completeness. To eliminate these limitations, morphological and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram (EPD) method. Morphological features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the EPD method can support high throughput crystallization experiments in its data amount as well as its data complexity. Copyright © 2018. Published by Elsevier Inc.

Long Range Debye-Hückel Correction for Computation of Grid-based Electrostatic Forces Between Biomacromolecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mereghetti, Paolo; Martinez, M.; Wade, Rebecca C.

Brownian dynamics (BD) simulations can be used to study very large molecular systems, such as models of the intracellular environment, using atomic-detail structures. Such simulations require strategies to contain the computational costs, especially for the computation of interaction forces and energies. A common approach is to compute interaction forces between macromolecules by precomputing their interaction potentials on three-dimensional discretized grids. For long-range interactions, such as electrostatics, grid-based methods are subject to finite size errors. We describe here the implementation of a Debye-Hückel correction to the grid-based electrostatic potential used in the SDA BD simulation software that was applied to simulatemore » solutions of bovine serum albumin and of hen egg white lysozyme.« less

Structural studies of the effect that dimethyl sulfoxide (DMSO) has on cisplatin and carboplatin binding to histidine in a protein.

PubMed

Tanley, Simon W M; Schreurs, Antoine M M; Kroon-Batenburg, Loes M J; Meredith, Joanne; Prendergast, Richard; Walsh, Danielle; Bryant, Patrick; Levy, Colin; Helliwell, John R

2012-05-01

The anticancer complexes cisplatin and carboplatin target the DNA major groove, forming intrastrand and interstrand cross-links between guanine bases through their N7 atoms, causing distortion of the DNA helix and apoptotic cell death. A major side effect of these drugs is toxicity, which is caused via binding to many proteins in the body. A range of crystallographic studies have been carried out involving the cocrystallization of hen egg-white lysozyme (HEWL) as a test protein with cisplatin and carboplatin in aqueous and dimethyl sulfoxide (DMSO) conditions. Different cryoprotectants, glycerol and Paratone, were used for each of the cisplatin and carboplatin cocrystallization cases, while silicone oil was used for studies involving N-acetylglucosamine (NAG). Both cisplatin and carboplatin do not bind to HEWL in aqueous media on the timescales of the conditions used here, but upon addition of DMSO two molecules of cisplatin or carboplatin bind either side of His15, which is the only His residue in lysozyme and is assumed to be an imidazolyl anion or a chemical resonance moiety, i.e. both imidazole N atoms are chemically reactive. To identify the platinum-peak positions in the 'with DMSO conditions', anomalous scattering maps were calculated as a cross-check with the F(o) - F(c) OMIT maps. Platinum-occupancy σ values were established using three different software programs in each case. The use of EVAL15 to process all of the diffraction data sets provided a consistent platform for a large ensemble of data sets for the various protein and platinum-compound model refinements with REFMAC5 and then SHELXTL. Overall, this extensive set of crystallization and cryoprotectant conditions allowed a systematic evaluation of cisplatin and carboplatin binding to lysozyme as a test protein via detailed X-ray crystal structure characterizations. DMSO is used as a super-solvent for drug delivery as it is deemed to cause no effect upon drug binding. However, these results show that addition of DMSO causes the platinum anticancer drugs to bind to HEWL. This effect should be considered in toxicity assessments of these drugs and perhaps more widely. © 2012 International Union of Crystallography

Effects of environmental conditions on growth and survival of Salmonella in pasteurized whole egg.

PubMed

Jakočiūnė, Džiuginta; Bisgaard, Magne; Hervé, Gaëlle; Protais, Jocelyne; Olsen, John Elmerdahl; Chemaly, Marianne

2014-08-01

This study investigated the influence of three parameters (time, temperature and NaCl concentration) on survival and four parameters (temperature, NaCl and lysozyme concentrations and pH) on growth of Salmonella enterica serovar Enteritidis (S. Enteritidis) in pasteurized whole egg (PWE). Doehlert uniform shell design was employed to choose conditions for trials and data was fitted to polynomial models and were presented as estimated response surfaces. A model for prediction of reduction of S. Enteritidis in PWE within temperatures between 50 and 58°C, NaCl concentrations of 0-12%, and heating times between 30 and 210s and a model for prediction of growth rate of S. Enteritidis in PWE in the temperature range of 1-25°C, NaCl concentration of 0-12%, pH between 5 and 9, and lysozyme concentrations of 107-1007 U/mg proteins were developed. The maximum reduction condition was 58°C, 0% of NaCl at a fixed heating time of 120s, while maximum growth rate was estimated at 25°C and 0% of NaCl. pH and lysozyme concentration were shown not to influence growth performance significantly in the range of values studied. Results inform industry of the optimal pasteurization and storage parameters for liquid whole egg. Copyright © 2014 Elsevier B.V. All rights reserved.

Two-step relaxation mode analysis with multiple evolution times applied to all-atom molecular dynamics protein simulation.

PubMed

Karasawa, N; Mitsutake, A; Takano, H

2017-12-01

Proteins implement their functionalities when folded into specific three-dimensional structures, and their functions are related to the protein structures and dynamics. Previously, we applied a relaxation mode analysis (RMA) method to protein systems; this method approximately estimates the slow relaxation modes and times via simulation and enables investigation of the dynamic properties underlying the protein structural fluctuations. Recently, two-step RMA with multiple evolution times has been proposed and applied to a slightly complex homopolymer system, i.e., a single [n]polycatenane. This method can be applied to more complex heteropolymer systems, i.e., protein systems, to estimate the relaxation modes and times more accurately. In two-step RMA, we first perform RMA and obtain rough estimates of the relaxation modes and times. Then, we apply RMA with multiple evolution times to a small number of the slowest relaxation modes obtained in the previous calculation. Herein, we apply this method to the results of principal component analysis (PCA). First, PCA is applied to a 2-μs molecular dynamics simulation of hen egg-white lysozyme in aqueous solution. Then, the two-step RMA method with multiple evolution times is applied to the obtained principal components. The slow relaxation modes and corresponding relaxation times for the principal components are much improved by the second RMA.

Structure of Bordetella pertussis peptidoglycan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folkening, W.J.; Nogami, W.; Martin, S.A.

1987-09-01

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing (/sup 3/H)diaminopimelic acid and treated by a hot (96/sup 0/C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60/sup 6/). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and <2% protein. Radiochemical analyses indicated that /sup 3/H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelicmore » acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived pepidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of >95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine.« less

Two-step relaxation mode analysis with multiple evolution times applied to all-atom molecular dynamics protein simulation

NASA Astrophysics Data System (ADS)

Karasawa, N.; Mitsutake, A.; Takano, H.

2017-12-01

Proteins implement their functionalities when folded into specific three-dimensional structures, and their functions are related to the protein structures and dynamics. Previously, we applied a relaxation mode analysis (RMA) method to protein systems; this method approximately estimates the slow relaxation modes and times via simulation and enables investigation of the dynamic properties underlying the protein structural fluctuations. Recently, two-step RMA with multiple evolution times has been proposed and applied to a slightly complex homopolymer system, i.e., a single [n ] polycatenane. This method can be applied to more complex heteropolymer systems, i.e., protein systems, to estimate the relaxation modes and times more accurately. In two-step RMA, we first perform RMA and obtain rough estimates of the relaxation modes and times. Then, we apply RMA with multiple evolution times to a small number of the slowest relaxation modes obtained in the previous calculation. Herein, we apply this method to the results of principal component analysis (PCA). First, PCA is applied to a 2-μ s molecular dynamics simulation of hen egg-white lysozyme in aqueous solution. Then, the two-step RMA method with multiple evolution times is applied to the obtained principal components. The slow relaxation modes and corresponding relaxation times for the principal components are much improved by the second RMA.

Silver nano particles ameliorate learning and spatial memory of male Wistar rats by prevention of amyloid fibril-induced neurotoxicity.

PubMed

Ramshini, H; Moghaddasi, A-S; Aldaghi, L-S; Mollania, N; Ebrahim-Habibi, A

2017-12-08

Alzheimer's disease (AD) is a chronic degenerative disease characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs), which results into memory and learning impairments. In the present study, we showed that the aggregates formed by a protein that has no link with Alzheimer's disease, namely the hen egg white lysozyme (HEWL), were cytotoxic and decreased spatial learning and memory in rats. The effect of Ag-nano particles (Ag-NPs) was investigated on disruption of amyloid aggregation and preservation of cognitive behavior of rats. Twenty-four male Wistar rats were divided into 4 groups including a control group, and injected with either scopolamine, lysozyme or aggregates pre-incubated with Ag-NPs. Rats' behavior was monitored using Morris water maze (MWM) twenty days after injections. HEWL aggregation in the presence and absence of the Ag-NPs was assayed by Thioflavin T binding, atomic force microscopy and cell-based cytotoxicity assay. Ag-NPs were capable to directly disrupt HEWL oligomerization and the resulting aggregates were non-toxic. We also showed that rats of the Ag-NPs group found MWM test platform in less time and with less distance traveled, in comparison with lysozyme group. Ag-NPs also increased the percentage of time elapsed and the distance swum in the target quadrant in the rat model of AD, in probe test. These observations suggest that Ag-NPs improved spatial learning and memory by inhibiting amyloid fibril-induced neurotoxicity. Furthermore, we suggest using model proteins as a valid tool to investigate the pathogenesis of Alzheimer's disease.

Effects of Microheterogeneity in Hen Egg-White Lysozyme Crystallization

NASA Technical Reports Server (NTRS)

Thomas, B. R.; Vekilov, P. G.; Rosenberger, F.

1998-01-01

In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.

21 CFR 160.150 - Frozen egg whites.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Frozen egg whites. 160.150 Section 160.150 Food... HUMAN CONSUMPTION EGGS AND EGG PRODUCTS Requirements for Specific Standardized Eggs and Egg Products § 160.150 Frozen egg whites. (a) Frozen egg whites, frozen egg albumen is the food prepared by freezing...

21 CFR 160.150 - Frozen egg whites.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 2 2011-04-01 2011-04-01 false Frozen egg whites. 160.150 Section 160.150 Food... HUMAN CONSUMPTION EGGS AND EGG PRODUCTS Requirements for Specific Standardized Eggs and Egg Products § 160.150 Frozen egg whites. (a) Frozen egg whites, frozen egg albumen is the food prepared by freezing...

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for themore » HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.« less

Fluorescence Studies of Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Smith, Lori

1998-01-01

Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each other. The results of these and other studies will be discussed.

Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme.

PubMed

Gaudana, Ripal; Gokulgandhi, Mitan; Khurana, Varun; Kwatra, Deep; Mitra, Ashim K

2013-01-01

Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.

Physicochemical and functional properties of ultrasonic-treated tragacanth hydrogels cross-linked to lysozyme.

PubMed

Koshani, Roya; Aminlari, Mahmoud

2017-10-01

The purpose of this study was to prepare, characterize and investigate physiochemical and functional attributes of hen egg white lysozyme (LZM) cross-linked with ultrasonic-treated tragacanth (US-treated TGC) under mild Maillard reactions conditions. FT-IR spectroscopy together with OPA assay revealed that covalent attachment of LZM with TCG's. Under optimum condition (pH=8.5, 60°C, RH=79%, 8 days), only one of the free amino group of LZM was blocked by TGC whereas under the same condition, US treated-TGC's blocked about three amino groups. The thermal stability of the LZM-TGC conjugates differed depending on the lengths of the main and branch chains. The microstructure of LZM-TGC conjugates was characterized by scanning electron microscopy. US-treated TGC-LZM exhibited improved solubility, emulsion properties, foam capacity and stability as compared with the native LZM. Since this gum is extensively used in food industry and application of LZM as a natural antimicrobial agents in different food systems is recommended and practiced in some countries, the results of this study indicates that a conjugated product of these two polymers combines different properties into one macromolecule and improves the property of each. These properties may make the conjugate an attractive food ingredient. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential of mean force for human lysozyme camelid vhh hl6 antibody interaction studies

NASA Astrophysics Data System (ADS)

Wang, Yeng-Tseng; Liao, Jun-Min; Chen, Cheng-Lung; Su, Zhi-Yuan; Chen, Chang-Hung; Hu, Jeu-Jiun

2008-04-01

Calculating antigen-antibody interaction energies is crucial for understanding antigen-antibody associations in immunology. To shed further light into this equation, we study a separation of human lysozyme-camelid vhh hl6 antibody (cAb-HuL6) complex. The c-terminal end-to-end stretching of the lysozyme-antibody complex structures have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and explicit water model. For the lysozyme-antibody complex, there are six important intermediates in the c-terminal extensions process. Inclusion of our simulations may help to understand the binding mechanics of lysozyme-cAb-HuL6 antibody complex.

Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine.

PubMed

Grizard, Stéphanie; Versteegh, Maaike A; Ndithia, Henry K; Salles, Joana F; Tieleman, B Irene

2015-01-01

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components--pH, lysozyme and ovotransferrin--during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon's α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.

Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

PubMed Central

Grizard, Stéphanie; Versteegh, Maaike A.; Ndithia, Henry K.; Salles, Joana F.; Tieleman, B. Irene

2015-01-01

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment. PMID:25880684

Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.

PubMed

Guyot, N; Réhault-Godbert, S; Slugocki, C; Harichaux, G; Labas, V; Helloin, E; Nys, Y

2016-12-01

Egg white is an important contributor to the protection of eggs against bacterial contaminations during the first half of incubation (day zero to 12), prior to the egg white transfer into the amniotic fluid to be orally absorbed by the embryo. This protective system relies on an arsenal of antimicrobial proteins and on intrinsic physicochemical properties that are generally unfavorable for bacterial multiplication and dissemination. Some changes in these parameters can be observed in egg white during egg storage and incubation. The aim of this work was to characterize changes in the antibacterial potential of egg white in embryonated eggs (FE) during the first half of incubation using unfertilized eggs (UF) as controls. Egg white samples were collected at day zero, 4, 8, and 12 and analyzed for pH, protein concentration, and protein profile. Antibacterial properties of egg white proteins were evaluated against Listeria monocytogenes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis. During incubation, differential variations of egg white pH and protein concentrations were observed between UF and FE. At equal protein concentrations, similar activities against L. monocytogenes and S. uberis were observed for FE and UF egg white proteins. A progressive decline in these activities, however, was observed over incubation time, regardless of the egg group (UF or FE). SDS-PAGE analysis of egg white proteins during incubation revealed discrete changes in the profile of major proteins, whereas the stability of some less abundant antimicrobial proteins seemed more affected. To conclude, the antibacterial activity of egg white proteins progressively decreased during the first half of egg incubation, possibly resulting from the alteration of specific antimicrobial proteins. This apparent decline may be partly counterbalanced in embryonated eggs by the increase in egg white protein concentration. The antibacterial potential of egg white is very effective during early stages of embryonic development but its alteration during incubation suggests that extra-embryonic structures could then progressively ensure protective functions. © 2016 Poultry Science Association Inc.

Irradiation of shell egg on the physicochemical and functional properties of liquid egg white.

PubMed

Min, B; Nam, K C; Jo, C; Ahn, D U

2012-10-01

This study was aimed at determining the effect of irradiation of shell eggs on the physiochemical and functional properties of liquid egg white during storage. Color and textural parameters of irradiated liquid egg white after cooking were also determined. Shell eggs were irradiated at 0, 2.5, 5, or 10 kGy using a linear accelerator. Egg white was separated from yolk and stored in at 4°C up to 14 d. Viscosity, pH, turbidity, foaming properties, color, and volatile profile of liquid egg white, and color and texture properties of cooked egg white were determined at 0, 7, and 14 d of storage. Irradiation increased the turbidity but decreased viscosity of liquid egg white. Foaming capacity and foam stability were not affected by irradiation at lower dose (2.5 kGy), but were deteriorated at higher doses (≥5.0 kGy) of irradiation. Sulfur-containing volatiles were generated by irradiation and their amounts increased as the irradiation dose increased. However, the sulfur volatiles disappeared during storage under aerobic conditions. Lightness (L* value) and yellowness (b* value) decreased, but greenness (-a* value) increased in cooked egg white in irradiation dose-dependent manners. All textural parameters (hardness, adhesiveness, cohesiveness, chewiness, and resilience) of cooked egg white increased as the irradiation dose increased, but those changes were marginal. Our results indicated that irradiation of shell egg at lower doses (up to 2.5 kGy) had little negative impact on the physiochemical and functional properties of liquid egg white, but can improve the efficiency of egg processing due to its viscosity-lowering effect. Therefore, irradiation of shell eggs at the lower doses has high potential to be used by the egg processing industry to improve the safety of liquid egg without compromising its quality.

Spatial Extent of Charge Repulsion Regulates Assembly Pathways for Lysozyme Amyloid Fibrils

PubMed Central

Hill, Shannon E.; Miti, Tatiana; Richmond, Tyson; Muschol, Martin

2011-01-01

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path. PMID:21483680

Hydrogen-bond dynamics at the bio-water interface in hydrated proteins: a molecular-dynamics study.

PubMed

Nandi, Prithwish K; English, Niall J; Futera, Zdenek; Benedetto, Antonio

2016-12-21

Water is fundamental to the biochemistry of enzymes. It is well known that without a minimum amount of water, enzymes are not biologically active. Bare minimal solvation for biological function corresponds to about a single layer of water covering enzymes' surfaces. Many contradictory studies on protein-hydration-water-coupled dynamics have been published in recent decades. Following prevailing wisdom, a dynamical crossover in hydration water (at around 220 K for hydrated lysozymes) can trigger larger-amplitude motions of the protein, activating, in turn, biological functions. Here, we present a molecular-dynamics-simulation study on a solvated model protein (hen egg-white lysozyme), in which we determine, inter alia, the relaxation dynamics of the hydrogen-bond network between the protein and its hydration water molecules on a residue-per-residue basis. Hydrogen-bond breakage/formation kinetics is rather heterogeneous in temperature dependence (due to the heterogeneity of the free-energy surface), and is driven by the magnitude of thermal motions of various different protein residues which provide enough thermal energy to overcome energy barriers to rupture their respective hydrogen bonds with water. In particular, arginine residues exhibit the highest number of such hydrogen bonds at low temperatures, losing almost completely such bonding above 230 K. This suggests that hydration water's dynamical crossover, observed experimentally for hydrated lysozymes at ∼220 K, lies not at the origin of the protein residues' larger-amplitude motions, but rather arises as a consequence thereof. This highlights the need for new experimental investigations, and new interpretations to link protein dynamics to functions, in the context of key interrelationships with the solvation layer.

Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient

NASA Technical Reports Server (NTRS)

Thomas, B. R.; Chernov, A. A.

2000-01-01

Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

Synchrotron X-ray reciprocal-space mapping, topography and diffraction resolution studies of macromolecular crystal quality.

PubMed

Boggon, T J; Helliwell, J R; Judge, R A; Olczak, A; Siddons, D P; Snell, E H; Stojanoff, V

2000-07-01

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

Ionic liquids as refolding additives: N′-alkyl and N′-(ω-hydroxyalkyl) N-methylimidazolium chlorides

PubMed Central

Lange, Christian; Patil, Ganesh; Rudolph, Rainer

2005-01-01

The purpose of this work was to investigate the influence of a series of N′-alkyl and N′-(ω-hydroxy-alkyl)-N-methylimidazolium chlorides on the renaturation of two model proteins, namely hen egg white lysozyme and the single-chain antibody fragment ScFvOx. All tested ionic liquids acted as refolding enhancers, with varying efficacies and efficiencies. The results of the refolding screening could be interpreted by taking into account the effect of the studied ionic liquids on protein aggregation, together with the systematic variations of their influence on the stability of native proteins in solution. More hydrophobic imidazolium cations carrying longer alkyl chains were increasingly destabilizing, while terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability. The studied ionic liquids can be classified as preferentially bound, slightly to moderately chaotropic cosolvents for proteins. PMID:16195554

Synchrotron X-Ray Reciprocal Space Mapping, Topography and Diffraction Resolution Studies of Macromolecular Crystal Quality

NASA Technical Reports Server (NTRS)

Boggon, T. J.; Helliwell, J. R.; Judge, Russell A.; Siddons, D. P.; Snell, Edward H.; Stojanoff, V.

2000-01-01

A comprehensive study of microgravity and ground grown chicken egg white lysozyme crystals is presented using synchrotron X-ray reciprocal space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed, on average, reduced intrinsic mosaicities but no differences in terms of stress over their earth grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the earth case at the diffraction peak only a small volume of the crystal contributed to the intensity. The techniques prove to be highly complementary with the reciprocal space mapping providing a quantitative measure of the crystal mosaicity and stress (or variation in lattice spacing) and topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out both at the synchrotron and in the laboratory.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanders, L.K.; Xian, W.; Guaqueta, C.

The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozymemore » complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.« less

Chemical structure, comparison antioxidant capacity and separation antioxidant of hen, duck and quail egg white protein hydrolysate

NASA Astrophysics Data System (ADS)

Fatah, A.; Meihu, M.; Ning, Q.; Setiani, B. E.; Bintoro, V. P.

2018-01-01

Amino acid linkages as proteins are nutritional substance which important for diet intake. Purification protein procesing undergo heating procedure process followed by additional of proteolytic enzymes or acid had been resulting in protein hydrolysates. A protein hydrolysate describe as many free amino acids bound together through a complex mixture of peptides. Egg white protein hydrolysates is one of subject interested to study for human health or industry product. The objectives of the research are to determine and identification the antioxidant derived from egg white hydrolysate protein. Identification of chemical structure of albumen and albumen protein hydrolysate was examine using IR Spectrophotometry. While comparison of antioxidant capacity and antioxidant separation egg albumen was also investigate using FTIR method (Fourier Transform Infrared Spectroscopy). Hen, duck and quail albumen egg white and on hydrolisate form were used as research materials. The results were showing that different time and enzyme of hydrolysis were not influence at secondary structure of hydrolysate albumen protein. Phytochemical content such as alcohol and hydroxyl compound which have potential as functional group of antioxidant were detected in all of the samples. Their results of radical scavenging activities samples hydrolyzed by pepsin were respectively 89.40%, 50.25% and 85.13%. Whereas the radical scavenging activities of hydrolysates hydrolyzed by papain were 72.85%, 61% and 76.45% respectively.

[Lysozyme in the treatment of juvenile laryngeal papillomatosis. A new concept in its etiopathogenesis].

PubMed

Altamar-Ríos, J

1990-01-01

The A. inform about the results achieved with lysozyme chlorhydrate in the treatment of 15 patients with juvenile laryngeal papillomatosis. The lysozyme is an electropositive enzyme which synthesis is related to the degree of proteins and vitamin B complex ingestion. Lysozyme is a component of the immunitary inespecific system, serving to prevent against HPV-DNA at the level of the secretory film of the mucociliary apparatus of the respiratory mucous membrane. Furthermore, lysozyme hydrolyzes the mucopolysaccharide of the connective tissue and inhibits the virus-DNA replication. 100-300 mgr daily during 30-60 days simultaneously with hyperproteic diet and vitamin B complex (after correction of the nutrimental deficiencies) brought about the evanishment of papillomatosis. The A. suggest that the predisposition to infection by virus DNA is primarily of immunitary origin, because of lysozyme deficiency, and secondary due to a low intake of proteins and vitamin B complex.

Studies on immunocompetence status in two turkey varieties in India.

PubMed

Singh, N S; Kumar, S; Majumdar, S; Sivaraman, G K; Shivakumar, B M

2004-10-01

(1) Two hundred and twenty-seven adult turkeys of both sexes, of two varieties (104 Black and 123 White) were used to evaluate their immunocompetence status and body weights. (2) Response to sheep red blood cells (SRBC) (humoral immunity) was measured by Haemagglutination (HA) test 5 days post immunisation (dpi) and expressed as log2 values. Mercaptoethanol resistant (MER) antibodies representing IgG were determined by Mercaptoethanol HA test and Mercaptoethanol sensitive (MES) antibodies, representing IgM as the difference in total HA titre and IgG. Serum lysozyme concentrations were estimated by 'Lysoplate assay' and expressed in log2 values. (3) Least squares analysis of variance revealed that the White variety had higher adult body weight (4.788 +/- 0.040 kg) than the Black (3.774 +/- 0.044 kg). Sexual dimorphism was apparent and meals were heavier than females in both varieties. The interaction effect of variety and sex on body weight was also significant. (4) Least squares means for immunological traits, namely, total anti-SRBC antibodies, MER, MES titres and serum lysozyme were 7.161 +/- 0.189, 0.801 +/- 0.071, 6.362 +/- 0.160 and 1.766 +/- 0.043 microg/ml, respectively. The Black variety had a higher MES antibody titre than the White. (5) Sex had an effect on all the immunological traits except on MER titres. Females generally had higher anti-SRBC, MER and MES titres and serum lysozyme. The variety x sex interaction effect was significant for MES titres and serum lysozyme. White males had the lowest MES titres.

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Interactions of cisplatin analogues with lysozyme: a comparative analysis.

PubMed

Ferraro, Giarita; De Benedictis, Ilaria; Malfitano, Annamaria; Morelli, Giancarlo; Novellino, Ettore; Marasco, Daniela

2017-10-01

The biophysical characterization of drug binding to proteins plays a key role in structural biology and in the discovery and optimization of drug discovery processes. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding of metal-based drugs to their final target is challenging, due to the physicochemical properties of these agents. Different cisplatin derivatives have shown different citotoxicities in most common cancer lines, suggesting that they exert their biological activity via different mechanisms of action. Here we carried out a comparative analysis, by studying the behaviours of three Pt-compounds under the same experimental conditions and binding assays to properly deepen the determinants of the different MAOs. Indeed we compared the results obtained using surface plasmon resonance, isothermal titration calorimetry, fluorescence spectroscopy and thermal shift assays based on circular dichroism experiments in the characterization of the formation of adducts obtained upon reaction of cisplatin, carboplatin and iodinated analogue of cisplatin, cis-Pt (NH 3 ) 2 I 2 , with the model protein hen egg white lysozyme, both at neutral and acid pHs. Further we reasoned on the applicability of employed techniques for the study the thermodynamics and kinetics of the reaction of a metallodrug with a protein and to reveal which information can be obtained using a combination of these analyses. Data were discussed on the light of the existing structural data collected on the platinated protein.

Viability of murine norovirus in salads and dressings and its inactivation using heat-denatured lysozyme.

PubMed

Takahashi, Hajime; Tsuchiya, Tomoki; Takahashi, Michiko; Nakazawa, Moemi; Watanabe, Tomoka; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

2016-09-16

In recent years, a number of food poisoning outbreaks due to the contamination of norovirus in ready-to-eat (RTE) foods such as salads have been reported, and this issue is regarded as a global problem. The risk of contamination of fresh vegetables with norovirus has been previously reported, but the survivability of norovirus that contaminates salads remains unknown. In addition, there have been limited reports on the control of norovirus in food products by using inactivating agents. In this study, the viability of norovirus in various types of salads and dressings was examined using murine norovirus strain 1 (MNV-1) as a surrogate for the closely related human norovirus. In addition, the inactivation of MNV-1 in salads was examined using heat-denatured lysozyme, which had been reported to inactivate norovirus. MNV-1 was inoculated in 4 types of salads (coleslaw, thousand island salad, vinaigrette salad, egg salad) and 3 types of dressings (mayonnaise, thousand island dressing, vinaigrette dressing), stored at 4°C for 5days. The results revealed that in the vinaigrette dressing, the infectivity of MNV-1 decreased by 2.6logPFU/mL in 5days, whereas in the other dressings and salads, the infectivity of MNV-1 did not show any significant decrease. Next, 1% heat-denatured lysozyme was added to the 4 types of salads, and subsequently it was found that in 2 types of salads (thousand island salad, vinaigrette salad), the infectivity of MNV-1 decreased by >4.0logPFU/g, whereas in coleslaw salad, a decrease of 3.0logPFU/g was shown. However, in egg salads, the infectivity of MNV-1 did not show such decrease. These results suggest that norovirus can survive for 5days in contaminated salads. Further, these findings also indicated that heat-denatured lysozyme had an inactivating effect on norovirus, even in salads. In the future, heat-denatured lysozyme can be used as a novel norovirus-inactivating agent, although it is essential to investigate the mechanism of inactivating effect of heat-denatured lysozyme against norovirus. Copyright © 2016 Elsevier B.V. All rights reserved.

Doxycycline depletion and residues in eggs after oral administration to laying hens.

PubMed

Gajda, Anna; Posyniak, Andrzej

2015-01-01

The depletion of doxycycline (DC) residues in eggs was determined after oral drug administration by drinking water to laying hens. The antibiotic was supplied to birds for 5 consecutive days and the eggs were collected during medication and 18 days after withdrawal. DC residues were determined by LC-MS/MS. DC was isolated from eggs with a solution of 0.02 M of oxalic acid (pH 4), 0.1 M Na2EDTA and acetonitrile. The limit of detection (LOD) and limit of quantification (LOQ) of the method were 2 and 5 µg kg(-1), respectively. Analyses were performed on whole egg, egg white and yolk separately. DC was detectable 24 h after the beginning of administration. The concentration of antibiotic increased daily, resulting in the highest DC concentration in whole eggs at the first day of the withdrawal period. Thirteen days after withdrawal, the content of DC in whole eggs was below the LOQ of the method. However, some differences were found in the depletion curve of DC between egg white and yolk. Residues of DC in egg white were much higher during treatment and 1 day after withdrawal, but later the concentration in egg white decreased fairly rapidly and a higher DC content in egg yolk was observed. The depletion period was shorter for egg white than for yolk, and DC was detected in the egg white until 12 days after withdrawal and 2 days more in egg yolk than in white. DC reached a peak faster in egg white, but the residues were detectable for longer period in the yolk.

Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

PubMed

Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

2016-04-19

The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein-polyelectrolyte systems. Hence, the present protein-PEGylated poly(amino acid) mixture provides an ideal water-soluble model system to study the important role of electrostatic interaction in the complexation between proteins and polymers, leading to important new knowledge on the protein-polymer interactions. Moreover, the polyelectrolyte complex micelle formed between protein and PEGylated polymer may provide a good drug delivery vehicle for therapeutic proteins.

Improving foaming properties of yolk-contaminated egg albumen by basic soy protein.

PubMed

Wang, Guang; Wang, Tong

2009-10-01

Yolk contamination of egg white is a common problem in the egg breaking industry. Foaming properties of egg white protein are affected by such contamination, but proteins of basic nature may restore the foaming properties of the yolk-contaminated egg white protein. The purpose of this study was to chemically modify a soy protein, that is, to esterify the acidic groups on the protein and to study the potential of such modified protein in improving foaming. We showed that the modification changed the isoelectric point of soy protein isolate (SPI) from 4.5 to about 10. Sonication was proven to be a very effective means to redisperse the methanol-denatured soy protein during reaction, as shown by the improved solubility profile. Such modified basic protein, that is, the sonicated-modified SPI (SMSPI), when added to the yolk-contaminated (at 0.4% level, as-is basis) egg white, gave significantly improved foaming properties. We have shown that the slight change in pH due to the addition of SMSPI was not the reason for improved foaming performance; instead, the modified protein itself was the main reason for such improvement. Addition of SMSPI increased the foaming performance of both pure egg white and yolk-contaminated egg white. SMSPI consistently performed better than the unmodified SPI for improving foaming. Addition of SMSPI (16%, based on dry egg white, and 1.6% based on liquid egg white) fully restored foam expansion and foam liquid stability of 0.4% yolk-contaminated egg white, and it even out-performed the foaming of pure white protein. Therefore, a feasible solution to restore the foaming properties of yolk-contaminated egg white has been identified. It is expected that such modified SPI can be used as an additive or ingredient in foaming formulation, especially when the egg white protein is suspected of lipid contamination.

Anodic Oxidative Modification of Egg White for Heat Treatment.

PubMed

Takahashi, Masahito; Handa, Akihiro; Yamaguchi, Yusuke; Kodama, Risa; Chiba, Kazuhiro

2016-08-31

A new functionalization of egg white was achieved by an electrochemical reaction. The method involves electron transfer from thiol groups of egg white protein to form disulfide bonds. The oxidized egg white produced less hydrogen sulfide during heat treatment; with sufficient application of electricity, almost no hydrogen sulfide was produced. In addition, gels formed by heating electrochemically oxidized egg white exhibited unique properties, such as a lower gelation temperature and a softened texture, presumably due to protein aggregation and electrochemically mediated intramolecular disulfide bond formation.

Egg labeling methods for gastric emptying scintigraphy are not equivalent in producing a stable solid meal.

PubMed

Knight, Linda C; Kantor, Steven; Doma, Siva; Parkman, Henry P; Maurer, Alan H

2007-11-01

A wide range of radiolabeled test meals have been used for gastric emptying scintigraphy. The purpose of this study was to test whether (99m)Tc-sulfur colloid-labeled liquid egg white is as stable as 2 fresh whole eggs labeled with (99m)Tc-sulfur colloid and whether the cooking method is important. Whole eggs and liquid egg white were mixed with (99m)Tc-sulfur colloid and cooked by either microwaving or frying on a griddle. The cooked eggs were tested for breakdown after 2 and 4 h of incubation in gastric fluid or HCl. Labeled liquid egg white, prepared by either method of cooking, exhibited less breakdown in gastric fluid than whole eggs. Whole eggs cooked in the microwave exhibited significantly more breakdown than liquid egg white. (99m)Tc-Sulfur colloid binds better to egg whites compared with whole eggs. These results emphasize the need to evaluate the stability of new radiolabeled test meal preparations, including the method of cooking.

Global Gene-expression Analysis of the Response of Salmonella Enteritidis to Egg White Exposure Reveals Multiple Egg White-imposed Stress Responses

PubMed Central

Baron, Florence; Bonnassie, Sylvie; Alabdeh, Mariah; Cochet, Marie-Françoise; Nau, Françoise; Guérin-Dubiard, Catherine; Gautier, Michel; Andrews, Simon C.; Jan, Sophie

2017-01-01

Chicken egg white protects the embryo from bacterial invaders by presenting an assortment of antagonistic activities that combine together to both kill and inhibit growth. The key features of the egg white anti-bacterial system are iron restriction, high pH, antibacterial peptides and proteins, and viscosity. Salmonella enterica serovar Enteritidis is the major pathogen responsible for egg-borne infection in humans, which is partly explained by its exceptional capacity for survival under the harsh conditions encountered within egg white. However, at temperatures up to 42°C, egg white exerts a much stronger bactericidal effect on S. Enteritidis than at lower temperatures, although the mechanism of egg white-induced killing is only partly understood. Here, for the first time, the impact of exposure of S. Enteritidis to egg white under bactericidal conditions (45°C) is explored by global-expression analysis. A large-scale (18.7% of genome) shift in transcription is revealed suggesting major changes in specific aspects of S. Enteritidis physiology: induction of egg white related stress-responses (envelope damage, exposure to heat and alkalinity, and translation shutdown); shift in energy metabolism from respiration to fermentation; and enhanced micronutrient provision (due to iron and biotin restriction). Little evidence of DNA damage or redox stress was obtained. Instead, data are consistent with envelope damage resulting in cell death by lysis. A surprise was the high degree of induction of hexonate/hexuronate utilization genes, despite no evidence indicating the presence of these substrates in egg white. PMID:28553268

Study on the interaction between cinnamic acid and lysozyme

NASA Astrophysics Data System (ADS)

Zhang, Hong-Mei; Chen, Jian; Zhou, Qiu-Hua; Shi, Yue-Qin; Wang, Yan-Qing

2011-02-01

The interaction between lysozyme and cinnamic acid was investigated systematically by ultraviolet-vis absorbance, circular dichroism, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques at pH 7.40. The binding constants, quenching mechanism, and the number of binding sites were determined by the quenching of lysozyme fluorescence in presence of cinnamic acid. The results showed that the fluorescence quenching of lysozyme by cinnamic acid was a result of the formation of cinnamic acid-lysozyme complex. The hydrophobic and electrostatic interactions played major roles in stabilizing the complex; the distance r between donor and acceptor was obtained to be 2.07 nm according to Förster's theory; the effect of cinnamic acid on the conformation of lysozyme was analyzed using synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectra.

Induction of Food Allergy in Mice by Allergen Inhalation

DTIC Science & Technology

2015-10-01

whether and how inhaling egg white can sensitize for the development of egg white food allergy. Despite difficulties related to the complicated pregnancy...and illness of a key researcher, considerable progress has been made: 1) We cannot induce an allergic response to aerosolized egg white, even if...have shown that egg yolk acts as an adjuvant for the development of allergic lung disease tp egg white and induction of food allergy; 3) We have

Changes of the antigenic and allergenic properties of a hen's egg albumin in a cake with gamma-irradiated egg white

NASA Astrophysics Data System (ADS)

Lee, Ju.-Woon; Seo, Ji.-Hyun; Kim, Jae.-Hun; Lee, Soo.-Young; Kim, Kwan.-Soo; Byun, Myung.-Woo

2005-04-01

Changes of the antigenicity and allergenicity of a hen's egg albumin (ovalbumin, OVA) in white layer cakes containing egg white gamma-irradiated with 10 or 20 kGy were monitored by an enzyme-linked immunosorbent assay (ELISA), individually formatted with mouse anti-OVA IgG (mouse IgG) and with egg allergic patients' IgE. Mouse IgG recognized OVA in the cakes with irradiated egg white better than that in the control with a non-irradiated one. Whereas, the detected concentrations of intact OVA in the control significantly decreased in the treatments, when determined by IgE-based ELISA. The results appeared to indicate that the antigenicity of the OVA increased, but that the allergenicity was decreased by irradiation and processing. Egg white irradiated for reducing the egg allergy could be used for producing a safer cake from the egg allergy.

Clinical and immunological profile of children aged 5-9 years with persistent egg allergy before oral immunotherapy with egg. A multicenter, randomized controlled trial of the Spanish Society of Pediatric Allergy, Asthma and Clinical Immunology (SEICAP).

PubMed

Echeverria, L; Martin-Muñoz, M F; Martorell, C; Belver, M T; Alonso Lebrero, E; Zapatero, L; Fuentes, V; Piqué, M; Plaza, A; Muñoz, C; Martorell, A; Blasco, C; Villa, B; Gómez, C; Nevot, S; García, J M; Madero, R

2018-05-24

In children with egg protein allergy (EA), the probability of overcoming the allergy decreases with age, and the possibility of suffering severe adverse reactions as a consequence of dietetic transgressions results in worsened quality of life. One treatment option in such cases is oral immunotherapy (OIT) with foods. We present a cohort of children with EA scheduled for OIT with pasteurized raw egg white, describing their clinical and allergic characteristics before the start of OIT. The median age was six years, and 93% of the patients also suffered other allergies (58% asthma and 38.6% allergy to more than two food groups). In the last year, 14.8% had suffered a severe reaction due to dietetic transgression with egg. The median IgE specific of egg white titer was 38.5kU/l. A double-blind placebo-controlled food challenge with cooked egg white was performed, and if the test proved positive, it was repeated with pasteurized raw egg white. The mean symptoms-provoking dose was 1.26g and 0.55g for cooked egg white and raw egg white, respectively. An IgE specific of ovomucoid titer of <2.045kU/l differentiated those patients that tolerated cooked egg white. OIT with egg is regarded as an option in patients with persistent egg allergy. In the previous challenge test, an IgE specific of ovomucoid titer of <2.045kU/l differentiates those patients that tolerate cooked egg white. Copyright © 2018 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

Rheological behaviour of egg white and egg yolk from different poultry specimen

NASA Astrophysics Data System (ADS)

Kumbár, V.; Nedomová, Š.; Votava, J.; Buchar, J.

2017-01-01

The main goal of this study is differences in rheological behaviour of hen (ISA BROWN), goose (Anser anser f. domestica) and Japanese quail (Coturnix japonica) egg white and egg yolk. The rheological behaviour of egg white and egg yolk was studied using a concentric cylinder viscometer. Rheological behaviour was pseudoplastic and flow curves were fitted by the Herschel-Bulkley model and Ostwald-de Waele model with high values of coeficients of determination R2. The meaning of rheological parameters on friction factors during flow of egg white and egg yolk in real tube has been shown. Preliminary information on time-dependent behaviour of tested liquids has been also obtained.

[Effect of the alimentary factor on the immunobiologic reactivity of children's bodies].

PubMed

Voznesenskaia, F M; Panshinskaia, N M

1976-01-01

Observations covered 66 healthy six-year old children of a childrens' home. The actual alimentation of the children was studied according to tabulated values for one year and 112 apportionoments. In the rations of actual nutrition a disturbed correlation of proteins, fats and carbohydrates was noted. Seasonal variations of the salival lysozyme activity were revealed against the background of the actual alimentation. The lowest antimicrobial activity of the lysozyme was recorded in the winter and spring seasons of the year. The low lysozyme activity of the saliva in spring may be explained by deficiency of the animal protein in the ration. In winter time added to the insufficient content of the animal protein were features specific for the day's routine, typical of this season. An addition of the animal protein to the actual nutritional rations of the children, in the form of eggs and nonfat dry milk and a correction of the proteins, fats and carbohydrates proportions in the rations led to a statistically significant rise in the lysozyme activity in the saliva of children during all the months of observation.

Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

PubMed

Carballo, Sergio M; Haas, Linda; Krueger, Christian G; Reed, Jess D

2017-09-20

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in macrophages.

Characterization of the c-type lysozyme gene family in Anopheles gambiae.

PubMed

Li, Bin; Calvo, Eric; Marinotti, Osvaldo; James, Anthony A; Paskewitz, Susan M

2005-11-07

Seven new c-type lysozyme genes were found using the Anopheles gambiae genome sequence, increasing to eight the total number of genes in this family identified in this species. The eight lysozymes in An. gambiae have considerable variation in gene structure and expression patterns. Lys c-6 has the most unusual primary amino acid structure as the predicted protein consists of five lysozyme-like domains. Transcript abundance of each c-type lysozyme was determined by semiquantitative RT-PCR. Lys c-1, c-6 and c-7 are expressed constitutively in all developmental stages from egg to adult. Lys c-2 and c-4 also are found in all stages, but with relatively much higher levels in adults. Conversely, Lys c-3 and c-8 transcripts are highest in larvae. Lys c-1, c-6 and c-7 transcripts are found in nearly all the adult tissue samples examined while Lys c-2 and Lys c-4 are more restricted in their expression. Lys c-1 and c-2 transcripts are clearly immune responsive and are increased significantly 6-12 h post challenge with bacteria. The functional adaptive changes that may have evolved during the expansion of this gene family are briefly discussed in terms of the expression patterns, gene and protein structures.

Using modified soy protein to enhance foaming of egg white protein.

PubMed

Wang, Guang; Troendle, Molly; Reitmeier, Cheryll A; Wang, Tong

2012-08-15

It is well known that the foaming properties of egg white protein are significantly reduced when a small amount of yolk is mixed in the white. To improve foaming properties of yolk-contaminated egg white protein, soy protein isolate (SPI) and egg proteins were modified to make basic proteins, and effects of these modified proteins on egg white foaming were evaluated in a model and an angel cake system. SPI and egg yolk proteins were modified to have an isoelectric point of 10, and sonication was used to increase protein dispersibility after the ethyl esterification reaction. However, only the addition of sonicated and modified SPI (SMSPI) showed improvement of foaming in the 5% egg protein model system with 0.4% yolk addition. SMSPI was then used in making angel food cake to examine whether the cake performance reduction due to yolk contamination of the white would be restored by such alkaline protein. Cake performance was improved when cream of tartar was used together with SMSPI. Basic soy protein can be made and used to improve egg white foaming properties and cake performance. Copyright © 2012 Society of Chemical Industry.

Reorientational Dynamics of Enzymes Adsorbed on Quartz: A Temperature-Dependent Time-Resolved TIRF Anisotropy Study

PubMed Central

Czeslik, C.; Royer, C.; Hazlett, T.; Mantulin, W.

2003-01-01

The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20–80°C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized and the fractional amplitudes for the whole-body rotation, which are related to the order parameter for the local rotational freedom of the Trp residues, remain constant and do not approach zero. This behavior indicates that the angular range of the Trp reorientation within the adsorbed proteins is largely restricted even at high temperatures, in contrast to that of the dissolved proteins. The results of this study thus provide a deeper understanding of protein activity at solid surfaces. PMID:12668461

Temperature-dependent physical properties of egg white for HIFU applications

NASA Astrophysics Data System (ADS)

Liu, Yunbo; Maruvada, Subha; Herman, Bruce A.; Harris, Gerald R.

2012-10-01

Because egg white denatures at elevated temperature due to its protein content, it has the potential for use as a blood coagulation surrogate in pre-clinical evaluations of thermal therapy procedures such as high intensity focused ultrasound (HIFU) surgery. We therefore have measured the relevant physical properties of egg white, including coagulation temperature, frequency-dependent attenuation, sound speed, viscosity, and thermal properties, as a function of temperature (20 - 95°C). Thermal coagulation and attenuation (5-12 MHz) of cow blood, pig blood, and human blood also were assessed and compared with egg white. For a 30 s thermal exposure, both egg white and blood samples started to denature at 65°C and coagulate into an elastic gel at 85°C. The temperature-dependent parameters were found to be similar to that of the blood samples. For example, the attenuation of egg white ranged from 0.23f1.09 to 2.7f0.5 dB/cm over the 20°C - 95°C range. These results suggest that egg white would make a useful blood mimic for bench testing of therapeutic ultrasound devices.

9 CFR 590.510 - Classifications of shell eggs used in the processing of egg products.

Code of Federal Regulations, 2014 CFR

2014-01-01

... to include black rots, white rots, mixed rots, green whites, eggs with diffused blood in the albumen... any other filthy and decomposed eggs including the following: (1) Any egg with visible foreign matter...

9 CFR 590.510 - Classifications of shell eggs used in the processing of egg products.

Code of Federal Regulations, 2013 CFR

2013-01-01

... to include black rots, white rots, mixed rots, green whites, eggs with diffused blood in the albumen... any other filthy and decomposed eggs including the following: (1) Any egg with visible foreign matter...

Body weight, egg production, and egg quality traits of gray, brown, and white varieties of Japanese quail (Coturnix coturnix japonica) in coastal climatic condition of Odisha.

PubMed

Bagh, Jessy; Panigrahi, B; Panda, N; Pradhan, C R; Mallik, B K; Majhi, B; Rout, S S

2016-08-01

The present study was conducted to evaluate the performance of gray, brown, and white varieties of Japanese quail (Coturnix coturnix japonica) with respect to body weight, egg production, and egg quality traits in the coastal climatic condition of Odisha. A total of 500-day-old straight run Japanese quail chicks of three varieties, viz., gray, brown, and white were randomly selected and reared in deep litter system at Central Poultry Development Organization, Eastern Region, Bhubaneswar. The weekly body weight of the birds was recorded till their egg production stage (up to 6 weeks of age). The average egg production was recorded every biweekly from 6(th) to 20(th) week. Exterior and interior quality of eggs from each variety was determined at 6 weeks of age. The initial average weekly body weight of three varieties did not differ (p>0.05) among the varieties. However, from 1(st) to 6(th) week significantly higher body weight was observed in gray than white and brown. Brown varieties had reached 50% egg production 1 week earlier than gray and white. Brown had higher peak hen day (HD) production or hen-housed egg production followed by white and gray. External quality such as: Egg weight, egg length, egg width, volume, shape index, shell weight, shell thickness depicted no significant difference among the varieties except circumference length and circumference width, which were significantly higher (p≤0.05) in gray varieties than brown varieties. Internal egg characteristics such as: Albumen length, albumen width, albumen height, albumen index, yolk length, yolk width, yolk height, yolk index, albumen weight, yolk weight, Haugh unit revealed no significance difference among the varieties. It may be summarized from the findings that gray excelled in body weight followed by white and brown. Egg production potential in terms of hen house egg production or HD egg production was higher for brown followed by white and gray in the coastal climatic condition of Odisha.

Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

PubMed

Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

2003-09-01

The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

Surface Plasmon Resonance of Counterions coated Charged Silver Nanoparticles and Application in Bio-interaction

NASA Astrophysics Data System (ADS)

Ghosh, Goutam; Panicker, Lata; Naveen Kumar, N.; Mallick, Vivek

2018-05-01

Silver nanoparticles (SNPs) play very significant roles in biomedical applications, e.g., biosensors in numerous assays for quantitative detection, and the surface chemistry adds an important factor in that. In this investigation, we coated SNPs either by anionic citrates, like tri-lithium citrate (TLC) or tri-potassium citrate (TKC) which are associated with Li+ or K+ counterions, respectively; or by cationic surfactants, like cetylpyridinium chloride (CPC) or cetylpyridinium iodide (CPI) which are associated with Cl‑ or I‑ counterions, respectively, at the surface of nanoparticles. Our aim was to study (i) how the counterions affect the optical property of SNPs and (ii) the interaction of coated SNPs with a protein, hen egg white lysozyme (HEWL). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques were used to measure the size, and UV absorption spectroscopy was used to characterize the surface plasmon resonance (SPR) band of SNPs. ζ-potential, fluorescence quenching and circular dichroism (CD) spectroscopy techniques were used for characterizing the protein-nanoparticles interaction.

High-level expression of Camelid nanobodies in Nicotiana benthamiana.

PubMed

Teh, Yi-Hui Audrey; Kavanagh, Tony A

2010-08-01

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative 'universal' nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.

Effects of buoyancy-driven convection on nucleation and growth of protein crystals.

PubMed

Nanev, Christo N; Penkova, Anita; Chayen, Naomi

2004-11-01

Protein crystallization has been studied in presence or absence of buoyancy-driven convection. Gravity-driven flow was created, or suppressed, in protein solutions by means of vertically directed density gradients that were caused by generating suitable temperature gradients. The presence of enhanced mixing was demonstrated directly by experiments with crustacyanin, a blue-colored protein, and other materials. Combined with the vertical tube position the enhanced convection has two main effects. First, it reduces the number of nucleated hen-egg-white lysozyme (HEWL) crystals, as compared with those in a horizontal capillary. By enabling better nutrition from the protein in the solution, convection results in growth of fewer larger HEWL crystals. Second, we observe that due to convection, trypsin crystals grow faster. Suppression of convection, achieved by decreasing solution density upward in the capillary, can to some extent mimic conditions of growth in microgravity. Thus, impurity supply, which may have a detrimental effect on crystal quality, was avoided.

Semi-classical statistical description of Fröhlich condensation.

PubMed

Preto, Jordane

2017-06-01

Fröhlich's model equations describing phonon condensation in open systems of biological relevance are reinvestigated within a semi-classical statistical framework. The main assumptions needed to deduce Fröhlich's rate equations are identified and it is shown how they lead us to write an appropriate form for the corresponding master equation. It is shown how solutions of the master equation can be numerically computed and can highlight typical features of the condensation effect. Our approach provides much more information compared to the existing ones as it allows to investigate the time evolution of the probability density function instead of following single averaged quantities. The current work is also motivated, on the one hand, by recent experimental evidences of long-lived excited modes in the protein structure of hen-egg white lysozyme, which were reported as a consequence of the condensation effect, and, on the other hand, by a growing interest in investigating long-range effects of electromagnetic origin and their influence on the dynamics of biochemical reactions.

Structural differences between native Hen egg white lysozyme and its fibrils under different environmental conditions

NASA Astrophysics Data System (ADS)

Bhattacharya, Susmita; Ghosh, Sudeshna; Dasgupta, Swagata; Roy, Anushree

2013-10-01

The difference in molecular structure of native HEWL and its fibrils, grown at a pH value near physiological pH 7.4 and at a pH value just above the pI, 10.7 in presence and absence of Cu(II) ions, is discussed. We focus on differences between the molecular structure of the native protein and fibrils using principal component analysis of their Raman spectra. The overlap areas of the scores of each species are used to quantify the difference in the structure of the native HEWL and fibrils in different environments. The overall molecular structures are significantly different for fibrils grown at two pH values. However, in presence of Cu(II) ions, the fibrils have similarities in their molecular structures at these pH environments. Spectral variation within each species, as obtained from the standard deviations of the scores in PCA plots, reveals the variability in the structure within a particular species.

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen.

PubMed

Gugasyan, R; Velazquez, C; Vidavsky, I; Deck, B M; van der Drift, K; Gross, M L; Unanue, E R

2000-09-15

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.

Protein renaturation by the liquid organic salt ethylammonium nitrate.

PubMed Central

Summers, C. A.; Flowers, R. A.

2000-01-01

The room-temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured-reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility. PMID:11106174

Avian P1 antigens inhibit agglutination mediated by P fimbriae of uropathogenic Escherichia coli.

PubMed Central

Johnson, J R; Swanson, J L; Neill, M A

1992-01-01

Whole egg white from pigeon, dove, and cockatiel eggs, as well as the ovomucoid fraction of pigeon egg white, exhibited strong P1 antigenic activities and inhibited agglutination of human P1 erythrocytes and of digalactoside-coated latex beads by P-fimbriated Escherichia coli strains. In contrast, chicken egg white exhibited only weak P1 antigenic activity and had little impact on P-fimbrial agglutination. These preparations did not affect hemagglutination by E. coli strains expressing mannose-resistant adhesins other than P fimbriae, i.e., Dr, F1845, and S adhesins. Human anti-P1 serum diminished the P-fimbrial inhibitory activities of pigeon egg white and pigeon ovomucoid. Pigeon ovomucoid was equipotent on a molar basis with globoside, and the pigeon, dove, and cockatiel egg white preparations were equipotent with each other in P-fimbrial inhibition. Incubation of p erythrocytes in whole egg whites or in pigeon ovomucoid did not render them agglutinable by P-fimbriated bacteria, whereas incubation in globoside did. These data demonstrate that whole egg whites (and their ovomucoid fraction) from members of the families Columbidae (pigeons and doves) and Psittacidae (parrots) specifically and potently inhibit P-fimbrial agglutination, probably by providing P1 antigen as a receptor for the P-fimbrial adhesin. Avian egg white preparations may facilitate adhesin characterization of wild-type uropathogenic strains and may useful in preventing upper urinary tract infections due to P-fimbriated E. coli. PMID:1346125

Phenanthrene binding by humic acid-protein complexes as studied by passive dosing technique.

PubMed

Zhao, Jian; Wang, Zhenyu; Ghosh, Saikat; Xing, Baoshan

2014-01-01

This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Copyright © 2013 Elsevier Ltd. All rights reserved.

From Egg to Crystal: A Practical on Purification, Characterization, and Crystallization of Lysozyme for Bachelor Students

ERIC Educational Resources Information Center

Olieric, Vincent; Schreiber, Angelique; Lorber, Bernard; Putz, Joern

2007-01-01

A practical hands-on course encompassing enzyme purification, biochemical characterization, and crystallization that completed the course work of 350 second-year bachelor students enrolled in molecular biology/biochemistry was given at the Universite Louis Pasteur of Strasbourg (France). The experimental part of the practical dealt entirely with…

Shelf Stable Egg-Based Products Processed By Ultra High Pressure Technology

DTIC Science & Technology

2008-07-03

nonpathogenic flat-sour thermophilic organism have been used as one of the surrogate organisms for Clostridium botulinum in thermal processing (IFT, 2000...treated with lysozyme (100 μg/ml for 30 min) and trypsin (200 μg/ml for 2 h) to minimize interfering cell debris. After enzyme treatment the spore

Acidogenic fermentation characteristics of different types of protein-rich substrates in food waste to produce volatile fatty acids.

PubMed

Shen, Dongsheng; Yin, Jun; Yu, Xiaoqin; Wang, Meizhen; Long, Yuyang; Shentu, Jiali; Chen, Ting

2017-03-01

In this study, tofu and egg white, representing typical protein-rich substrates in food waste based on vegetable and animal protein, respectively, were investigated for producing volatile fatty acids (VFAs) by acidogenic fermentation. VFA production, composition, conversion pathways and microbial communities in acidogenesis from tofu and egg white with and without hydrothermal (HT) pretreatment were compared. The results showed HT pretreatment could improve the VFA production of tofu but not for egg white. The optimum VFA yields were 0.46g/gVS (tofu with HT) and 0.26g/gVS (egg white without HT), respectively. Tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30-40% of total protein remained in all groups after fermentation. Up to 50% of the unconverted soluble protein in the HT groups was protease. More lactate-producing bacteria, mainly Leuconostoc and Lactobacillus, were present during egg white fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complex coacervate core micelles with a lysozyme-modified corona.

PubMed

Danial, Maarten; Klok, Harm-Anton; Norde, Willem; Stuart, Martien A Cohen

2007-07-17

This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.

Extraction and Analysis of V-Type Agents (VX, RVX, CVX, and VM) from Various Food Matrices by Ultra-Performance Liquid Chromatography-Time-Of-Flight Mass Spectrometry

DTIC Science & Technology

2015-12-01

2% reduced fat milk, Egg Beaters egg whites, tomato sauce, and several meats, including hamburger meat (80% lean and 20% fat), hot dogs, chicken... Egg Whites and Tomato Sauce .....................................................3 2.3.3 Group 3: Hot Dogs, Chicken Nuggets, Turkey Deli Meat...Apple juice, orange juice, whole milk, 2% reduced fat milk, Egg Beaters processed egg whites, tomato sauce, precooked turkey deli meat (99% fat free

Impact of casein and egg white proteins on the structure of wheat gluten-based protein-rich food.

PubMed

Wouters, Arno G B; Rombouts, Ine; Lagrain, Bert; Delcour, Jan A

2016-02-01

There is a growing interest in texturally and nutritionally satisfying vegetable alternatives to meat. Wheat gluten proteins have unique functional properties but a poor nutritional value in comparison to animal proteins. This study investigated the potential of egg white and bovine milk casein with well-balanced amino acid composition to increase the quality of wheat gluten-based protein-rich foods. Heating a wheat gluten (51.4 g)-water (100.0 mL) blend for 120 min at 100 °C increased its firmness less than heating a wheat gluten (33.0 g)-freeze-dried egg white (16.8 g)-water (100.0 mL) blend. In contrast, the addition of casein to the gluten-water blend negatively impacted firmness after heating. Firmness was correlated with loss of protein extractability in sodium dodecyl sulfate containing medium during heating, which was higher with egg white than with casein. Even more, heat-induced polymerization of the gluten-water blend with egg white but not with casein was greater than expected from the losses in extractability of gluten and egg white on their own. Structure formation was favored by mixing gluten with egg white but not with casein. These observations were linked to the intrinsic polymerization behavior of egg white and casein, but also to their interaction with gluten. Thus not all nutritionally suitable proteins can be used for enrichment of gluten-based protein-rich foods. © 2015 Society of Chemical Industry.

Comparison of fatty acid, cholesterol, vitamin A and E composition, and trans fats in eggs from brown and white egg strains that were molted or nonmolted.

PubMed

Anderson, Kenneth E

2013-12-01

The impact of egg color, hen strain, and molting on the nutritional composition of eggs is limited. Therefore, this study compared nutritional composition and component percentages of cage-produced shell eggs with respect to egg color, hen strain, and molt. Four strains were selected from the North Carolina Layer Performance and Management Test: Hy-Line Brown (HB) and Bovans Brown (BB), and Hy-Line W-36 (HW) and Bovans White (BovW) were selected. Two groups from each strain were selected and 2 groups of molted HW and BovW were selected and compared with their nonmolted counterparts to examine the molt's impact. Two sets of eggs from each replicate were collected simultaneously at 101 wk of age. One sample of eggs was broken into a 12-egg pool stomached for 3 min (n = 12 samples), then divided into six 50-mL tubes, sealed, and frozen to be sent for cholesterol, n-3 fatty acids, saturated fat, monounsaturated fats, polyunsaturated fats, β-carotene, vitamin A, and vitamin E analyses. The other set of 12 eggs was then assessed for component percentages. The HW eggs had a greater (P < 0.05) percentage of yolk than the BovW eggs of 28.12 versus 27.00%, respectively; however, the BovW eggs had 1.0% more albumen. The HB and BB egg components were not different. Brown eggs were heavier (P < 0.01) than white eggs. White eggs had greater (P < 0.0001) percent yolk and the brown eggs had greater (P < 0.0001) percent albumen. The eggs from molted hens had a greater (P < 0.001) percent shell. Total fat content in the samples was (P < 0.05) higher in white eggs by 0.70% than brown eggs due to increased saturated and polyunsaturated fats. The molting of hens reduced (P < 0.01) saturated fats by 0.21% in the egg. Vitamin A levels were higher (P < 0.0001) in white eggs, and vitamin E was higher (P < 0.0001) in brown eggs. Strain and molt appear to influence nutrient composition and component percentages in eggs produced from laying hens.

Signals of monocyte activation in patients with SLE.

PubMed Central

Kávai, M; Zsindely, A; Sonkoly, I; Major, M; Demján, I; Szegedi, G

1983-01-01

The Fc receptor mediated reaction, the beta-glucuronidase and the lactic dehydrogenase activities of monocytes and the serum lysozyme level were tested together with the circulating immune complex content of patients with systemic lupus erythematosus. Simultaneously with the increasing FC receptor-mediated reaction and the elevated enzyme activities of patient monocytes, the secretion of lysozyme and the immune complex content of the sera were higher than those of the controls. A positive correlation was demonstrated between the Fc receptor-mediated reaction, the beta-glucuronidase activity, the lysozyme secretion and the immune complex content of the sera. Thus, the monocytes of patients appeared to be activated by the circulating immune complexes. PMID:6839541

Effects of salting treatment on the physicochemical properties, textural properties, and microstructures of duck eggs.

PubMed

Xu, Lilan; Zhao, Yan; Xu, Mingsheng; Yao, Yao; Nie, Xuliang; Du, Huaying; Tu, Yong-Gang

2017-01-01

In order to illuminate the forming process of salted egg, the effects of the brine solution with different salt concentrations on the physicochemical properties, textural properties, and microstructures of duck eggs were evaluated using conventional physicochemical property determination methods. The results showed that the moisture contents of both the raw and cooked egg whites and egg yolks, the springiness of the raw egg yolks and cooked egg whites exhibited a decreasing trend with the increase in the salting time and salt concentration. The salt content, oil exudation and the hardness of the raw egg yolks showed a constantly increasing trend. Viscosity of the raw egg whites showed an overall trend in which it first deceased and then increased and decreased again, which was similar to the trend of the hardness of the cooked egg whites and egg yolks. As the salting proceeded, the pH value of the raw and cooked egg whites declined remarkably and then declined slowly, whereas the pH of the raw and cooked egg yolks did not show any noticeable changes. The effect of salting on the pH value varied significantly with the salt concentration in the brine solution. Scanning electron microscopy (SEM) revealed that salted yolks consist of spherical granules and embedded flattened porosities. It was concluded that the treatment of salt induces solidification of yolk, accompanied with higher oil exudation and the development of a gritty texture. Different salt concentrations show certain differences.

Effects of salting treatment on the physicochemical properties, textural properties, and microstructures of duck eggs

PubMed Central

Xu, Lilan; Zhao, Yan; Xu, Mingsheng; Yao, Yao; Nie, Xuliang; Du, Huaying

2017-01-01

In order to illuminate the forming process of salted egg, the effects of the brine solution with different salt concentrations on the physicochemical properties, textural properties, and microstructures of duck eggs were evaluated using conventional physicochemical property determination methods. The results showed that the moisture contents of both the raw and cooked egg whites and egg yolks, the springiness of the raw egg yolks and cooked egg whites exhibited a decreasing trend with the increase in the salting time and salt concentration. The salt content, oil exudation and the hardness of the raw egg yolks showed a constantly increasing trend. Viscosity of the raw egg whites showed an overall trend in which it first deceased and then increased and decreased again, which was similar to the trend of the hardness of the cooked egg whites and egg yolks. As the salting proceeded, the pH value of the raw and cooked egg whites declined remarkably and then declined slowly, whereas the pH of the raw and cooked egg yolks did not show any noticeable changes. The effect of salting on the pH value varied significantly with the salt concentration in the brine solution. Scanning electron microscopy (SEM) revealed that salted yolks consist of spherical granules and embedded flattened porosities. It was concluded that the treatment of salt induces solidification of yolk, accompanied with higher oil exudation and the development of a gritty texture. Different salt concentrations show certain differences. PMID:28797071

Preliminary Work in Obtaining Site-Directed Mutants of Hen Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Holmes, Leonard D.

1996-01-01

Protein crystal growth studies are recognized as a critical endeavor in the field of molecular biotechnology. The scientific applications of this field include the understanding of how enzymes function and the accumulation of accurate information of atomic structures, a key factor in the process of rational drug design. NASA has committed substantial investment and resources to the field of protein crystal growth and has conducted many microgravity protein crystal growth experiments aboard shuttle flights. Crystals grown in space tend to be larger, denser and have a more perfect habit and geometry. These improved properties gained in the microgravity environment of space result largely from the reduction of solutal convection, and the elimination of sedimentation at the growing crystal surface. Shuttle experiments have yielded many large, high quality crystals that are suitable for high resolution X-ray diffraction analysis. Examples of biologically important macromolecules which have been successfully crystallized during shuttle missions include: lysozyme, isocitrate lyase, gamma-interferon, insulin, human serum albumin and canavalin. Numerous other examples are also available. In addition to obtaining high quality crystals, investigators are also interested in learning the mechanisms by which the growth events take place. Crystallization experiments indicate that for the enzyme HEWL, measured growth rates do not follow mathematical models for 2D nucleation and dislocation-led growth of tetragonal protein crystals. As has been suggested by the laboratory of Marc L. Pusey, a possible explanation for the disagreement between observation and data is that HEWL tetraconal crystals form by aggregated units of lysozyme in supersaturated solutions. Surface measurement data was shown to fit very well with a model using an octamer unit cell as the growth unit. According to this model, the aggregation pathway and subsequent crystal growth is described by: monomer < ------ > dimer < ------- > tetramer < ------ > octamer < ------ > higher order. It is believed that multimer aggregation of lysozyme occurs by interaction at specific binding sites on the surface of the protein crystals. If the presence of discrete binding sites and the aggregation hypothesis is true, then it follows that the alteration of the binding site(s) should have significant effect on the measurements obtained during growth experiments. Site-directed mutagenesis allows the specific alteration of proteins by replacement, deletion or addition of specific amino acid residues. This report outlines the approach for this strategy and the progress made thus far toward that end.

Geographical Variation in Egg Mass and Egg Content in a Passerine Bird

PubMed Central

Ruuskanen, Suvi; Siitari, Heli; Eeva, Tapio; Belskii, Eugen; Järvinen, Antero; Kerimov, Anvar; Krams, Indrikis; Moreno, Juan; Morosinotto, Chiara; Mänd, Raivo; Möstl, Erich; Orell, Markku; Qvarnström, Anna; Salminen, Juha-Pekka; Slater, Fred; Tilgar, Vallo; Visser, Marcel E.; Winkel, Wolfgang; Zang, Herwig; Laaksonen, Toni

2011-01-01

Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated. PMID:22110579

Hen genetic strain and extended cold storage influence on physical egg quality from cage-free aviary housing system.

PubMed

Jones, D R; Karcher, D M; Regmi, P; Robison, C O; Gast, R K

2018-04-02

In the United States, there is an increase in need for cage-free eggs in retail and food manufacturing sectors. Understanding the impact of cage-free systems and the corresponding management on egg quality is pertinent as the U.S. industry adapts existing housing and builds new cage-free housing structures. A study was conducted comparing 2 brown shell and 2 white shell hen strains housed in a cage-free aviary system. Each set of eggs were placed in cold storage and assessed at 0, 2, 4, 8, and 12 wk. Eggs were collected at 21, 31, 42, and 60 wk of hen age. A full profile of physical quality measurements was conducted on up to 18 intact eggs for each hen strain/egg storage/hen age combination. Egg weight increased approximately 10 g for brown shell and 14 g for white shell eggs as hens aged. Many of the properties monitored were significantly impacted by all 3 main effects (hen strain, egg storage, and hen age) resulting in 3-way interactions. A brown and a white shell strain had stronger shells (44 N; P < 0.0001) than the remaining brown and white shell strains (42 N and 39 N, respectively). The current study also determined volume of shell, total length, maximum width, and percent length at maximum width to more accurately indicate egg shape than shape index. One brown shell strain produced eggs with the most consistent shape characteristics over the hen ages monitored. White shell eggs from the cage-free aviary housing produced the highest whole-egg total solids between 31 to 60 wk of hen age, whereas brown shell eggs resulted in the most consistent level of whole-egg total solids (22-23.5%). The brown and white shell strains in the current study produce cage-free aviary eggs with distinctive physical quality attributes. The outcomes from this study can be utilized by the U.S. egg industry in planning management strategies and market placement of cage-free eggs.

Preparation of Egg White Liquid Hydrolysate (ELH) and Its Radical-Scavenging Activity

PubMed Central

Noh, Dong Ouk; Suh, Hyung Joo

2015-01-01

In the present study, an optimum protease was selected to hydrolyze the egg white liquid protein for the antioxidant peptides. Alcalase treatment yielded the highest amount of α-amino groups (15.27 mg/mL), while the control (no enzymatic hydrolysis) showed the lowest amount of α-amino groups (1.53 mg/mL). Alcalase also gave the highest degree of hydrolysis (DH) value (43.2%) and was more efficient for egg white liquid hydrolysis than the other enzymes. The Alcalase hydrolysate had the highest radical-scavenging activity (82.5%) at a concentration of 5.0 mg/mL. The conditions for enzymatic hydrolysis of egg white liquid with Alcalase were selected as substrate : water ratio of 2:1. Five percent Alacalse treatment did not show significant (P>0.05) increases of DH and α-amino nitrogen content after 24 h-hydrolysis. Thirty two hour-hydrolysis with 5% Alcalase is sufficient to make antioxidative egg white liquid hydrolysate from egg white liquid. DPPH and ABTS radical-scavenging activities were significantly (P<0.05) higher after enzymatic digestion. These results suggest that active peptides released from egg-white protein are effective radical-scavengers. Thus, this approach may be useful for the preparation of potent antioxidant products. PMID:26451355

Organochlorine contaminants in white-faced ibis eggs in southern Texas

USGS Publications Warehouse

Custer, T.W.; Mitchell, C.A.

1989-01-01

White-faced ibis eggs collected from 2 colonies in southern Texas in 1985 had low mean concentrations of DDE. DDD, the only other organochlorine contaminant detected, was found in only 1 of 20 eggs. DDE concentrations in eggs were not significantly correlated with eggshell thickness. Mean DDE concentrations were significantly higher in eggs collected from nests where not all of the remaining eggs hatched than in eggs collected from nests where all the remaining eggs hatched.

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

X-ray solution scattering combined with computation characterizing protein folds and multiple conformational states : computation and application.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, S.; Park, S.; Makowski, L.

Small angle X-ray scattering (SAXS) is an increasingly powerful technique to characterize the structure of biomolecules in solution. We present a computational method for accurately and efficiently computing the solution scattering curve from a protein with dynamical fluctuations. The method is built upon a coarse-grained (CG) representation of the protein. This CG approach takes advantage of the low-resolution character of solution scattering. It allows rapid determination of the scattering pattern from conformations extracted from CG simulations to obtain scattering characterization of the protein conformational landscapes. Important elements incorporated in the method include an effective residue-based structure factor for each aminomore » acid, an explicit treatment of the hydration layer at the surface of the protein, and an ensemble average of scattering from all accessible conformations to account for macromolecular flexibility. The CG model is calibrated and illustrated to accurately reproduce the experimental scattering curve of Hen egg white lysozyme. We then illustrate the computational method by calculating the solution scattering pattern of several representative protein folds and multiple conformational states. The results suggest that solution scattering data, when combined with a reliable computational method, have great potential for a better structural description of multi-domain complexes in different functional states, and for recognizing structural folds when sequence similarity to a protein of known structure is low. Possible applications of the method are discussed.« less

Physical properties of microencapsulated gamma-3 salmon oil with egg white powder

USDA-ARS?s Scientific Manuscript database

Microencapsulated salmon oil with egg white powders are a good source of high quality protein and amino acids including leucine and omega-3 fatty acids, which may be beneficial for athletes. The study demonstrated that egg white powders containing omega-3 salmon oil can be effectively produced by sp...

Effects of salting processes and time on the chemical composition, textural properties, and microstructure of cooked duck egg.

PubMed

Kaewmanee, Thammarat; Benjakul, Soottawat; Visessanguan, Wonnop

2011-03-01

Chemical composition, textural properties, and microstructure of cooked duck egg salted by 2 methods (coating and immersing) were determined during 4 wk of salting. As the salting time increased, moisture content increased and salt content decreased for both cooked salted egg white and yolk. Oil exudation of cooked yolk and expressible water content of cooked egg white obtained from both salting methods increased as salting proceeded (P < 0.05). After cooking, oil exudation accompanied by the solubilized pigments, especially at the outer layer of yolk, was obtained. At week 3 of salting, egg yolk from coating method had the higher egg exudation than that from immersing method. As the salting times increased, the lower hardness, springiness, gumminess, chewiness, and resilience with higher adhesiveness and cohesiveness were generally found in cooked salted egg white (P < 0.05), irrespective of salting methods. Conversely, the hardness of cooked yolk increased continuously and reached the maximum at week 2 and 2 to 3 for immersing and coating method (P < 0.05), respectively. Confocal laser scanning micrographs revealed the smaller yolk granules with more release of free lipid in salted egg after heating, compared with the fresh counterpart. As visualized by scanning electron microscope, gel of cooked salted egg white was coagulum type with larger voids. Salting methods determined oil exudation in egg yolk and texture profile of egg white gel after cooking; however, those attributes were also governed by the salting time. Salted duck egg can be made by 2 methods (coating and immersing) affecting the characteristic of salted egg white and yolk after cooking. Desirable cooked salted egg having the red yolk with hardness and high oil exudation could be obtained when salting was carried out for 3 and 4 wk for immersing and coating method, respectively.

Protein Conformation and Supercharging with DMSO from Aqueous Solution

NASA Astrophysics Data System (ADS)

Sterling, Harry J.; Prell, James S.; Cassou, Catherine A.; Williams, Evan R.

2011-07-01

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3-5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation.

PubMed

Morshedi, Dina; Ebrahim-Habibi, Azadeh; Moosavi-Movahedi, Ali Akbar; Nemat-Gorgani, Mohsen

2010-04-01

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein-protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97. Copyright 2009 Elsevier B.V. All rights reserved.

Unscrambling Egg Allergy: The Diagnostic Value of Specific IgE Concentrations and Skin Prick Tests for Ovomucoid and Egg White in the Management of Children with Hen's Egg Allergy.

PubMed

Marriage, D E; Erlewyn-Lajeunesse, M; Unsworth, D J; Henderson, A J

2012-01-01

Resolution of egg allergy occurs in the majority of egg allergic children. Positive specific IgE antibodies to ovomucoid (OVM) have been suggested to be of greater predictive value for persistent egg allergy than specific IgE to egg white. The performance of OVM-specific IgE antibody levels in a cohort of children referred for a routine egg challenge was compared with egg white specific IgE levels in predicting a positive egg challenge. 24/47 subjects had persistent egg allergy. Receiver operating characteristic analysis showed that OVM-specific IgE testing was the most useful test for the diagnosis of persistent egg allergy. The optimal decision points for the prediction of persistent egg allergy were >0.35 kUA/L for specific IgE levels to both EW and OVM, and ≥3 mm for SPT. Children with specific IgE levels suggestive of persistent egg allergy need not be subject to an egg provocation challenge, reducing both costs and risks to the child.

Anaphylactic reaction to probiotics. Cow's milk and hen's egg allergens in probiotic compounds.

PubMed

Martín-Muñoz, María Flora; Fortuni, Monserrat; Caminoa, Magdalena; Belver, Teresa; Quirce, Santiago; Caballero, Teresa

2012-12-01

Probiotics are used in the treatment of allergic diseases. We investigated the safety of probiotics for subjects with food allergy. Labels of probiotics commercially available in Spain were examined to assess their content of cow's milk or hen's egg. Skin prick tests with these compounds (20 mg/ml) were performed in five children allergic to cow's milk, five children allergic to hen's white egg, and five control subjects non-allergic to food. Three serum pools: I (positive-specific IgE to cow's milk and hen's egg white proteins), II (positive-specific IgE to cow's milk and negative to hen's egg white proteins), and III (negative-specific IgE to cow's milk and positive to hen's egg white proteins) were used to detect cow's milk and hen's egg white allergens in probiotics. ImmunoCAP(®) (Phadia), in-house ELISA, SDS-PAGE immunoblotting, and inhibition studies of these assays were performed. Proteins were quantified by enzyme-immunoassay. Eleven probiotics were studied. No label advertised about egg content, eight labels warned about lactose, lactic acid or cow's milk, one label claimed to be milk-free, and two gave no information. Cow's milk proteins were detected, by at least one lab technique, in 10/11 probiotics, three over 2.5 mg/kg (21, 52, 112 mg/kg). Hen's egg white proteins were detected in 3/11 probiotics, only one had more than 2.5 mg/kg (47 mg/kg). Probiotic compounds may contain hidden allergens of food and may not be safe for subjects with allergy to cow's milk or hen's egg. © 2012 John Wiley & Sons A/S.

Egg white hydrolysate inhibits oxidation in mayonnaise and a model system.

PubMed

Kobayashi, Hideaki; Sasahara, Ryou; Yoda, Shoichi; Kotake-Nara, Eiichi

2017-06-01

The flavor deterioration of mayonnaise is induced by iron, which is released from egg yolk phosvitin under acidic conditions and promotes lipid oxidation. To prevent oxidative deterioration, natural components, rather than synthetic chemicals such as ethylenediaminetetraacetic acid have been required by consumers. In the present study, we evaluated the inhibitory effects of three egg white components with the same amino acid composition, namely egg white protein, hydrolysate, and the amino acid mixture, on lipid oxidation in mayonnaise and an acidic egg yolk solution as a model system. We found that the hydrolysate had the strongest inhibitory effect on lipid oxidation among the three components. The mechanism underlying the antioxidant effect was associated with Fe 2+ -chelating activity. Thus, egg white hydrolysate may have the potential as natural inhibitors of lipid oxidation in mayonnaise.

A male sexually dimorphic trait provides antimicrobials to eggs in blenny fish.

PubMed

Giacomello, Eva; Marchini, Daniela; Rasotto, Maria B

2006-09-22

Predation and microbial infections are the major causes of natural mortality for early life stages of oviparous species. The parental traits reducing the effects of predation are rather well described, whereas antimicrobial mechanisms enhancing offspring survival are largely unexplored. In this paper, we report that a male sexually dimorphic trait, the anal glands, of the redlip blenny (Ophioblennius atlanticus atlanticus) and the peacock blenny (Salaria pavo), two fish species with paternal egg care, produce a mucus enriched with antimicrobial substances. Histological and histochemical analyses showed that the anal glands of these species are characterized by the massive presence of mucus-secreting cells. Anal gland extracts, from both the hydrophilic and the hydrophobic protein fraction, exhibited a lysozyme-like activity. Field observations demonstrated that redlip blenny males, while performing egg care, rub the anal region over the nest internal surface, probably facilitating the transfer of mucus to eggs. These results strongly indicate that this sexually dimorphic trait is involved in egg defence against microbial infections.

Interaction of nucleic acids with Coomassie Blue G-250 in the Bradford assay.

PubMed

Wenrich, Broc R; Trumbo, Toni A

2012-09-15

The Bradford assay has been used reliably for decades to quantify protein in solution. The analyte is incubated in acidic solution of Coomassie Blue G-250 dye, during which reversible ionic and nonionic binding interactions form. Bradford assay color yields were determined for salmon, bovine, shrimp, and kiwi fruit genomic DNA; baker's yeast RNA; bovine serum albumin (BSA); and hen egg lysozyme. Pure DNA and RNA bound the dye, with color yields of 0.0017 mg⁻¹ cm⁻¹ and 0.0018 mg⁻¹ cm⁻¹, respectively. The nucleic acid-Coomassie Blue response was significant, at roughly 9% of that for BSA and 18% of that for lysozyme. Copyright © 2012 Elsevier Inc. All rights reserved.

Dynamic analysis of growth of Salmonella Enteritidis in liquid egg whites

USDA-ARS?s Scientific Manuscript database

Salmonella Enteritidis (SE) is a common foodborne pathogen associated with eggs and egg products. This research was conducted to study the kinetics of growth and survival of SE in liquid egg whites (LEW). A dynamic temperature profile that exposed SE to suboptimal temperatures and below the minimu...

Biological and Clinical Implications of Lysozyme Deposition on Soft Contact Lenses

PubMed Central

Omali, Negar Babaei; Subbaraman, Lakshman N.; Coles-Brennan, Chantal; Fadli, Zohra; Jones, Lyndon W.

2015-01-01

ABSTRACT Within a few minutes of wear, contact lenses become rapidly coated with a variety of tear film components, including proteins, lipids, and mucins. Tears have a rich and complex composition, allowing a wide range of interactions and competitive processes, with the first event observed at the interface between a contact lens and tear fluid being protein adsorption. Protein adsorption on hydrogel contact lenses is a complex process involving a variety of factors relating to both the protein in question and the lens material. Among tear proteins, lysozyme is a major protein that has both antibacterial and anti-inflammatory functions. Contact lens materials that have high ionicity and high water content have an increased affinity to accumulate lysozyme during wear, when compared with other soft lens materials, notably silicone hydrogel lenses. This review provides an overview of tear film proteins, with a specific focus on lysozyme, and examines various factors that influence protein deposition on contact lenses. In addition, the impact of lysozyme deposition on various ocular physiological responses and bacterial adhesion to lenses and the interaction of lysozyme with other tear proteins are reviewed. This comprehensive review suggests that deposition of lysozyme on contact lens materials may provide a number of beneficial effects during contact lens wear. PMID:26002002

Heat resistance of Salmonella in various egg products.

PubMed

Garibaldi, J A; Straka, R P; Ijichi, K

1969-04-01

The heat-resistance characteristics of Salmonella typhimurium Tm-1, a reference strain in the stationary phase of growth, were determined at several temperatures in the major types of products produced by the egg industry. The time required to kill 90% of the population (D value) at a given temperature in specific egg products was as follows: at 60 C (140 F), D = 0.27 min for whole egg; D = 0.60 min for whole egg plus 10% sucrose; D = 1.0 min for fortified whole egg; D = 0.20 min for egg white (pH 7.3), stabilized with aluminum; D = 0.40 min for egg yolk; D = 4.0 min for egg yolk plus 10% sucrose; D = 5.1 min for egg yolk plus 10% NaCl; D = 1.0 min for scrambled egg mix; at 55 C (131 F), D = 0.55 min for egg white (pH 9.2); D = 1.2 min for egg white (pH 9.2) plus 10% sucrose. The average Z value (number of degrees, either centigrade or fahrenheit, for a thermal destruction time curve to traverse one logarithmic cycle) was 4.6 C (8.3 F) with a range from 4.2 to 5.3 C. Supplementation with 10% sucrose appeared to have a severalfold greater effect on the heat stabilization of egg white proteins than on S. typhimurium Tm-1. This information should be of value in the formulation of heat treatments to insure that all egg products be free of viable salmonellae.

Physicochemical properties of anti Vibrio harveyi egg yolk antibody (IgY) and its immunological influence in Indian white shrimp Fenneropenaeus indicus.

PubMed

Kumaran, Thankamani; Thirumalaikumar, Eswaramoorthy; Lelin, Chinnadurai; Palanikumar, Pandi; Michaelbabu, Mariavincent; Citarasu, Thavasimuthu

2018-03-01

Edible antibodies specific to host pathogens is an attractive approach to establish protective immunity, especially against gastrointestinal pathogens both in humans and animals. The edible antibody of anti-Vibrio harveyi IgY (anti-V. h IgY) was produced by antigen mixed with immunoadjuvant Asparagus racemosus and Glycine max. Hens were immunized and eggs were collected five weeks after the immunization. Anti-V. harveyi IgY stability in different digestive enzymes such as trypsin and chymotrypsin were evaluated to determine its ability to withstand in the gastrointestinal tract of F. indicus. Specific binding activity and concentration (average 9.5% of total IgY content) of the anti-V. h IgY were determined by the ELISA using V. harveyi antigen. Further the anti-V. h IgY diets including V.h wo, V.h A, V.h G and control diets were fed to F. indicus for 60 days. After 30 and 60 of feeding, group of shrimps were challenged with virulent V. harveyi. After the respective days of feeding, haematological and immunological changes were studied. The parameters including total haemocyte count (THC), coagulase activity, oxyhaemocyanin level, prophenoloxidase, intracellular superoxide anion production, lysozyme, phagocytosis and bacterial agglutinin had significantly (P ≤ .001) increased in the experimental groups in comparission with the control diet fed shrimps. The anti-V. h IgY coated diets helped to reduce the Vibrio load and boosted the immune system in F. indicus's against V. harveyi challenge. The research work shows the potential applications of egg yolk antibodies as anti-bacterial prophylactic uses for infectious diseases and suggests an edible antibody concept as an alternative to conventional antibiotics. Copyright © 2018 Elsevier Ltd. All rights reserved.

Organochlorine contaminants in white-faced ibis eggs in southern Texas

USGS Publications Warehouse

Custer, T.W.; Mitchell, C.A.

1989-01-01

White-faced Ibis (Plegadis chihi ) eggs collected from two colonies in southern Texas in 1985 had low mean concentrations of DDE (0.14-0.27 ppm wet weight). DDD, the only other organochlorine contaminant detected, was found in only 1 of 20 eggs. DDE concentrations in eggs were not significantly correlated with eggshell thickness. Mean DDE concentrations were significantly higher in eggs collected from nests where not all of the remaining eggs hatched (1.0 ppm) than in eggs collected from nests where all the remaining eggs hatched (0.15 ppm).

The Role of Wheat and Egg Constituents in the Formation of a Covalent and Non-covalent Protein Network in Fresh and Cooked Egg Noodles.

PubMed

Lambrecht, Marlies A; Rombouts, Ine; Nivelle, Mieke A; Delcour, Jan A

2017-01-01

Noodles of constant protein content and flour-to-egg protein ratio were made with whole egg, egg white, or egg yolk. The optimal cooking time, water absorption, and cooking loss of salted whole egg noodles was respectively lower and higher than of egg white and egg yolk noodles. However, cooked whole egg noodles showed the best Kieffer-rig extensibility. Differences in noodle properties were linked to protein network formation. Disulfide bonds in whole egg noodles developed faster and to a larger extent during cooking than in egg yolk noodles but slower and to a lower extent than in egg white noodles. The balance between the rate of protein cross-linking and starch swelling determines cooked noodle properties. Ionic and hydrophobic protein interactions increase the optimum cooking time and total work in Kieffer-rig extensibility testing of fresh noodles. Hydrogen bonds and covalent cross-links are probably the main determinants of the extensibility of cooked noodles. © 2016 Institute of Food Technologists®.

Role of specific IgE and skin-prick testing in predicting food challenge results to baked egg.

PubMed

Cortot, Catherine F; Sheehan, William J; Permaul, Perdita; Friedlander, James L; Baxi, Sachin N; Gaffin, Jonathan M; Dioun, Anahita F; Hoffman, Elaine B; Schneider, Lynda C; Phipatanakul, Wanda

2012-01-01

Previous studies suggest that children with egg allergy may be able to tolerate baked egg. Reliable predictors of a successful baked egg challenge are not well established. We examined egg white-specific IgE levels, skin-prick test (SPT) results, and age as predictors of baked egg oral food challenge (OFC) outcomes. We conducted a retrospective chart review of children, aged 2-18 years, receiving an egg white-specific IgE level, SPT, and OFC to baked egg from 2008 to 2010. Fifty-two oral baked egg challenges were conducted. Of the 52 challenges, 83% (n = 43) passed and 17% (n = 9) failed, including 2 having anaphylaxis. Median SPT wheal size was 12 mm (range, 0-35 mm) for passed challenges and 17 mm (range, 10-30 mm) for failed challenges (p = 0.091). The negative predictive value for passing the OFC was 100% (9 of 9) if SPT wheal size was <10 mm. Median egg white-specific IgE was 2.02 kU/L (range, <0.35-13.00 kU/L) for passed challenges and 1.52 kU/L (range, 0.51-6.10 kU/L) for failed challenges (p = 0.660). Receiver operating characteristic (ROC) curve analysis for SPT revealed an area under the curve (AUC) of 0.64. ROC curve analysis for egg white-specific IgE revealed an AUC of 0.63. There was no significant difference in age between patients who failed and those who passed (median = 8.8 years versus 7.0 years; p = 0.721). Based on our sample, SPT, egg white-specific IgE and age are not good predictors of passing a baked egg challenge. However, there was a trend for more predictability with SPT wheal size.

Determination of yolk contamination in liquid egg white using Raman spectroscopy.

PubMed

Cluff, K; Konda Naganathan, G; Jonnalagada, D; Mortensen, I; Wehling, R; Subbiah, J

2016-07-01

Purified egg white is an important ingredient in a number of baked and confectionary foods because of its foaming properties. However, yolk contamination in amounts as low as 0.01% can impede the foaming ability of egg white. In this study, we used Raman spectroscopy to evaluate the hypothesis that yolk contamination in egg white could be detected based on its molecular optical properties. Yolk contaminated egg white samples (n = 115) with contamination levels ranging from 0% to 0.25% (on weight basis) were prepared. The samples were excited with a 785 nm laser and Raman spectra from 250 to 3,200 cm(-1) were recorded. The Raman spectra were baseline corrected using an optimized piecewise cubic interpolation on each spectrum and then normalized with a standard normal variate transformation. Samples were randomly divided into calibration (n = 77) and validation (n = 38) data sets. A partial least squares regression (PLSR) model was developed to predict yolk contamination levels, based on the Raman spectral fingerprint. Raman spectral peaks, in the spectral region of 1,080 and 1,666 cm(-1), had the largest influence on detecting yolk contamination in egg white. The PLSR model was able to correctly predict yolk contamination levels with an R(2) = 0.90 in the validation data set. These results demonstrate the capability of Raman spectroscopy for detection of yolk contamination at very low levels in egg white and present a strong case for development of an on-line system to be deployed in egg processing plants. © 2016 Poultry Science Association Inc.

Surfactant sodium lauryl sulfate enhances skin vaccination: molecular characterization via a novel technique using ultrafiltration capillaries and mass spectrometric proteomics.

PubMed

Huang, Chun-Ming; Wang, Chao-Cheng; Kawai, Mikako; Barnes, Stephen; Elmets, Craig A

2006-03-01

The skin is a highly accessible organ and thus provides an attractive immune environment for cost-effective, simple, and needle-free delivery of vaccines and immunomodulators. In this study, we pretreated mouse skin with an anionic surfactant, sodium lauryl sulfate (SLS), for a short period of time (10 min) followed by epicutaneous vaccination with hen egg lysozyme antigen. We demonstrated for the first time that pretreatment of skin with surfactant SLS significantly enhances the production of antibody to hen egg lysozyme. Short term pretreatment with SLS disorganized the stratum corneum, extracted partial lamellar lipids, induced the maturation of Langerhans cells, and did not result in epidermis thickening. To reveal the mechanism underlying these changes, particularly at the molecular level, we used a novel proteomic technique using ultrafiltration capillaries and mass spectrometry to identify in vivo proteins/peptides secreted in the SLS-pretreated skin. Two secretory proteins, named as calcium-binding protein S100A9 and thymosin beta4, were identified by this novel technique. These two proteins thus may provide new insight into the enhancing effect of surfactants on skin vaccination.

On the development of multifunctional luminescent supramolecular hydrogel of gold and egg white

NASA Astrophysics Data System (ADS)

Patra, Sudeshna; Ravulapalli, Sathyavathi; Hahm, Myung Gwan; Tadi, Kiran Kumar; Narayanan, Tharangattu N.

2016-10-01

Highly stable, luminescent, and printable/paintable supramolecular egg white hydrogel-based surface enhanced Raman scattering (SERS) matrix is created by an in situ synthesis of gold clusters inside a luminescent egg white hydrogel (Au-Gel). The synthesis of stable luminescent egg-white-based hydrogel, where the hydrogel can act as a three dimensional (3D) matrix, using a simple cross-linking chemistry, has promising application in the biomedical field including in 3D cell culturing. Furthermore, this functional hydrogel is demonstrated for micromolar-level detection of Rhodamine 6G using the SERS technique, where Au-Gel is painted over a flexible cellulose pad.

A study of the interaction between malachite green and lysozyme by steady-state fluorescence.

PubMed

Ding, Fei; Liu, Wei; Liu, Feng; Li, Zhi-Yuan; Sun, Ying

2009-09-01

The interaction of a N-methylated diaminotriphenylmethane dye, malachite green, with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results revealed that malachite green caused the fluorescence quenching of lysozyme through a static quenching procedure. The thermodynamic parameters like DeltaH and DeltaS were calculated to be -15.33 kJ mol(-1) and 19.47 J mol(-1) K(-1) according to van't Hoff equation, respectively, which proves main interaction between malachite green and lysozyme is hydrophobic forces and hydrogen bond contact. The distance r between donor (lysozyme) and acceptor (malachite green) was obtained to be 3.82 nm according to Frster's theory. The results of synchronous fluorescence, UV/vis and three-dimensional fluorescence spectra showed that binding of malachite green with lysozyme can induce conformational changes in lysozyme. In addition, the effects of common ions on the constants of lysozyme-malachite green complex were also discussed.

Is it possible to make a diagnosis of raw, heated, and baked egg allergy in children using cutoffs? A systematic review.

PubMed

Calvani, Mauro; Arasi, Stefania; Bianchi, Annamaria; Caimmi, Davide; Cuomo, Barbara; Dondi, Arianna; Indirli, Giovanni Cosimo; La Grutta, Stefania; Panetta, Valentina; Verga, Maria Carmen

2015-09-01

The diagnosis of IgE-mediated egg allergy lies both on a compatible clinical history and on the results of skin prick tests (SPTs) and IgEs levels. Both tests have good sensitivity but low specificity. For this reason, oral food challenge (OFC) is the ultimate gold standard for the diagnosis. The aim of this study was to systematically review the literature in order to identify, analyze, and synthesize the predictive value of SPT and specific IgEs both to egg white and to main egg allergens and to review the cutoffs suggested in the literature. A total of 37 articles were included in this systematic review. Studies were grouped according to the degree of cooking of the egg used for OFC, age, and type of allergen used to perform the allergy workup. In children <2 years, raw egg allergy seems very likely when SPTs with egg white extract are ≥4 mm or specific IgEs are ≥1.7 kUA /l. In children ≥2 years, OFC could be avoided when SPTs with egg white extract are ≥10 mm or prick by prick with egg white is ≥14 mm or specific IgE is ≥7.3 kUA /l. Likewise, heated egg allergy can be diagnosed if SPTs with egg white extract are >5 and >11 mm in children <2 and ≥2 years, respectively. Further and better-designed studies are needed to determine the remaining diagnostic cutoff of specific IgE and SPT for heated and baked egg allergy. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Complexation of lysozyme with adsorbed PtBS-b-SCPI block polyelectrolyte micelles on silver surface.

PubMed

Papagiannopoulos, Aristeidis; Christoulaki, Anastasia; Spiliopoulos, Nikolaos; Vradis, Alexandros; Toprakcioglu, Chris; Pispas, Stergios

2015-01-20

We present a study of the interaction of the positively charged model protein lysozyme with the negatively charged amphiphilic diblock polyelectrolyte micelles of poly(tert-butylstyrene-b-sodium (sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) on the silver/water interface. The adsorption kinetics are monitored by surface plasmon resonance, and the surface morphology is probed by atomic force microscopy. The micellar adsorption is described by stretched-exponential kinetics, and the micellar layer morphology shows that the micelles do not lose their integrity upon adsorption. The complexation of lysozyme with the adsorbed micellar layers depends on the micelles arrangement and density in the underlying layer, and lysozyme follows the local morphology of the underlying roughness. When the micellar adsorbed amount is small, the layers show low capacity in protein complexation and low resistance in loading. When the micellar adsorbed amount is high, the situation is reversed. The adsorbed layers both with or without added protein are found to be irreversibly adsorbed on the Ag surface.

Heat Resistance of Salmonella in Various Egg Products

PubMed Central

Garibaldi, J. A.; Straka, R. P.; Ijichi, K.

1969-01-01

The heat-resistance characteristics of Salmonella typhimurium Tm-1, a reference strain in the stationary phase of growth, were determined at several temperatures in the major types of products produced by the egg industry. The time required to kill 90% of the population (D value) at a given temperature in specific egg products was as follows: at 60 C (140 F), D = 0.27 min for whole egg; D = 0.60 min for whole egg plus 10% sucrose; D = 1.0 min for fortified whole egg; D = 0.20 min for egg white (pH 7.3), stabilized with aluminum; D = 0.40 min for egg yolk; D = 4.0 min for egg yolk plus 10% sucrose; D = 5.1 min for egg yolk plus 10% NaCl; D = 1.0 min for scrambled egg mix; at 55 C (131 F), D = 0.55 min for egg white (pH 9.2); D = 1.2 min for egg white (pH 9.2) plus 10% sucrose. The average Z value (number of degrees, either centigrade or fahrenheit, for a thermal destruction time curve to traverse one logarithmic cycle) was 4.6 C (8.3 F) with a range from 4.2 to 5.3 C. Supplementation with 10% sucrose appeared to have a severalfold greater effect on the heat stabilization of egg white proteins than on S. typhimurium Tm-1. This information should be of value in the formulation of heat treatments to insure that all egg products be free of viable salmonellae. Images PMID:4890741

Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

NASA Astrophysics Data System (ADS)

Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography.

PubMed

Coughlan, H D; Darmanin, C; Kirkwood, H J; Phillips, N W; Hoxley, D; Clark, J N; Vine, D J; Hofmann, F; Harder, R J; Maxey, E; Abbey, B

2017-01-01

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed.

Specific chemical and structural damage to proteins produced by synchrotron radiation.

PubMed

Weik, M; Ravelli, R B; Kryger, G; McSweeney, S; Raves, M L; Harel, M; Gros, P; Silman, I; Kroon, J; Sussman, J L

2000-01-18

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Broecker, Jana; Klingel, Viviane; Ou, Wei-Lin

In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometerbased X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble andmore » membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.« less

Control of Protein Crystal Nucleation and Growth Using Stirring Solution

NASA Astrophysics Data System (ADS)

Niino, Ai; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Kinoshita, Takayoshi; Warizaya, Masaichi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

2004-11-01

We have previously developed a protein crystallization technique using a stirring protein solution and revealed that (i) continuous stirring prevents excess spontaneous nucleation and accelerates the growth of protein crystals and (ii) prestirring (solution stirring in advance) promotes the crystal nucleation of hen egg-white lysozyme. In bovine adenosine deaminase (ADA) crystallization, continuous stirring improves the crystal quality but elongates the nucleation time. In this paper, in order to control both the crystal nucleation and growth of ADA using a Micro-Stirring technique, we carried out five different stirring patterns such as (i) no stirring, (ii) continuous stirring, (iii) prestirring, (iv) poststirring (stirring late in the growth period) and (v) restirring (combined pre- and poststirring). The results showed that high-quality well-shaped crystals were obtained under the continuous stirring and restirring conditions and the nucleation time under the prestirring and restirring conditions was shorter than that under the continuous stirring and poststirring conditions. Consequently, high-quality crystals were promptly obtained under the restirring condition. These results suggest that we are able to control both the nucleation and growth of protein crystals with the stirring techniques.

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

DOE PAGES

Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.; ...

2017-01-01

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to themore » diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.« less

Graphene-based microfluidics for serial crystallography.

PubMed

Sui, Shuo; Wang, Yuxi; Kolewe, Kristopher W; Srajer, Vukica; Henning, Robert; Schiffman, Jessica D; Dimitrakopoulos, Christos; Perry, Sarah L

2016-08-02

Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to themore » diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.« less

Changes in the Molar Ellipticities of HEWL Observed by Circular Dichroism and Quantitated by Time Resolved Fluorescence Anisotropy Under Crystallizing Conditions

NASA Technical Reports Server (NTRS)

Sumida, John

2002-01-01

Fluid models for simple colloids predict that as the protein concentration is increased, crystallization should occur at some sufficiently high concentration regardless of the strength of attraction. However, empirical measurements do not fully support this assertion. Measurements of the second virial coefficient (B22) indicate that protein crystallization occurs only over a discrete range of solution parameters. Furthermore, observations of a strong correlation between protein solubility and B22, has led to an ongoing debate regarding the relationship between the two. Experimental work in our lab, using Hen Egg White Lysozyme (HEWL), previously revealed that the rotational anisotropy of the protein under crystallizing conditions changes systematically with pH, ionic strength and temperature. These observations are now supported by recent work revealing that small changes in the molar ellipticity also occur systematically with changes in ionic strength and temperature. This work demonstrates that under crystallization conditions, the protein native state is characterized by a conformational heterogeneity that may prove fundamental to the relationship between protein crystallization and protein solubility.

Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station.

PubMed

Fujiwara, Takahisa; Suzuki, Yoshihisa; Yoshizaki, Izumi; Tsukamoto, Katsuo; Murayama, Kenta; Fukuyama, Seijiro; Hosokawa, Kouhei; Oshi, Kentaro; Ito, Daisuke; Yamazaki, Tomoya; Tachibana, Masaru; Miura, Hitoshi

2015-08-01

The normal growth rates of the {110} faces of tetragonal hen egg-white lysozyme crystals, R, were measured as a function of the supersaturation σ parameter using a reflection type interferometer under μG at the International Space Station (NanoStep Project). Since water slightly evaporated from in situ observation cells during a long-term space station experiment for several months, equilibrium temperature T(e) changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration C(s). To correct σ, the actual C(s) and protein concentration C(p), which correctly represent the measured T(e) value in space, were first calculated. Second, a new solubility curve with the corrected C(s) was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the C(s) and C(p) of the solution.

Thermal Optimization of Growth and Quality in Protein Crystals

NASA Technical Reports Server (NTRS)

Wiencek, John M.

1996-01-01

Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints.

Exploratory and descriptive study on nutritional characteristics and quality of eggs from Chilean partridge (Nothoprocta perdicaria).

PubMed

Arias, José Luis; Matthei, Alberto; Valenzuela, Carolina

2018-01-01

This work aims to contribute more information on tinamou eggs by performing an exploratory and descriptive study of some of their nutritional and quality characteristics. The chemical composition of tinamou egg showed a high protein content in white (85% dry basis) and high lipid concentration in yolk (52% db). The iron (Fe) content in white was higher than hen egg (0.47 mg/100 g) and this could be associated with the observed pinkish color of the white. As in the hen egg, the major fatty acids in tinamou yolk were: oleic (39%), linoleic (23%) and palmitic (20%). The cholesterol content of tinamou was 21.2 mg/g of yolk, and 100 g of whole egg provides 589 mg of cholesterol. As in the hen egg, tinamou egg white showed high levels of lysine, sulfur-containing amino acids, threonine and valine with respect to the recommended allowance for an adult man. All essential amino acids with the exception of histidine cover the adult requirements. The shell inorganic composition of these eggs is calcium carbonate and the morphology was similar to other avian eggs. Tinamou egg is small and elongated, with a dark brown color. The eggshell is thinner and experiences more deformation but less breaking strength than hen eggshell. © 2017 Japanese Society of Animal Science.

Unfolding and refolding details of lysozyme in the presence of β-casein micelles.

PubMed

Wu, Fu-Gen; Luo, Jun-Jie; Yu, Zhi-Wu

2011-02-28

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.

Genome-wide association study reveals novel variants for growth and egg traits in Dongxiang blue-shelled and White Leghorn chickens.

PubMed

Liao, R; Zhang, X; Chen, Q; Wang, Z; Wang, Q; Yang, C; Pan, Y

2016-10-01

This study was designed to investigate the genetic basis of growth and egg traits in Dongxiang blue-shelled chickens and White Leghorn chickens. In this study, we employed a reduced representation sequencing approach called genotyping by genome reducing and sequencing to detect genome-wide SNPs in 252 Dongxiang blue-shelled chickens and 252 White Leghorn chickens. The Dongxiang blue-shelled chicken breed has many specific traits and is characterized by blue-shelled eggs, black plumage, black skin, black bone and black organs. The White Leghorn chicken is an egg-type breed with high productivity. As multibreed genome-wide association studies (GWASs) can improve precision due to less linkage disequilibrium across breeds, a multibreed GWAS was performed with 156 575 SNPs to identify the associated variants underlying growth and egg traits within the two chicken breeds. The analysis revealed 32 SNPs exhibiting a significant genome-wide association with growth and egg traits. Some of the significant SNPs are located in genes that are known to impact growth and egg traits, but nearly half of the significant SNPs are located in genes with unclear functions in chickens. To our knowledge, this is the first multibreed genome-wide report for the genetics of growth and egg traits in the Dongxiang blue-shelled and White Leghorn chickens. © 2016 Stichting International Foundation for Animal Genetics.

Sensitizing and Eliciting Capacity of Egg White Proteins in BALB/c Mice As Affected by Processing.

PubMed

Pablos-Tanarro, Alba; Lozano-Ojalvo, Daniel; Martínez-Blanco, Mónica; López-Fandiño, Rosina; Molina, Elena

2017-06-07

This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential.

Simultaneous Measurement of Thermophysical Properties of Tissue-Mimicking Phantoms for High Intensity Focused Ultrasound (HIFU) Exposures

NASA Astrophysics Data System (ADS)

Gao, Jing; You, Jiang; Huang, Zhihong; Cochran, Sandy; Corner, George

2012-03-01

Tissue-mimicking phantoms, including bovine serum albumin phantoms and egg white phantoms, have been developed for, and in laboratory use for, real-time visualization of high intensity focused ultrasound-induced thermal coagulative necrosis since 2001. However, until now, very few data are available concerning their thermophysical properties. In this article, a step-wise transient plane source method has been used to determine the values of thermal conductivity, thermal diffusivity, and specific heat capacity of egg white phantoms with elevated egg white concentrations (0 v/v% to 40 v/v%, by 10 v/v% interval) at room temperature (~20 °C). The measured thermophysical properties were close to previously reported values; the thermal conductivity and thermal diffusivity were linearly proportional to the egg white concentration within the investigation range, while the specific heat capacity decreased as the egg white concentration increased. Taking account of large differences between real experiment and ideal model, data variations within 20 % were accepted.

Ontogenetic profile of innate immune related genes and their tissue-specific expression in brown trout, Salmo trutta (Linnaeus, 1758).

PubMed

Cecchini, Stefano; Paciolla, Mariateresa; Biffali, Elio; Borra, Marco; Ursini, Matilde V; Lioi, Maria B

2013-09-01

The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1β, TGF-β and lysozyme c-type). The constitutive expression of β-actin was detected in all developmental stages. IL-1β and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-β transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1β and TGF-β transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout. Copyright © 2013 Elsevier Ltd. All rights reserved.

Formation of crystalline nanoparticles by iron binding to pentapeptide (Asp-His-Thr-Lys-Glu) from egg white hydrolysates.

PubMed

Sun, Na; Cui, Pengbo; Li, Dongmei; Jin, Ziqi; Zhang, Shuyu; Lin, Songyi

2017-09-20

A novel peptide from egg white, Asp-His-Thr-Lys-Glu (DHTKE), contains specific amino acids associated with iron binding. The present study aims to better understand the molecular basis of interactions between the DHTKE peptide and iron ions. The ultraviolet-visible and fluorescence spectra indicate an interaction between the DHTKE peptide and iron ions, which leads to the formation of a DHTKE-iron complex. Notably, Asp, Glu, His, and Lys in the DHTKE peptide play crucial roles in the formation of the DHTKE-iron complex, and the iron-binding site of the DHTKE peptide corresponds primarily to the amide and carboxyl groups. The DHTKE peptide can bind iron ions in a 1 : 2 ratio with a binding constant of 1.312 × 10 5 M -1 . Moreover, the DHTKE-iron complex belongs to thermodynamically stable nanoparticles that are present in the crystalline structure, which might be attributed to peptide folding induced by iron binding. Meanwhile, the DHTKE-iron complex exhibits a relatively high iron-releasing percentage and exerts excellent solubility in the human gastrointestinal tract in vitro. This suggests a potential application of peptides containing Asp, Glu, His, or Lys residues as potential iron supplements.

Compositional analysis and structural elucidation of glycosaminoglycans in chicken eggs

PubMed Central

Liu, Zhangguo; Zhang, Fuming; Li, Lingyun; Li, Guoyun; He, Wenqing; Linhardt, Robert J.

2014-01-01

Glycosaminoglycans (GAGs) have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods. GAGs are also critically important in the developmental biology of all multicellular animals. GAGs were isolated from chicken egg components including yolk, thick egg white, thin egg white, membrane, calcified shell matrix supernatant, and shell matrix deposit. Disaccharide compositional analysis was performed using ultra high-performance liquid chromatography-mass spectrometry. The results of these analyses showed that all four families of GAGs were detected in all egg components. Keratan sulfate was found in egg whites (thick and thin) and shell matrix (calcified shell matrix supernatant and deposit) with high level. Chondroitin sulfates were much more plentiful in both shell matrix components and membrane. Hyaluronan was plentiful in both shell matrix components and membrane, but were only present in a trace of quantities in the yolk. Heparan sulfate was plentiful in the shell matrix deposit but was present in a trace of quantities in the egg content components (yolk, thick and thin egg whites). Most of the chondroitin and heparan sulfate disaccharides were present in the GAGs found in chicken eggs with the exception of chondroitin and heparan sulfate 2,6-disulfated disaccharides. Both CS and HS in the shell matrix deposit contained the most diverse chondroitin and heparan sulfate disaccharide compositions. Eggs might provide a potential new source of GAGs. PMID:25218438

Preparation and Characterization of Fluorescent Derivatives of Chicken Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Sumida, John; Forsythe, Elizabeth; Pusey, Marc

2000-01-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow, cascade blue, and 5-(2-aminoethyl)aminonapthalene-l-sulfonic acid (EDANS) have been attached to the side chain carboxyl of asp101 using a carbodiimide coupling procedure. asp101 lies within the active site cleft, and it is believed that the probes are at least partially "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to hisl5, located on the "back side" of the molecule relative to the active site. The fluorescently labeled protein is readily purified from the starting material by cation exchange chromatography. All the derivatives fluoresce in both the solution and the crystalline states. Fluorescence characterization has focused on determining the bound probe quantum yields, lifetimes, absorption and emission spectra, and quenching by added solutes in comparison to the free probe. No appreciable changes are found in the lifetimes of any of the probes except for cascade blue, where Tau(sub free) = 3.52 ns vrs Tau(sub bound) = 2.8 ns. Spectral shifts are found in most cases. Particularly strong quenching upon binding is found in the case of the cascade blue derivative, likely due to probe interactions with the active site cleft. While none of the asp101 bound probes are well quenched by commonly employed solutes, such as potassium and sodium iodide, acrylamide, primuline, the chloride salts of manganese, cesium, and cobalt, trifluoroacetamide, trichloroethanol, and thallium iodide, in those cases where quenching is observed the bound probe is less efficiently quenched relative to the free probe. This indicates that the bound probes are less accessible to the bulk solution, an expected finding for attachment within the active site cleft. Attempts have been made to bind other molecules to these sites, with varying success. Interestingly, all three probes contain one or more sulfonate ((Ar-S03)-) groups. We have not been successful in binding analogous probes without sulfate groups such as pyrene, or with derivatized sulfonate groups such as dansyl type probes, analogous to MANS but where the sulfonate group is derivatized, Ar-S02-N2C2H7. None of the probes is rigidly bound to the protein, i.e., they all have a probe motion superimposed on that of the protein.

Effect of the combination of white and red LED lighting during incubation on layer, broiler, and Pekin duck hatchability.

PubMed

Archer, G S; Jeffrey, D; Tucker, Z

2017-08-01

Previous research has shown that providing light during incubation can have positive effects on hatchability and chick quality; however, white light alone has been observed to improve these factors only in pigmented broiler eggs and non-pigmented white layer eggs. Monochromatic red light has been shown to improve hatchability in layer eggs. Therefore the objective of this study was to utilize one light fixture that emitted both white and monochromatic red light to determine if this one light source could improve hatchability in both types of chicken eggs and pigmented Pekin duck egg. To determine this, 3 experiments were conducted, the first using White Leghorn eggs (N = 6912), the second using commercial broiler eggs (N = 4608), and the third using commercial Pekin duck eggs (N = 3564) in which eggs were incubated with 12 h of light and 12 h of darkness (LED) or complete darkness (DARK); the light level was 250 lux. Hatchability, embryo mortality, and hatchling quality were measured. In Experiment 1, LED had fewer early dead embryos (P = 0.03), less overall embryo mortality (P = 0.05), fewer chicks with unhealed navels (P < 0.001), fewer chicks with defects (P < 0.001), and a higher percentage of fertile eggs that hatched (P = 0.05) than DARK. In Experiment 2, LED had fewer chicks with unhealed navels (P = 0.003), fewer chicks with defects (P = 0.001), and a higher percentage of fertile eggs that hatched (P = 0.04) than DARK. In Experiment 3, LED had fewer early dead embryos (P = 0.05), lower overall embryo mortality (P = 0.04), and a higher percentage of fertile eggs that hatched (P = 0.05), and had ducklings with lower bodyweights at hatch (P = 0.04) than DARK. These results indicate that providing both white and red light during incubation can improve chick quality across poultry varieties. This type of fixture could be used to improve commercial hatchery efficiency and chick quality. © 2017 Poultry Science Association Inc.

The Oral Provocation Test for Raw Egg in Patients with Hen Egg Allergy.

PubMed

Kido, Jun; Nishi, Natsuko; Matsumoto, Tomoaki

2018-06-06

Many researchers have made efforts to develop diagnostic tools for predicting the outcome of oral food challenges (OFCs). The aim of this study was to assess the diagnostic value of the skin prick test (SPT) and blood-specific IgE concentrations based on the outcome of the OFCs for heated and raw hen egg. This study included 103 children with suspected hen egg allergy (HEA; median age 23 months, range 10-155; 72 boys, 31 girls). Forty-three patients were diagnosed with HEA by OFC. Of 60 patients who tolerated heated egg white (HEW), 22 underwent the OFC for raw hen egg and 7 developed adverse reactions after ingesting raw egg. Their wheal diameters and specific IgE levels for egg white and ovomucoid were determined. Wheal diameters as well as blood-specific IgE levels for egg white and ovomucoid were significantly larger in children with positive OFC results for HEW than in those with negative results. However, there were no significant differences between the positive and negative test results for raw hen egg white (REW) in wheal diameter or blood-specific IgE levels. The SPT and blood-specific IgE can be used to diagnose HEA. However, the provocation test for REW in children without HEW allergy is important because the values of SPT and specific IgE were not significantly different between children with and without raw egg allergy. © 2018 S. Karger AG, Basel.

Passive Immune-Protection of Litopenaeus vannamei against Vibrio harveyi and Vibrio parahaemolyticus Infections with Anti-Vibrio Egg Yolk (IgY)-Encapsulated Feed

PubMed Central

Gao, Xiaojian; Zhang, Xiaojun; Lin, Li; Yao, Dongrui; Sun, Jingjing; Du, Xuedi; Li, Xiumei; Zhang, Yue

2016-01-01

Vibrio spp. are major causes of mortality in white shrimp (Litopenaeus vannamei) which is lacking adaptive immunity. Passive immunization with a specific egg yolk antibody (IgY) is a potential method for the protection of shrimp against vibriosis. In this study, immune effects of the specific egg yolk powders (IgY) against both V. harveyi and V. parahaemolyticus on white shrimp were evaluated. The egg yolk powders against V. harveyi and V. parahaemolyticus for passive immunization of white shrimp were prepared, while a tube agglutination assay and an indirect enzyme-linked immunosorbent assay (ELISA) were used for detection of IgY titer. Anti-Vibrio egg yolk was encapsulated by β-cyclodextrin, which could keep the activity of the antibody in the gastrointestinal tract of shrimp. The results showed that the anti-Vibrio egg powders had an inhibiting effect on V. harveyi and V. parahaemolyticus in vitro. Lower mortality of infected zoeae, mysis, and postlarva was observed in groups fed with anti-Vibrio egg powders, compared with those fed with normal egg powders. The bacterial load in postlarva fed with specific egg powders in seeding ponds was significantly lower than those fed with normal egg powders in seeding ponds. These results show that passive immunization by oral administration with specific egg yolk powders (IgY) may provide a valuable protection of vibrio infections in white shrimp. PMID:27196895

LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

PubMed Central

Huang, Peng; Li, Wenshu; Yang, Zhifang; Zhang, Ning; Xu, Yixin; Bao, Jianying; Jiang, Deke; Dong, Xianping

2017-01-01

Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract. PMID:28182716

The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

1998-01-01

Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

Stability and activity of lysozyme in stoichiometric and non-stoichiometric protic ionic liquid (PIL)-water systems

NASA Astrophysics Data System (ADS)

Wijaya, Emmy C.; Separovic, Frances; Drummond, Calum J.; Greaves, Tamar L.

2018-05-01

There has been a substantial increase in enzyme applications within the biochemical and pharmaceutical industries, for example, as industrial biocatalysts. However, enzymes have narrow marginal stability which makes them prone to become inactive and/or denature with a slight change in the solvent environment. Typically industrial applications require harsher solvent environments than enzyme native environments, and hence there is a need to understand solvent-protein interactions in order to develop strategies to maintain, or enhance, the enzymatic activity under industrially relevant solvent conditions. Previously we have shown that protic ionic liquids (PILs) with water can have a stabilising effect on lysozyme, with a large variation dependent on which PIL ions are present, and the water concentration [E. C. Wijaya et al., Phys. Chem. Chem. Phys. 18(37), 25926-25936 (2016)]. Here we extend on this work using non-stoichiometric aqueous PIL solvents to investigate, and isolate, the role of pH and ionicity on enzymes. We have used the PILs ethylammonium nitrate (EAN) and ethanolammonium formate (EOAF) since our previous work has identified these as good solvents for lysozyme. Solvent libraries were made from these two PILs with an additional precursor acid or base to modify the acidity/basicity of the neutral stoichiometric PIL, and with water added, to have solutions with 4-17 mol. % of the PIL ions in water. Molar ratios of base:acid were varied between 1:1.05 and 2:1 for EAN and 1:1.25 and 2:1 for EOAF, which enabled from highly basic to highly acidic solutions to be obtained. This was to modify the acidity/basicity of the neutral stoichiometric PILs, without the addition of buffers. The structure and stability of hen egg white lysozyme (HEWL) were explored under these solvent conditions using synchrotron small angle X-ray scattering (SAXS), Fourier transform infrared (FTIR), and activity assays. The radius of gyration and Kratky plots obtained from the SAXS data showed little change with varying ionicity or acid:base ratio. FTIR showed that α-helix was maintained in all, except for the most acidic solvent conditions. The activity data show that HEWL was active between pH 0 and 11 for the EA:N-water system and pH 4.4 and 11 for the EOA:F-water system. This work indicates that ionic liquids have the potential to enable enzymes to maintain activity across a broader range of solvent conditions.

A role for mitochondria in antigen processing and presentation

PubMed Central

Bonifaz, Laura C; Cervantes-Silva, Mariana P; Ontiveros-Dotor, Elizabeth; López-Villegas, Edgar O; Sánchez-García, F Javier

2015-01-01

Immune synapse formation is critical for T-lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen-presenting cells (APCs) and T lymphocytes is a two-way signalling process. However, the role of mitochondria in APCs during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing and -presentation process. Here we show that hen egg white lysozyme (HEL) -loaded B lymphocytes, as a type of APC, undergo a small but significant mitochondrial depolarization by 1–2 hr following antigen exposure, suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca2+ uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analysed. Oligomycin treatment reduced the amount of specific MHC–peptide complexes but not total MHC II on the cell membrane of B lymphocytes, which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes, which endogenously express HEL and by B lymphocytes loaded with the HEL48–62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taken together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APC mitochondria were found to re-organize towards the APC–T immune synapse. PMID:25251370

Constant-pH Hybrid Nonequilibrium Molecular Dynamics–Monte Carlo Simulation Method

PubMed Central

2016-01-01

A computational method is developed to carry out explicit solvent simulations of complex molecular systems under conditions of constant pH. In constant-pH simulations, preidentified ionizable sites are allowed to spontaneously protonate and deprotonate as a function of time in response to the environment and the imposed pH. The method, based on a hybrid scheme originally proposed by H. A. Stern (J. Chem. Phys.2007, 126, 164112), consists of carrying out short nonequilibrium molecular dynamics (neMD) switching trajectories to generate physically plausible configurations with changed protonation states that are subsequently accepted or rejected according to a Metropolis Monte Carlo (MC) criterion. To ensure microscopic detailed balance arising from such nonequilibrium switches, the atomic momenta are altered according to the symmetric two-ends momentum reversal prescription. To achieve higher efficiency, the original neMD–MC scheme is separated into two steps, reducing the need for generating a large number of unproductive and costly nonequilibrium trajectories. In the first step, the protonation state of a site is randomly attributed via a Metropolis MC process on the basis of an intrinsic pKa; an attempted nonequilibrium switch is generated only if this change in protonation state is accepted. This hybrid two-step inherent pKa neMD–MC simulation method is tested with single amino acids in solution (Asp, Glu, and His) and then applied to turkey ovomucoid third domain and hen egg-white lysozyme. Because of the simple linear increase in the computational cost relative to the number of titratable sites, the present method is naturally able to treat extremely large systems. PMID:26300709

Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.

PubMed Central

Jeruzalmi, D; Steitz, T A

1998-01-01

The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold. PMID:9670025

Effect of combination of chitosan coating and irradiation on physicochemical and functional properties of chicken egg during room-temperature storage

NASA Astrophysics Data System (ADS)

Liu, Xian De; Jang, Aera; Kim, Dong Hun; Lee, Bong Duk; Lee, Mooha; Jo, Cheorun

2009-07-01

The effect of combination of chitosan coating and irradiation on quality and storage stability of shell egg was investigated. Salmonella typhimurium inoculated on eggshell was not detected by irradiation of 2.0 kGy at day 0 and/or chitosan coating (1%, pH 5.0) after 3 days of storage. One-day-old fresh chicken egg was chitosan coated and irradiated at 0, 0.5, 1.0, 1.5 and 2.0 kGy by gamma ray. The egg samples were stored at room temperature for 14 days and the effects of the combination treatment on internal physicochemical and functional properties were investigated. The Haugh unit of egg was decreased by irradiation even at 0.5 kGy. Irradiation increased the lipid oxidation in egg yolk at 2 kGy but the egg with chitosan coating reduced the level of lipid oxidation. Irradiation increased the foaming ability of egg white and decreased viscosity of egg yolk and white. Results suggested that combination of irradiation and chitosan coating can improve safety of shell egg but irradiation treatment may reduce the egg quality for direct consumption. However, an improved functional property for further processing and efficient separation of egg white and yolk can be expected for egg processing industry using irradiation.

Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

PubMed Central

Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

2013-01-01

In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+pe) of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/+pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos. PMID:24027530

7 CFR 57.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... eggs, eggs with green whites, eggs with stuck yolks, moldy eggs, musty eggs, eggs showing blood rings... processing plant means one or more buildings or parts thereof comprising a single plant in which the plant...

Effect of Guci powder on toe swelling induced by egg white in rats

NASA Astrophysics Data System (ADS)

Xie, Guoqi; Hao, Shaojun; Shen, Huiling; Ma, Zhenzhen; Zhang, Xuehui; Zhang, Zhengchen

2018-04-01

To observe the effect of Guci Powder on foot swelling induced by egg white in rats. 50 male rats were randomly divided into normal saline group (n=10), white vinegar group (n=10) and Guning lotion group (n=10). There were 10 rats in the high-dose group and 10 in the low-dose group. The rats in each group were treated with the drug on the left and right feet of the rats. 0.5 hours after the last administration, the rats in each group were inflamed. The left hindsole plantar volume was measured respectively, so that the difference of the posterior toe volume before inflammation was taken as the swelling degree, and the swelling degree of each group was calculated. Compared with physiological saline group, the rats' egg white toe swelling (P<0.01) was significantly inhibited at 0.5˜6h after administration. The swelling of egg white toe in rats was inhibited at 0.5˜2h (P<0.05). Bone spur powder has a good intervention effect on the model of toe swelling induced by egg white in rats, and the external application of bone spur powder has anti-inflammatory and swelling effect.

Dietary egg whites for phosphorus control in maintenance haemodialysis patients: a pilot study.

PubMed

Taylor, Lynn M; Kalantar-Zadeh, Kamyar; Markewich, Theodore; Colman, Sara; Benner, Debbie; Sim, John J; Kovesdy, Csaba P

2011-03-01

High dietary protein intake is associated with greater survival in maintenance haemodialysis (MHD) patients. High-protein foods may increase dietary phosphorus burden, which is associated with increased mortality in these patients. Hypothesis is: an egg white based diet with low phosphorus to protein ratio (<1.4 mg/g) will lower serum phosphorus without deteriorating the nutritional status in MHD patients. We assessed serum phosphorus and albumin levels in MHD patients who agreed to ingest one meal per day with pasteurised liquid egg whites without phosphorus additives, as principal protein source. Thirteen otherwise stable MHD patients with serum phosphorus >4.0 mg/dl agreed to consume eight ounces (225 g) of pasteurised liquid egg whites one meal per day for six weeks. Recipes were suggested to improve diet variety. Thirteen participating patients included seven women, three African Americans and five diabetics. Twelve patients exhibited drop in serum phosphorus. Mean population fall in serum phosphorus was 0.94 mg/dl, i.e. from 5.58 ± 1.34 (mean ± SD) to 4.63 ± 1.18 (p = 0.003). Serum albumin showed an increase by 0.19 g/dl, i.e. from 4.02 ± 0.29 to 4.21 ± 0.36 g/dl (p = 0.014). Changes in phosphorus pill count were not statistically significant (p = 0.88). The egg white diet was well tolerated, and recipe variety appreciated. Pasteurised liquid egg whites may be an effective diet component lowering serum phosphorus without risking malnutrition. Controlled trials are indicated to examine egg white based dietary interventions in MHD patients at home or during haemodialysis treatment. © 2011 European Dialysis and Transplant Nurses Association/European Renal Care Association.

Salmonella enterica serovar Enteridis (SE) infection in 5 commercial white egg and 3 commercial brown egg strains of laying hens: comparison of SE colonization with immune responsiveness

USDA-ARS?s Scientific Manuscript database

Different chicken breeds have been shown to exhibit varying susceptibility to infection with paratyphoid salmonellae. The current study was undertaken to compare the course of infection with SE in crops and intestines of 5 commercial white egg strains (W1-W5) and 3 commercial brown egg strains (B1-...

Comparing in vitro lymphocyte reactivity to Salmonella enterica serovar Enteridis (SE) antigens 5 commercial white egg and 3 commercial brown egg strains of laying hens following SE infection.

USDA-ARS?s Scientific Manuscript database

Different chicken breeds have been shown to exhibit varying susceptibility to infection with paratyphoid salmonellae. The current study was undertaken to compare the course of infection with SE in crops and intestines of 5 commercial white egg strains (W1-W5) and 3 commercial brown egg strains (B1-...

Antioxidant activity in cooked and simulated digested eggs.

PubMed

Remanan, M K; Wu, J

2014-07-25

The avian egg is an excellent source of nutrients consisting of components with beneficial properties but there is limited knowledge on the effect of cooking methods and gastrointestinal digestion on the antioxidant activity of eggs. The present study was focused on the effect of cooking and simulated gastrointestinal digestion on antioxidant activity of eggs using ORAC, ABTS and DPPH assays. The results suggest that fresh egg yolk has higher antioxidant activity than fresh egg white and whole eggs. Cooking reduced whereas simulated gastrointestinal digestion increased the antioxidant activity of eggs. Boiled egg white hydrolysate showed the highest antioxidant activity; a total of 63 peptides were identified, indicative of the formation of novel antioxidant peptides upon simulated gastrointestinal digestion. This study suggests the potential role of eggs as a dietary source of antioxidants.

21 CFR 160.145 - Dried egg whites.

Code of Federal Regulations, 2010 CFR

2010-04-01

... whites. The quantity used and the time of reaction are sufficient to substantially reduce the glucose... time of reaction are sufficient to substantially reduce the glucose content. (ii) Bacterial procedure. The liquid egg whites are subjected to the action of a culture of glucose-fermenting bacteria either...

Effect of Egg Size on Predation by White-Footed Mice

Treesearch

R. M. DeGraaf; T. J. Maier

1996-01-01

We compared predation by wild-trapped, caged white-footed mice (Peromyscus leucopus) on eggs of Japanese Quail (Coturnix coturnix) and Zebra Finches (Poephila guttata) to test the effect of egg size. Nine male and nine female mice were weighed, acclimated to cages for 24 h, and presented with two wicker nests,...

Efficacy of hydrogen peroxide in controlling mortality associated with saprolegniasis on walleye, white sucker, and paddlefish eggs

USGS Publications Warehouse

Gaikowski, M.P.; Rach, J.J.; Drobish, M.; Hamilton, J.; Harder, T.; Lee, L.A.; Moen, C.; Moore, A.

2003-01-01

The efficacy of hydrogen peroxide in controlling saprolegniasis on eggs of walleye Stizostedion vitreum, white sucker Catostomus commersoni, and paddlefish Polyodon spathula was evaluated at four private, state, and federal production hatcheries participating in an Investigational New Animal Drug efficacy study (experiment 1; walleyes) and in a laboratory-based miniature egg jar incubation system (experiment 2; walleyes, white suckers, and paddlefish). Naturally occurring fungal infestations (saprolegniasis) were observed on eggs in both experiments. Confirmatory diagnosis of infested eggs from one hatchery in experiment 1 identified the pathogen as Saprolegnia parasitica. During experiment 1, eggs were treated daily for 15 min with either 0, 500, or 750 mg/L of hydrogen peroxide, and one trial compared a 500-mg/L hydrogen peroxide treatment with a formalin treatment at 1,667 mg/L. Saprolegniasis infestation was observed in control egg jars, whereas treatment with either formalin or hydrogen peroxide virtually eliminated the infestation. Hydrogen peroxide treatments of 500 mg/L either increased egg hatch or were as effective as physical removal of infested eggs in controlling mortality. Although treatment with formalin at 1,667 mg/L significantly increased the percent eye-up of walleye eggs compared with that of those treated with hydrogen peroxide at 500 mg/L, the difference was only 1.9-2.6%. In experiment 2, noneyed eggs were treated for 15 min every other day with 0, 283, 565, or 1,130 mg/L of hydrogen peroxide until the viable eggs hatched. Saprolegniasis infestation engulfed most control eggs, whereas infestation of treated eggs was either reduced or not visible. Hydrogen peroxide significantly increased egg hatch for all three species tested in experiment 2. Although hydrogen peroxide treatments as low as 283 mg/L significantly increased walleye and white sucker hatch, treatments between 500 and 1,000 mg/L are more likely to be effective in production egg incubation systems.

A contribution to the rational design of Ru(CO)3Cl2L complexes for in vivo delivery of CO.

PubMed

Seixas, João D; Santos, Marino F A; Mukhopadhyay, Abhik; Coelho, Ana C; Reis, Patrícia M; Veiros, Luís F; Marques, Ana R; Penacho, Nuno; Gonçalves, Ana M L; Romão, Maria J; Bernardes, Gonçalo J L; Santos-Silva, Teresa; Romão, Carlos C

2015-03-21

A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)3Cl2L] (L = DMSO (3), L-H3CSO(CH2)2CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)2CH(NH2)CO2H (6b); 3-NC5H4(CH2)2SO3Na (7); 4-NC5H4(CH2)2SO3Na (8); PTA (9); DAPTA (10); H3CS(CH2)2CH(OH)CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWN) shows the addition of Ru(II)(CO)(H2O)4 at the His15 binding site. Soakings with 7(4UWN) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)3](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO(-) on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.

Egg hatching response to a range of ultraviolet-B (UV-B) radiation doses for four predatory mites and the herbivorous spider mite Tetranychus urticae.

PubMed

Koveos, Dimitrios S; Suzuki, Takeshi; Terzidou, Anastasia; Kokkari, Anastasia; Floros, George; Damos, Petros; Kouloussis, Nikos A

2017-01-01

Egg hatchability of four predatory mites-Phytoseiulus persimilis Athias-Henriot, Iphiseius [Amblyseius] degenerans Berlese, Amblyseius swirskii Athias-Henriot, and Euseius finlandicus Oudemans (Acari: Phytoseiidae)-and the spider mite Tetranychus urticae Koch (Acari: Tetranychidae) was determined under various UV-B doses either in constant darkness (DD) or with simultaneous irradiation using white light. Under UV-B irradiation and DD or simultaneous irradiation with white light, the predator's eggs hatched in significantly lower percentages than in the control non-exposed eggs, which indicates deleterious effects of UV-B on embryonic development. In addition, higher hatchability percentages were observed under UV-B irradiation and DD in eggs of the predatory mites than in eggs of T. urticae. This might be caused by a higher involvement of an antioxidant system, shield effects by pigments or a mere shorter duration of embryonic development in predatory mites than in T. urticae, thus avoiding accumulative effects of UV-B. Although no eggs of T. urticae hatched under UV-B irradiation and DD, variable hatchability percentages were observed under simultaneous irradiation with white light, which suggests the involvement of a photoreactivation system that reduces UV-B damages. Under the same doses with simultaneous irradiation with white light, eggs of T. urticae displayed higher photoreactivation and were more tolerant to UV-B than eggs of the predatory mites. Among predators variation regarding the tolerance to UV-B effects was observed, with eggs of P. persimilis and I. degenerans being more tolerant to UV-B radiation than eggs of A. swirskii and E. finlandicus.

21 CFR 160.145 - Dried egg whites.

Code of Federal Regulations, 2011 CFR

2011-04-01

... fermentation procedures—(i) Yeast procedure. Food-grade baker's yeast (Saccharomyces cerevisiae) is added to the liquid egg whites and controlled fermentation is maintained. The quantity of yeast used and the...

21 CFR 160.145 - Dried egg whites.

Code of Federal Regulations, 2013 CFR

2013-04-01

... fermentation procedures—(i) Yeast procedure. Food-grade baker's yeast (Saccharomyces cerevisiae) is added to the liquid egg whites and controlled fermentation is maintained. The quantity of yeast used and the...

21 CFR 160.145 - Dried egg whites.

Code of Federal Regulations, 2014 CFR

2014-04-01

... fermentation procedures—(i) Yeast procedure. Food-grade baker's yeast (Saccharomyces cerevisiae) is added to the liquid egg whites and controlled fermentation is maintained. The quantity of yeast used and the...

21 CFR 160.145 - Dried egg whites.

Code of Federal Regulations, 2012 CFR

2012-04-01

... fermentation procedures—(i) Yeast procedure. Food-grade baker's yeast (Saccharomyces cerevisiae) is added to the liquid egg whites and controlled fermentation is maintained. The quantity of yeast used and the...

X-Ray Topography of Tetragonal Lysozyme Grown by the Temperature-Controlled Technique

NASA Technical Reports Server (NTRS)

Stojanoff, V.; Siddons, D. P.; Monaco, Lisa A.; Vekilov, Peter; Rosenberger, Franz

1997-01-01

Growth-induced defects in lysozyme crystals were observed by white-beam and monochromatic X-ray topography at the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL). The topographic methods were non-destructive to the extent that traditional diffraction data collection could be performed to high resolution after topography. It was found that changes in growth parameters, defect concentration as detected by X-ray topography, and the diffraction quality obtainable from the crystals were all strongly correlated. In addition, crystals with fewer defects showed lower mosaicity and higher diffraction resolution as expected.

Culture of chicken embryo in interspecific surrogate egg white.

PubMed

Liu, C; Zu, J; Baskar, V; Wernery, U; Chang, I K

2012-11-01

The effect of interspecific egg white on the development of chicken embryos was investigated in a surrogate eggshell culture system. Egg yolks were separated from fertile White Leghorn chicken eggs and cultured in different egg whites from turkey (group TK), guineafowl (group GF), and duck (group DK), and chicken (group CK) was used as control. The viability of chicken embryos in groups CK, TK, GF, and DK after 3 d culture in system II was 98.3, 90.2, 96.1, and 91.1%. The whole contents (egg yolk and surrogate egg white) were further transferred into an eggshell from a 1.5 times heavier chicken egg with air space (system III), and incubated for further 16 d, before moving them to a hatcher. No significant difference between the 4 groups was found in their viabilities, which ranged between 72.9 and 81.3%, until 14 d postincubation (P > 0.05). After 21 d, the viability decreased to 60.4, 57.4, 50.0, and 27.7% in groups CK, TK, GF, and DK. The viability in group DK was significantly lower than in the other groups (P < 0.05). Weight loss in system III was approximately 12% in all the 4 groups without significant difference (P > 0.05). Hatchability of the chicken embryo was 60.4, 55.3, 47.9, and 19.1% in groups CK, TK, GF, and DK, respectively, and that in group DK was significantly lower than in the other groups (P < 0.05). There was no difference between the other groups (P > 0.05). These results show that chicken embryos can develop to hatch in duck, guineafowl, and turkey egg whites. However, the hatchability decreases according to the phylogenetic distance. The present study will provide a tool for manipulation of avian embryos and eventual conservation of endangered wild birds.

Oatmeal Pecan Waffles (or Pancakes)

MedlinePlus

... Whip egg whites to medium peaks. Gently fold egg whites into batter (for pancakes, see note below). Pour batter into preheated waffle iron, and cook until the waffle iron light signals it's done ...

Ovomucoid (Gal d 1) specific IgE detected by microarray system predict tolerability to boiled hen's egg and an increased risk to progress to multiple environmental allergen sensitisation.

PubMed

Alessandri, C; Zennaro, D; Scala, E; Ferrara, R; Bernardi, M Livia; Santoro, M; Palazzo, P; Mari, A

2012-03-01

Egg allergy is a very common finding in early childhood. Detecting hen's egg (HE) allergy outgrowing and reintroduction of food containing egg is a task for the allergist. We sought to evaluate the suitability of boiled egg food challenge compared with IgE to allergenic molecules from HE white using a microarray system. Sixty-eight children referring to our centre by the family paediatricians for a suspected egg allergy were enrolled. Patients underwent double-blind, placebo-controlled food challenge with boiled and raw eggs. Challenge outcomes were compared with skin tests performed using egg white and yolk commercial extracts, to prick-prick test with boiled and raw egg white and yolk, total IgE, egg white specific IgE detected using ImmunoCAP and IgE to egg allergens available on the immunosolid phase allergen chip (ISAC) 103 microarray. Nineteen subjects (28%) were reactive to both raw and boiled egg, 14 (20.5%) to raw egg only and 35 (51.4%) tolerated both boiled and raw egg. Efficiency analysis was carried out using both raw and boiled egg challenges as gold standard. Forty four of 47 Gal d 1 negative patients tolerated boiled egg (94%). Conversely, 20 of 21 Gal d 1 positive patients reacted to raw egg (95%). None of the other tests was able to discriminate patients' response to HE challenge. Furthermore, Gal d 1 positivity seems to lead to broader environmental allergen IgE sensitization. The Gal d 1 IgE reactivity appears to be a very good predictor of HE clinical allergy. Gal d 1 positive children have a high frequency of HE allergy, whereas Gal d 1 negative children have a high frequency of tolerance to boiled egg. Multiple specific IgE detection by means of ISAC improves the diagnostic approach in HE allergic children, disclosing other food and inhalant allergic sensitizations, anyhow requiring a comprehensive clinical evaluation. © 2011 Blackwell Publishing Ltd.

Egg Component-Composited Inverse Opal Particles for Synergistic Drug Delivery.

PubMed

Liu, Yuxiao; Shao, Changmin; Bian, Feika; Yu, Yunru; Wang, Huan; Zhao, Yuanjin

2018-05-23

Microparticles have a demonstrated value in drug delivery systems. The attempts to develop this technology focus on the generation of functional microparticles by using innovative but accessible materials. Here, we present egg component-composited microparticles with a hybrid inverse opal structure for synergistic drug delivery. The egg component inverse opal particles were produced by using egg yolk to negatively replicate colloid crystal bead templates. Because of their huge specific surface areas, abundant nanopores, and complex nanochannels of the inverse opal structure, the resultant egg yolk particles could be loaded with different kinds of drugs, such as hydrophobic camptothecin (CPT), by simply immersing them into the corresponding drug solutions. Attractively, additional drugs, such as the hydrophilic doxorubicin (DOX), could also be encapsulated into the particles through the secondary filling of the drug-doped egg white hydrogel into the egg yolk inverse opal scaffolds, which realized the synergistic drug delivery for the particles. It was demonstrated that the egg-derived inverse opal particles were with large quantity and lasting releasing for the CPT and DOX codelivery, and thus could significantly reduce cell viability, and enhance therapeutic efficacy in treating cancer cells. These features of the egg component-composited inverse opal microparticles indicated that they are ideal microcarriers for drug delivery.

Improvement of foaming ability of egg white product by irradiation and its application

NASA Astrophysics Data System (ADS)

Song, Hyun-Pa; Kim, Binna; Choe, Jun-Ho; Jung, Samooel; Kim, Kyong-Su; Kim, Dong-Ho; Jo, Cheorun

2009-03-01

To investigate the enhancement of foaming abilities of liquid egg white (LEW) and egg white powder (EWP) by irradiation and its application for bakery product, LEW and EWP were irradiated at 0, 1, 2, and 5 kGy by Co-60 gamma ray. There was no pH change found among treatments in both LEW and EWP. The viscosity of LEW decreased significantly by irradiation ( P<0.05), whereas that of EWP was not affected by irradiation. The foaming ability of LEW and EWP was significantly increased by irradiation as a dose-dependent manner ( P<0.05). The volume and the height of angel cake baked with irradiated LEW were significantly higher than those of unirradiated control ( P<0.05). For EWP, the volume and the height of angel cake were greater at 2 kGy only than those of control. A significant decrease in hardness, chewiness, and gumminess values and an increase in Hunter L* value were observed in the angel cakes prepared from irradiated egg white products ( P<0.05). Results indicated that irradiation of egg white could offer advantages in increasing foaming ability and improving quality of final bakery products.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

PubMed

Sethi, Deepti; Mahajan, Sahil; Singh, Chaahat; Lama, Amrita; Hade, Mangesh Dattu; Gupta, Pawan; Dikshit, Kanak L

2016-02-05

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Relation between IgG antibodies to foods and IgE antibodies to milk, egg, cat, dog and/or mite in a cross-sectional study.

PubMed

Eysink, P E; De Jong, M H; Bindels, P J; Scharp-Van Der Linden, V T; De Groot, C J; Stapel, S O; Aalberse, R C

1999-05-01

Because IgG antibodies to foods can be detected before IgE antibodies to inhalants, increased levels of IgG antibodies to foods might be used as a predictor of IgE-mediated allergy in initially nonatopic children. To examine the cross-sectional relation between IgG to foods (i.e. mixture of wheat and rice, mixture of soybean and peanut, egg white, cow's milk, meat, orange and potato) and specific IgE to cat, dog, mite, milk and egg white in 1-year-old children. All atopic children (n = 120; 58 with and 62 without eczema) and a random sample of the nonatopic children (n = 144) of the Bokaal study were tested on their IgG response to foods. The IgG results of the food assays were dichotomized high or low using the 66th centile as a cut-off value. Atopic children more often had high IgG levels to foods than nonatopic children. IgG to egg white (OR = 7.50) and mixture of wheat and rice (OR = 4.79) were most strongly associated with positive specific IgE. In a stepwise logistic regression analysis egg white, mixture of wheat and rice, and orange were selected (OR = 3.76, OR = 2.43, and OR = 2.11, respectively). In children without eczema higher levels of IgG to foods were still significantly associated with atopy, which was most prominent for egg white, orange and cow's milk. An increased IgG antibody level to foods, especially to egg white, orange, and mixture of wheat and rice, indicates an increased risk of having IgE to cat, dog, mite, egg and/or milk allergens, even in the noneczematous group. Therefore, in another prospective study we are currently investigating the usefulness of IgG in early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing allergic diseases in the future.

Effect of Different Heat Treatments on In Vitro Digestion of Egg White Proteins and Identification of Bioactive Peptides in Digested Products.

PubMed

Wang, Xuefen; Qiu, Ning; Liu, Yaping

2018-04-01

Chicken eggs are ingested by people after a series of processes, but to date, only a few studies have explored the nutrient variations caused by different heat treatments. In this work, the impacts of different heat treatments (4, 56, 65, and 100 °C on the in vitro digestibility of egg white proteins were investigated by hydrolysis with pepsin or pepsin + pancreatin to simulate human gastrointestinal digestion, and the digested products were identified using Nano-LC-ESI-MS/MS. Egg white proteins treated at 65 °C had the highest in vitro pepsin digestibility value whereas the pepsin + pancreatin digestibility increased significantly (P < 0.05) as the cooking temperature was raised. The molecular weight distribution of the digested products indicated that, when compared to pepsin-treated samples, pepsin + pancreatin-treated samples contained more low-molecular-weight peptides (m/z < 849.2) with higher signal intensities. The number of unique peptides identified in every digestion product showed a positive correlation with their in vitro digestibility. Additionally, bioactive peptides such as antioxidant, antimicrobial and antihypertensive peptides were found present in egg white digested products, especially in samples treated at 4 and 100 °C. These findings may facilitate a better understanding of nutritive values of egg white proteins and their digested products under different cooking temperatures, such as antibacterial and antioxidant peptides identified in the digestion samples treated, respectively at 4 and 100 °C. This study also provided information for improving the applications of eggs in the food industry as well as a theoretical basis for egg consumption. © 2018 Institute of Food Technologists®.

Magnetic fluid - a novel approach to treat amyloid-related diseases

NASA Astrophysics Data System (ADS)

Antosova, Andrea; Siposova, Katarina; Koneracka, Martina; Zavisova, Vlasta; Daxnerova, Zuzana; Vavra, Ivo; Fedunova, Diana; Bagelova, Jaroslava; Kopcansky, Peter; Gazova, Zuzana

Protein amyloid aggregates are associated with several human pathologies termed amyloid-related diseases. We have investigated effect of two magnetic fluids (MFs) - electrostatically stabilized Fe3O4 magnetic nanoparticles (MF1) and sterically stabilized Fe3O4 magnetic nanoparticles by sodium oleate with adsorbed BSA (MF2) on amyloid aggregation of two proteins - human insulin and hen egg lysozyme. We have found that both MF1 and MF2 are able to interact with amyloid fibrils in vitro resulting into decreasing of amyloid aggregates. The extent of fibril disruption depends on MF concentration with extensive reduction of amyloid aggregates, 90% for lysozyme and 70% for insulin (ratio protein: MF=1:5). The obtained results suggest that magnetite component of MF play significant role in the process of amyloid fibril depolymerisation. Our findings indicate that MF1 and MF2 have potential to be used for treatment of amyloid diseases.

21 CFR 169.150 - Salad dressing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... liquid egg white or frozen egg white. (d) Starchy paste. It may be prepared from a food starch, food starch-modified, tapioca flour, wheat flour, rye flour, or any two or more of these. Water may be added...

21 CFR 169.150 - Salad dressing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... liquid egg white or frozen egg white. (d) Starchy paste. It may be prepared from a food starch, food starch-modified, tapioca flour, wheat flour, rye flour, or any two or more of these. Water may be added...

21 CFR 169.150 - Salad dressing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... liquid egg white or frozen egg white. (d) Starchy paste. It may be prepared from a food starch, food starch-modified, tapioca flour, wheat flour, rye flour, or any two or more of these. Water may be added...

Glutathione exposes sequential IgE-epitopes in ovomucoid relevant in persistent egg allergy.

PubMed

Roth-Walter, Franziska; Starkl, Philipp; Zuberbier, Torsten; Hummel, Karin; Nöbauer, Karin; Razzazi-Fazeli, Ebrahim; Brunner, Richard; Pali-Schöll, Isabella; Kinkel, Janis; Felix, Ferdinand; Jensen-Jarolim, Erika; Kinaciyan, Tamar

2013-03-01

Patients with persistent egg allergy have more immunoglobulin E (IgE) against sequential than conformational epitopes of ovomucoid (OVO). Here, we aimed to identify compounds capable to render sequential epitopes in egg. Glutathione was used for in vitro reduction of OVO and circular dichroism analyses were performed. Glutathione reduced OVO in a concentration-dependent manner. Egg white was analyzed for reduced proteins with a thiol probe and by MALDI-TOF/TOF. In unprocessed total egg white, several reduced proteins were detected by the thiol probe, among them reduced ovalbumin could be confirmed with MS analyses. Egg-allergics or sensitized controls were tested serologically (n = 19) for IgE against native and reduced OVO and in skin prick tests (n = 9). More patients had IgE against reduced than native OVO in Western blots. In skin prick test, five out of seven persistent egg-allergics and none of the controls reacted with reduced OVO. Reduced egg proteins are present in natural egg white. Glutathione, which is present in egg and furthermore is used as texture-improving additive in processed food, is capable of reducing OVO. Patients with persistent egg allergy reacted rather to reduce the native OVO. Hence, our data indicate that reduction is a novel natural and processing-associated principle, which contributes to the allergenicity of food. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry

NASA Astrophysics Data System (ADS)

Archer, Jieutonne J.; Karki, Santosh; Shi, Fengjian; Sistani, Habiballah; Levis, Robert J.

2018-04-01

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (Kd) for hen egg white lysozyme (HEWL) and N,N',N″-triacetylchitotriose (NAG3) revealed an apparent Kd value of 313.2 ± 25.9 μM for the ligand titration method. Similar measurements for N,N',N″,N″'-tetraacetylchitotetraose (NAG4) revealed an apparent Kd of 249.3 ± 13.6 μM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a Kd value of 9.8 ± 0.6 μM. In a second LEMS approach, a calibrated measurement was used to determine a Kd value of 6.8 ± 1.5 μM for NAG3. The capture efficiency of LEMS was measured to be 3.6 ± 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted Kd value was 11.3 μM for NAG3 and 9.0 μM for NAG4. The calibration method for measuring Kd developed in this study can be applied to solutions containing unknown analyte concentrations. [Figure not available: see fulltext.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

PubMed Central

Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

2016-01-01

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases. PMID:27230476

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

PubMed

Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

2016-05-27

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

PubMed Central

Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Razinkov, Vladimir; Chase, Susan F; Laue, Thomas M; Brems, David N

2011-01-01

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− > Cl− > Br− > NO3− ∼ I− > SCN− > ClO4− ≫ SO42−, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO42− anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface. PMID:21432935

Effect of Glycerol Water Binary Mixtures on the Structure and Dynamics of Protein Solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghattyvenkatakrishna, Pavan K; Carri, Gustavo A.

We have performed 20ns of fully atomistic molecular dynamics simulations of Hen Egg-White Lysozyme in 0, 10, 20, 30 and 100% by weight of glycerol in water to better understand the microscopic physics behind the bioprotection offered by glycerol to naturally occuring biological systems. The sovlent exposure of protein surface residues changes when glycerol is introduced. The dynamic behavior of the protein, as quantified by the Incoherent Intermediate Scattering Function, shows a non-monotonic dependence on glycerol content. The fluctuations of the protein residues with respect to each other were found to be similar in all water containing solvents; but differentmore » from the pure glycerol case. The increase in the number of protein glycerol hydrogen bonds in glycerol water binary mixtures explains the slowing down of protein dynamics as the glycerol content increases. We also explored the dynamic behavior of the hydration layer. We show that the short-length scale dynamics of this layer are insenstive to glycerol concentration. However, the long-length scale behavior shows a significant dependence on glycerol content. We also provide insights into the behavior of bound and mobile water molecules.« less

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.

PubMed

Clabbers, M T B; van Genderen, E; Wan, W; Wiegers, E L; Gruene, T; Abrahams, J P

2017-09-01

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm 3 , i.e. no more than 6 × 10 5 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

PubMed Central

Clabbers, M. T. B.; van Genderen, E.; Wiegers, E. L.; Gruene, T.; Abrahams, J. P.

2017-01-01

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures. PMID:28876237

Adaptation of in-situ microscopy for crystallization processes

NASA Astrophysics Data System (ADS)

Bluma, A.; Höpfner, T.; Rudolph, G.; Lindner, P.; Beutel, S.; Hitzmann, B.; Scheper, T.

2009-08-01

In biotechnological and pharmaceutical engineering, the study of crystallization processes gains importance. An efficient analytical inline sensor could help to improve the knowledge about these processes in order to increase efficiency and yields. The in-situ microscope (ISM) is an optical sensor developed for the monitoring of bioprocesses. A new application for this sensor is the monitoring in downstream processes, e.g. the crystallization of proteins and other organic compounds. This contribution shows new aspects of using in-situ microscopy to monitor crystallization processes. Crystals of different chemical compounds were precipitated from supersaturated solutions and the crystal growth was monitored. Exemplified morphological properties and different forms of crystals could be distinguished on the basis of offline experiments. For inline monitoring of crystallization processes, a special 0.5 L stirred tank reactor was developed and equipped with the in-situ microscope. This reactor was utilized to carry out batch experiments for crystallizations of O-acetylsalicyclic acid (ASS) and hen egg white lysozyme (HEWL). During the whole crystallization process, the in-situ microscope system acquired images directly from the crystallization broth. For the data evaluation, an image analysis algorithm was developed and implemented in the microscope analysis software.

Quantitative methods for structural characterization of proteins based on deep UV resonance Raman spectroscopy.

PubMed

Shashilov, Victor A; Sikirzhytski, Vitali; Popova, Ludmila A; Lednev, Igor K

2010-09-01

Here we report on novel quantitative approaches for protein structural characterization using deep UV resonance Raman (DUVRR) spectroscopy. Specifically, we propose a new method combining hydrogen-deuterium (HD) exchange and Bayesian source separation for extracting the DUVRR signatures of various structural elements of aggregated proteins including the cross-beta core and unordered parts of amyloid fibrils. The proposed method is demonstrated using the set of DUVRR spectra of hen egg white lysozyme acquired at various stages of HD exchange. Prior information about the concentration matrix and the spectral features of the individual components was incorporated into the Bayesian equation to eliminate the ill-conditioning of the problem caused by 100% correlation of the concentration profiles of protonated and deuterated species. Secondary structure fractions obtained by partial least squares (PLS) and least squares support vector machines (LS-SVMs) were used as the initial guess for the Bayessian source separation. Advantages of the PLS and LS-SVMs methods over the classical least squares calibration (CLSC) are discussed and illustrated using the DUVRR data of the prion protein in its native and aggregated forms. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Is the Quantification of Antigen-Specific Basophil Activation a Useful Tool for Monitoring Oral Tolerance Induction in Children With Egg Allergy?

PubMed

Gamboa, P M; Garcia-Lirio, E; Gonzalez, C; Gonzalez, A; Martinez-Aranguren R M; Sanz María, L

2016-01-01

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. 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. 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. 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.

[Determination of Cu in Shell of Preserved Egg by LIBS Coupled with PLS].

PubMed

Hu, Hui-qin; Xu, Xue-hong; Liu, Mu-hua; Tu, Jian-ping; Huang, Le; Huang, Lin; Yao, Ming-yin; Chen, Tian-bing; Yang, Ping

2015-12-01

In this work, the content of copper in the shell of preserved eggs were determined directly by Laser induced breakdown spectroscopy (LIBS), and the characteristics lines of Cu was obtained. The samples of eggshell were pretreated by acid wet digestion, and the real content of Cu was obtained by atomic absorption spectrophotometer (AAS). Due to the test precision and accuracy of LIBS was influenced by a serious of factors, for example, the complex matrix effect of sample, the enviro nment noise, the system noise of the instrument, the stability of laser energy and so on. And the conventional unvariate linear calibration curve between LIBS intensity and content of element of sample, such as by use of Schiebe G-Lomakin equation, can not meet the requirement of quantitative analysis. In account of that, a kind of multivariate calibration method is needed. In this work, the data of LIBS spectra were processed by partial least squares (PLS), the precision and accuracy of PLS model were compared by different smoothing treatment and five pretreatment methods. The result showed that the correlation coefficient and the accuracy of the PLS model were improved, and the root mean square error and the average relative error were reduced effectively by 11 point smoothing with Multiplicative scatter correction (MSC) pretreatment. The results of the study show that, heavy metal Cu in preserved egg shells can be direct detected accurately by laser induced breakdown spectroscopy, and the next step batch tests will been conducted to find out the relationship of heavy metal Cu content in the preserved egg between the eggshell, egg white and egg yolk. And the goal of the contents of heavy metals in the egg white, egg yolk can be knew through determinate the eggshell by the LIBS can be achieved, to provide new method for rapid non-destructive testing technology for quality and satety of agricultural products.

Crowding of white shrimp Litopenaeus vananmei depresses their immunity to and resistance against Vibrio alginolyticus and white spot syndrome virus.

PubMed

Lin, Yong-Chin; Chen, Jiann-Chu; Chen, Yu-Yuan; Yeh, Su-Tuen; Chen, Li-Li; Huang, Chien-Lun; Hsieh, Jen-Fang; Li, Chang-Che

2015-07-01

Immunity parameters and the expression levels of several immune-related proteins, including lipopolysaccharide and β-glucan binding protein (LGBP), peroxinectin (PX), intergin β (IB), prophenoloxidase (proPO) I, proPO II, α2-macroglobulin (α2-M), cytosolic mangangese superoxide dismutase (cytMnSOD), mitochondria manganese superoxide dismutase (mtMnSOD), catalase, glutathione peroxidase (GPx), lysozyme, and penaeidin 3a were examined in white shrimp Litopenaeus vannamei reared at stocking densities of 2, 10, 20, 30, and 40 shrimp L(-1) after 3, 6, and 12 h. All immune parameters including haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, lysozyme activity, and haemolymph protein were negatively related to density and time. The PO activity, SOD activity, and lysozyme activity of shrimp reared at 10 shrimp L(-1) after 12 h significantly decreased. The transcript levels of these immune-related proteins were down-regulated in shrimp reared at 20, 30, and 40 shrimp L(-1) after 12 h. Phagocytic activity and clearance efficiency to Vibrio alginolyticus were significantly lower in shrimp reared at 30 and 40 shrimp L(-1) after 12 h. The mortality rates of shrimp reared at 20 and 40 shrimp L(-1) were significantly higher than shrimp reared at 2 shrimp L(-1) over 12-144 h and 12-48 h, respectively. Shrimp reared at high densities (>10 shrimp L(-1)) exhibited decreased resistance against pathogens as evidenced by reductions in immune parameters together with decreased expression levels of immune-related proteins, indicating perturbations of the immune system. Copyright © 2015 Elsevier Ltd. All rights reserved.

Distribution of ingested americium in chickens and transport to eggs. Final report, 1975

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mullen, A.A.; Lloyd, S.R.; Mosley, R.E.

1976-05-01

The soluble citrate complex of americium-241 was orally administered to 20 white Leghorn laying hens daily for two weeks. The yolks, whites, and shells from the eggs were analyzed for their americium content. Yolk was the only egg fraction in which radioactivity was observed. The americium-241 activity in yolks reached a maximum on the 14th day of dosing. Biological half-times of 2.00 plus or minus 0.18 days and greater than 33 days were indicated by the average concentration values of americium-241 in yolks laid after the maximum activity was reached. The hens were serially sacrificed at 1, 10, and 20more » days after the final administration of americium-241. Tissue samples were collected and the americium content determined in the edible portions and feathers of the hens. Americium was detected in most tissues shortly after dosing; the main concentrations were found in the liver and the skeleton. The highest concentration per organ (3.03 X 0.001 percent of the dose) occurred in the liver of the hens sacrificed 10 days after final administration of americium-241. (GRA)« less

Effect of basic alkali-pickling conditions on the production of lysinoalanine in preserved eggs.

PubMed

Zhao, Yan; Luo, Xuying; Li, Jianke; Xu, Mingsheng; Tu, Yonggang

2015-09-01

During the pickling process, strong alkali causes significant lysinoalanine (LAL) formation in preserved eggs, which may reduce the nutritional value of the proteins and result in a potential hazard to human health. In this study, the impacts of the alkali treatment conditions on the production of LAL in preserved eggs were investigated. Preserved eggs were prepared using different times and temperatures, and alkali-pickling solutions with different types and concentrations of alkali and metal salts, and the corresponding LAL contents were measured. The results showed the following: during the pickling period of the preserved egg, the content of LAL in the egg white first rapidly increased and then slowly increased; the content of LAL in the egg yolk continued to increase significantly. During the aging period, the levels of LAL in both egg white and egg yolk slowly increased. The amounts of LAL in the preserved eggs were not significantly different at temperatures between 20 and 25ºC. At higher pickling temperatures, the LAL content in the preserved eggs increased. With the increase of alkali concentration in the alkali-pickling solution, the LAL content in the egg white and egg yolk showed an overall trend of an initial increase followed by a slight decrease. The content of LAL produced in preserved eggs treated with KOH was lower than in those treated with NaOH. NaCl and KCl produced no significant effects on the production of LAL in the preserved eggs. With increasing amounts of heavy metal salts, the LAL content in the preserved eggs first decreased and then increased. The LAL content generated in the CuSO4 group was lower than that in either the ZnSO4 or PbO groups. © 2015 Poultry Science Association Inc.

Predation on walleye eggs by fish on reefs in western Lake Erie

USGS Publications Warehouse

Roseman, E.F.; Taylor, W.W.; Hayes, D.B.; Jones, A.L.; Francis, J.T.

2006-01-01

We examined diets of fishes from gillnet and egg pump collections conducted on reefs in western Lake Erie during walleye (Sander vitreus) egg incubation periods from 1994–1999 and 2004 to assess incidence of walleye eggs in fish diets. We collected no potential egg predators in samples taken in 1994 but from 1995–1999 and in 2004 we caught 22 different species of fish on reefs in addition to spawning walleye. In most years, white perch (Morone americana) stomachs contained more walleye eggs than any other species on the reefs averaging 253 eggs per stomach. We also found lower numbers of walleye eggs in the stomachs of channel catfish (Ictalurus punctatus; 53 eggs/stomach), johnny darter (Etheostoma nigrum; 2 eggs/stomach), logperch (Percina caprodes; 10 eggs/stomach), quillback (Carpiodes cyprinus; 184 eggs/stomach), rock bass (Ambloplites rupestris; 3 eggs/stomach), round goby (Neogobius melanostomus; 4 eggs/stomach), sculpin (Cottidae; 21 eggs/stomach), silver chub (Macrhybopsis storeriana; 3 eggs/stomach), spottail shiner (Notropis hudsonius; 14 eggs/stomach), trout-perch (Percopsis omiscomaycus; 30 eggs/stomach), white sucker (Catastomus commersonii; 20 eggs/stomach), and yellow perch (Perca flavescens; 181 eggs/stomach). Similar to other studies of predation on walleye eggs, our results indicate that prolonged incubation periods increase the potential for egg loss due to predation.

Quality characteristic of spray-drying egg white powders.

PubMed

Ma, Shuang; Zhao, Songning; Zhang, Yan; Yu, Yiding; Liu, Jingbo; Xu, Menglei

2013-10-01

Spray drying is a useful method for developing egg process and utilization. The objective of this study was to evaluate effects on spray drying condition of egg white. The optimized conditions were spraying flow 22 mL/min, feeding temperature 39.8 °C and inlet-air temperature 178.2 °C. Results of sulfydryl (SH) groups measurement indicated conformation structure have changed resulting in protein molecule occur S-S crosslinking phenomenon when heating. It led to free SH content decreased during spray drying process. There was almost no change of differential scanning calorimetry between fresh egg white and spray-drying egg white powder (EWP). For a given protein, the apparent SH reactivity is in turn influenced by the physico-chemical characteristics of the reactant. The phenomenon illustrated the thermal denaturation of these proteins was unrelated to their free SH contents. Color measurement was used to study browning level. EWP in optimized conditions revealed insignificant brown stain. Swelling capacity and scanning electron micrograph both proved well quality characteristic of spray-drying EWP. Results suggested spray drying under the optimized conditions present suitable and alternative method for egg processing industrial implementation. Egg food industrialization needs new drying method to extend shelf-life. The purpose of the study was to provide optimal process of healthy and nutritional instant spray-drying EWP and study quality characteristic of spray-drying EWP.

Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies

NASA Astrophysics Data System (ADS)

Jing, Mingyang; Song, Wei; Liu, Rutao

2016-07-01

Although copper is essential to all living organisms, its potential toxicity to human health have aroused wide concerns. Previous studies have reported copper could alter physical properties of lysozyme. The direct binding of copper with lysozyme might induce the conformational and functional changes of lysozyme and then influence the body's resistance to bacterial attack. To better understand the potential toxicity and toxic mechanisms of copper, the interaction of copper with lysozyme was investigated by biophysical methods including multi-spectroscopic measurements, isothermal titration calorimetry (ITC), molecular docking study and enzyme activity assay. Multi-spectroscopic measurements proved that copper quenched the intrinsic fluorescence of lysozyme in a static process accompanied by complex formation and conformational changes. The ITC results indicated that the binding interaction was a spontaneous process with approximately three thermodynamical binding sites at 298 K and the hydrophobic force is the predominant driven force. The enzyme activity was obviously inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. This study helps to elucidate the molecular mechanism of the interaction between copper and lysozyme and provides reference for toxicological studies of copper.

Three-dimensional structure of an antibody-antigen complex.

PubMed

Sheriff, S; Silverton, E W; Padlan, E A; Cohen, G H; Smith-Gill, S J; Finzel, B C; Davies, D R

1987-11-01

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.

Development of a Novel Segmental Bone Defect Construct

DTIC Science & Technology

2016-10-01

from chicken egg white (90%) were purchased from Sigma Aldrich (St. Louis, MO). Ethylene glycol, sodium metabisulfite (SMS), urea, and hydrogen...HRP-conjugated polyclonal anti-LYZ (chicken egg white) was purchased from United States Biological (Swampscott, MA). N-(3-Sulfopropyl)- N

High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time*

PubMed Central

Umemoto, Ayaka; Yagi, Hisashi; So, Masatomo; Goto, Yuji

2014-01-01

Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0–5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ∼3.0 m, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation. PMID:25118286

Both positive and negative effects on immune responses by expression of a second class II MHC molecule.

PubMed

Ni, Peggy P; Wang, Yaming; Allen, Paul M

2014-11-01

It is perplexing why vertebrates express a limited number of major histocompatibility complex (MHC) molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A(+)) and B6.E(+) mice (I-A(+), I-E(+)), the latter expressing a second class II MHC molecule, I-E(b), due to a monomorphic Eα(k) transgene that pairs with the endogenous I-Eβ(b) chain. First, the naive T cell Vβ repertoire was altered in B6.E(+) thymi and spleens, potentially mediating different outcomes in T cell reactivity. Although the B6 and B6.E(+) responses to hen egg-white lysozyme (HEL) protein immunization remained similar, other immune models yielded differences. For viral infection, the quality of the T cell response was subtly altered, with diminished production of certain cytokines by B6.E(+) CD4(+) T cells. In alloreactivity, the B6.E(+) T cell response was significantly dampened. Finally, we observed markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E(+) mice. This correlated with decreased percentages of nTreg cells, supporting the concept of Tregs exhibiting differential susceptibility to negative selection. Altogether, our data suggest that expressing an additional class II MHC can produce diverse effects, with more severe autoimmunity providing a compelling explanation for limiting the expression of MHC molecules. Copyright © 2014 Elsevier Ltd. All rights reserved.

Altered virulence potential of Salmonella Enteritidis cultured in different foods: A cumulative effect of differential gene expression and immunomodulation.

PubMed

Jaiswal, Sangeeta; Sahoo, Prakash Kumar; Ryan, Daniel; Das, Jugal Kishore; Chakraborty, Eesha; Mohakud, Nirmal Kumar; Suar, Mrutyunjay

2016-08-02

Salmonella enterica serovars Enteritidis (S. Enteritidis) is one of the most common causes of food borne illness. Bacterial growth environment plays an important role in regulating gene expression thereby affecting the virulence profile of the bacteria. Different foods present diverse growth conditions which may affect the pathogenic potential of the bacteria. In the present study, the effect of food environments on the pathogenic potential of S. Enteritidis has been evaluated. S. Enteritidis was grown in different foods e.g. egg white, peanut butter and milk, and virulent phenotypes were compared to those grown in Luria Bertani broth. In-vivo experiments in C57BL/6 mice revealed S. Enteritidis grown in egg white did not induce significant (p<0.001) production of proinflammatory cytokines in mice and were unable to cause colitis despite efficient colonization in cecum, mesenteric lymph node, spleen and liver. Further studies revealed that bacteria grown in LB activated MAP Kinase and NFκB pathways efficiently, while those grown in egg white poorly activated the above pathways which can account for the decreased production of proinflammatory cytokines. qRT PCR analysis revealed SPI-1 effectors were downregulated in bacteria grown in egg white. Interestingly, bacteria grown in egg white showed reversal of phenotype upon change in growth media to LB. Additionally, bacteria grown in milk and peanut butter showed different degrees of virulence in mice as compared to those grown in LB media. Thus, the present study demonstrates that, S. Enteritidis grown in egg white colonizes systemic sites without causing colitis in a mouse model, while bacteria grown in milk and peanut butter show different pathogenicity profiles suggesting that food environments significantly affect the pathogenicity of S. Enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction and inhibitory influence of the azo dye carmoisine on lysozyme amyloid fibrillogenesis.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-07-25

The binding of the common food colorant carmoisine and its inhibitory effect on amyloid fibrillation in lysozyme have been investigated. Since humans are increasingly exposed to various food colorants like carmoisine, such studies are highly relevant. In the presence of lysozyme, the carmoisine absorption spectrum exhibited hypochromic changes. The intrinsic fluorescence of lysozyme was also quenched on interaction. Time-resolved fluorescence results suggested that the binding mechanism involved ground state complexation. The binding was predominantly dominated by non-polyelectrolytic forces. The molecular distance between the donor (lysozyme) and the acceptor (carmoisine), calculated from FRET theory, was found to be 3.37 nm, indicating that carmoisine binds close to Trp-62/63 residues in the β-domain of the protein. Information on alterations in the microenvironment surrounding the Trp-residues was also obtained from synchronous fluorescence data. Carmoisine binding induced significant loss in the alpha helical organization of lysozyme. The binding, nevertheless, did not influence the thermal stability of lysozyme significantly. The binding reaction was exothermic and driven by large negative enthalpy and small but favourable entropic contributions. Thioflavin T assay, far-UV circular dichroism studies and AFM imaging profiles testified that carmoisine had a significant inhibitory effect on amyloid fibrillogenesis in lysozyme. Carmoisine also had a definitive defibrillating effect on existing fibrils. The results may provide new insights for designing new small molecule inhibitors for amyloid related diseases.

TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods.

PubMed

Fox, C J; Taylor, M I; Pereyra, R; Villasana, M I; Rico, C

2005-03-01

Recent substantial declines in northeastern Atlantic cod stocks necessitate improved biological knowledge and the development of techniques to complement standard stock assessment methods (which largely depend on accurate commercial catch data). In 2003, an ichthyoplankton survey was undertaken in the Irish Sea and subsamples of 'cod-like' eggs were analysed using a TaqMan multiplex, PCR (polymerase chain reaction) assay (with specific probes for cod, haddock and whiting). The TaqMan method was readily applied to the large number of samples (n = 2770) generated during the survey and when combined with a manual DNA extraction protocol had a low failure rate of 6%. Of the early stage 'cod-like' eggs (1.2-1.75 mm diameter) positively identified: 34% were cod, 8% haddock and 58% whiting. As previous stock estimates based on egg surveys for Irish Sea cod assumed that the majority of 'cod-like' eggs were from cod, the TaqMan results confirm that there was probably substantial contamination by eggs of whiting and haddock that would have inflated estimates of the stock biomass.

Protein Structural Perturbation and Aggregation on Homogeneous Surfaces

PubMed Central

Sethuraman, Ananthakrishnan; Belfort, Georges

2005-01-01

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by α-helix to turns/random during the first 30 min and followed by a slow α-helix to β-sheet transition. Increase in intramolecular and intermolecular β-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with –CH3, –OPh, –CF3, –CN, –OCH3, and –OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces. PMID:15542559

Kinetics of temperature response of PEO-b-PNIPAM-b-PAA triblock terpolymer aggregates and of their complexes with lysozyme

DOE PAGES

Papagiannopoulos, Aristeidis; Meristoudi, Anastasia; Hong, Kunlun; ...

2015-12-18

We present the kinetics of temperature response of a PEO-b-PNIPAM-b-PAA triblock terpolymer and of its complexes with lysozyme in aqueous solution. It is found that during the coil-to-globule transition of PNIPAM new bonds within the polymer aggregates are created, making the transition of the aggregates partially irreversible. This effect is also found for the protein loaded PEO-b-PNIPAM-b-PAA aggregates whereas in this case protein globules appear to enhance the formation of bonds, making the transition totally irreversible. The internal dynamics of both aggregates and complexes are “frozen” once the temperature is increased upon PINIPAM's LCST in water and remain so evenmore » when the temperature drops below LCST. As a result, we investigate the complexation kinetics of lysozyme and PEO-b-PNIPAM-b-PAA and observe that it occurs in two stages, one where protein globules adsorb on single pre-formed aggregates and one where protein globules cause inter-aggregate clustering.« less

Kinetics of temperature response of PEO-b-PNIPAM-b-PAA triblock terpolymer aggregates and of their complexes with lysozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papagiannopoulos, Aristeidis; Meristoudi, Anastasia; Hong, Kunlun

We present the kinetics of temperature response of a PEO-b-PNIPAM-b-PAA triblock terpolymer and of its complexes with lysozyme in aqueous solution. It is found that during the coil-to-globule transition of PNIPAM new bonds within the polymer aggregates are created, making the transition of the aggregates partially irreversible. This effect is also found for the protein loaded PEO-b-PNIPAM-b-PAA aggregates whereas in this case protein globules appear to enhance the formation of bonds, making the transition totally irreversible. The internal dynamics of both aggregates and complexes are “frozen” once the temperature is increased upon PINIPAM's LCST in water and remain so evenmore » when the temperature drops below LCST. As a result, we investigate the complexation kinetics of lysozyme and PEO-b-PNIPAM-b-PAA and observe that it occurs in two stages, one where protein globules adsorb on single pre-formed aggregates and one where protein globules cause inter-aggregate clustering.« less

Organochlorine residues in six species of estuarine birds, South Carolina, 1971-75

USGS Publications Warehouse

Blus, L.J.; Lamont, T.G.

1979-01-01

In South Carolina between 1971 and 1975, authors evaluated the occurrence of organochlorine residues in the laughing gull (Larus atricilla), white ibis (Eudocimus albus), glossy ibis (Plegadis falcinellus), American oystercatcher (Haematopus palliatus), willet (Catoptrophorus semipalmatus), and ruddy turnstone (Arenaria interpres). Tissues of birds found dead and eggs were analyzed, eggshell thicknesses were measured, and incidental observations were made of reproductive success and population status. Eggshell thickness of the white bis, American oystercatcher, and laughing gull were not significantly different (P less than 0.05) from the pre-1947 norms. DDE and polychlorinated biphenyls (PCBs) were found most frequently and at the highest concentration in eggs. DDE residues declined significantly in oystercatcher eggs, and declined slightly in laughing gull eggs; no change was noted in white ibis eggs. No consistent trends were found for dieldrin and PCBs. Authors found no obvious problems with reproductive success of any species. Populations of the five species breeding in South Carolina appear stable. The white ibis and laughing gull in South Carolina have experienced population explosions over the past 50 years; the glossy ibis has increased substantially since the first documented breeding records in 1947.

Beneficial Effects of Simulated Gastro-Intestinal Digests of Fried Egg and Its Fractions on Blood Pressure, Plasma Lipids and Oxidative Stress in Spontaneously Hypertensive Rats

PubMed Central

Chakrabarti, Subhadeep; Morton, Jude S.; Panahi, Sareh; Kaufman, Susan; Davidge, Sandra T.; Wu, Jianping

2014-01-01

Abstract Background We have previously characterized several antihypertensive peptides in simulated digests of cooked eggs and showed blood pressure lowering property of fried whole egg digest. However, the long-term effects of this hydrolysate and its fractions on blood pressure are not known. Therefore, the objectives of the study were to determine the effects of long term administration of fried whole egg hydrolysate and its fractions (i.e. egg white and egg yolk) on regulation of blood pressure and associated factors in cardiovascular disease such as plasma lipid profile and tissue oxidative stress. Methods and Results We used spontaneously hypertensive rats (SHR), an animal model of essential hypertension. Hydrolysates of fried egg and its fractions were prepared by simulated gastro-intestinal digestion with pepsin and pancreatin. 16–17 week old male SHRs were orally administered fried whole egg hydrolysate, non-hydrolyzed fried whole egg, egg white hydrolysate or egg yolk hydrolysates (either defatted, or not) daily for 18 days. Blood pressure (BP) and heart rate were monitored by telemetry. Animals were sacrificed at the end of the treatment for vascular function studies and evaluating plasma lipid profile and tissue oxidative stress. BP was reduced by feeding fried whole egg hydrolysate but not by the non-hydrolyzed product suggesting a critical role for in vitro digestion in releasing anti-hypertensive peptides. Egg white hydrolysate and defatted egg yolk hydrolysate (but not egg yolk hydrolysate) also had similar effects. Reduction in BP was accompanied by the restoration of nitric oxide (NO) dependent vasorelaxation and reduction of plasma angiotensin II. Fried whole egg hydrolysate also reduced plasma levels of triglyceride although it was increased by the non-hydrolyzed sample. Additionally the hydrolyzed preparations attenuated tissue oxidative stress. Conclusion Our results demonstrate that fried egg hydrolysates exert anti-hypertensive effects, improve plasma lipid profile and attenuate tissue oxidative stress in vivo. PMID:25502445

Impact by condensed tannins with different mean degrees of polymerization on protein precipitation

USDA-ARS?s Scientific Manuscript database

Condensed tannins (CTs) isolated from white clover (Trifolium repens) flowers and big trefoil (Lotus pedunculatus) leaves were evaluated in precipitation studies with bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF). The CTs were of similar compositions, but differed in the...

Pentadecapeptide BPC 157 and anaphylactoid reaction in rats and mice after intravenous dextran and white egg administration.

PubMed

Duplancic, Bozidar; Stambolija, Vasilije; Holjevac, Jadranka; Zemba, Mladen; Balenovic, Igor; Drmic, Domagoj; Suran, Jelena; Radic, Bozo; Filipovic, Marinko; Blagaic, Alenka Boban; Brcic, Luka; Kolenc, Danijela; Grabarevic, Zeljko; Seiwerth, Sven; Sikiric, Predrag

2014-03-15

Anesthetized mice or rats received intravenously 6%, 10%, 20%, 40%, 60%, 80%, and 90% dextran and/or white egg (1ml/rat or 0.15ml/mouse) into their tails. Medication (/kg b.w., 5ml/kg) was given intraperitoneally (BPC 157 10µg, 1µg, 10ng, and 10pg/kg, chloropyramine 20mg/kg, and cimetidine 10mg/kg intraperitoneally, alone or in combination while controls received an equivolume of saline), immediately after challenge or, alternatively, at 5min after or 24 or 48h before challenge. The effect was assessed at 5, 10, 20 and 30min after dextran and/or white egg challenge. We commonly noted prominent edema involving the face, upper and lower lip, snout, paws and scrotum (presented with extreme cyanosis), poor respiration and the number of fatalities after dextran and/or white egg application. Contrary, BPC 157 regimens (10µg, 1µg, 10ng, and 10pg/kg) effectively, may both prevent anaphylactoid reactions that may arise from dextran and/or white egg application and furthermore, rescue already advanced reactions when given after the challenge. Chloropyramine and cimetidine given alone were only moderately effective. When given together with BPC 157, the observed effect correlates with the strong effect of BPC 157 given alone. Copyright © 2014 Elsevier B.V. All rights reserved.

The effect of organic quail egg supplementation on the blood lipid profile of white mice (Rattus Norvegicus L.) during the lactation period

NASA Astrophysics Data System (ADS)

lestari purba, Sri; Rini Saraswati, Tyas; Isdadiyanto, Sri

2018-05-01

Background: Quail eggs contain a considerable amount of complete nutritional sources such as carbohydrates, proteins, fats, and micronutrients. However, they also have a high cholesterol level, which can potentially cause atherosclerosis and chronic heart diseases. The response of the body to foods containing is influenced by factors such as ethnicity, genetics, and hormonal and nutrient status of the consumer. The cholesterol level of quail eggs can be reduced by manipulating the feed using supplemental organic feed. Organic quail eggs have been believed to correct the lipid profile of white mice during the lactation phase. Purpose: The aim of this study was to analyze the effect of feed containing organic quail eggs on the blood lipid profile of white mice (Rattus norvegicus L.) during the lactation phase. Materials and Methods: This experimental study was conducted using a completely randomized design with four experiments and five repetitions. Experimental mice: T0 mice were used as control; T1 mice were supplemented with quail eggs produced by quails that were fed with standard feed; T2 mice were supplemented with eggs produced by quails fed with standard organic feed; and T3 mice were supplemented with eggs produced by quails fed with organic feed with the addition of cassava leaf flour, mackerel flour, and turmeric powder. Quail egg supplementation was administered to the mice from the early pregnancy period till the end of the lactation phase. The acquired data were analyzed using ANOVA. SPSS version 16.0 software for Windows was used for data analyses. Results and summary: Feeding the white mice with different compositions of organic quail egg supplements had no effect on the consumption of feed and water, body weight, and lipid profile (including total cholesterol, LDL, HDL, and triglyceride) during the lactation phase (P > 0.05).

Spectrophotometric studies on the interaction between (-)-epigallocatechin gallate and lysozyme

NASA Astrophysics Data System (ADS)

Ghosh, Kalyan Sundar; Sahoo, Bijaya Ketan; Dasgupta, Swagata

2008-02-01

Various reported antibacterial activities of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea prompted us to study its binding with lysozyme. This has been investigated by fluorescence, circular dichroism (CD) and protein-ligand docking. The binding parameters were determined using a modified Stern-Volmer equation. The thermodynamic parameters are indicative of an initial hydrophobic association. The complex is, however, held together predominantly by van der Waals interactions and hydrogen bonding. CD studies do not indicate any significant changes in the secondary structure of lysozyme. Docking studies revealed that specific interactions are observed with residues Trp 62 and Trp 63.

Molecular dynamics simulations of lysozyme-lipid systems: probing the early steps of protein aggregation.

PubMed

Trusova, Valeriya M; Gorbenko, Galyna P

2017-07-10

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of β-structures. Along with this, the formation of protein-lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.

21 CFR 184.1498 - Microparticulated protein product.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Microparticulated protein product. 184.1498... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1498 Microparticulated protein product. (a) Microparticulated protein product is prepared from egg whites or milk protein or a combination of egg whites and...

21 CFR 184.1498 - Microparticulated protein product.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Microparticulated protein product. 184.1498... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1498 Microparticulated protein product. (a) Microparticulated protein product is prepared from egg whites or milk protein or a combination of egg whites and...

21 CFR 184.1498 - Microparticulated protein product.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Microparticulated protein product. 184.1498 Section... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1498 Microparticulated protein product. (a) Microparticulated protein product is prepared from egg whites or milk protein or a combination of egg whites and...

21 CFR 184.1498 - Microparticulated protein product.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Microparticulated protein product. 184.1498... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1498 Microparticulated protein product. (a) Microparticulated protein product is prepared from egg whites or milk protein or a combination of egg whites and...

Exposing broiler eggs to green, red and white light during incubation.

PubMed

Archer, G S

2017-07-01

Previous work has shown that exposing broiler eggs to white light during incubation can improve hatchability and post-hatch animal welfare. It was hypothesized that due to how different wavelengths of light can affect avian physiology differently, and how pigmented eggshells filter light that different monochromatic wavelengths would have differential effects on hatchability and post-hatch animal welfare indicators. To determine, we incubated chicken eggs (n=6912) under either no light (dark), green light, red light or white light; the light level was 250 lux. White and red light were observed to increase hatch of fertile (P0.05). Fear response of during isolation and tonic immobility was reduced (P0.05) from dark incubated broilers. All light incubated broilers had lower (P<0.05) plasma corticosterone and higher (P<0.05) plasma serotonin concentrations than dark incubated broilers. These results indicate that white light and red light that is a component of it are possibly the key spectrum to improving hatchability and lower fear and stress susceptibility, whereas green light is not as effective. Incubating broiler eggs under these spectrums could be used to improve hatchery efficiency and post-hatch animal welfare at the same time.

Microbial quality of industrial liquid egg white: assumptions on spoiling issues in egg-based chilled desserts.

PubMed

Techer, Clarisse; Daoud, Amina; Madec, Marie-Noëlle; Gautier, Michel; Jan, Sophie; Baron, Florence

2015-02-01

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French egg processing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However, Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This study emphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts. © 2015 Institute of Food Technologists®

Preparation and Characterization of Fluorescent Derivatives of Lysozyme

NASA Technical Reports Server (NTRS)

Smith, Lori; Pusey, Marc

1998-01-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached using a carbodiimide coupling procedure. ASP 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these results.

HHP treatment of liquid egg at 200-350 MPa

NASA Astrophysics Data System (ADS)

Tóth, A.; Németh, Cs; Palotás, P.; Surányi, J.; Zeke, I.; Csehi, B.; Castillo, L. A.; Friedrich, L.; Balla, Cs

2017-10-01

High hydrostatic pressure (HHP) treatment of egg proteins partially limits their sensitivity to pressure. According to the literature, at the 450 MPa level, denaturation of some proteins sets in to the extent that sensory and functional characteristics are impacted. This study involved treating liquid egg (egg white, yolk, and melange) at less than the above-mentioned value, after which the microbiological effect was examined. For the study, pressure pouches were filled with 100ml of raw liquid egg per pouch. Then the samples were treated at 200, 250, 300 and 350 MPa. In each case, the level was reached by increasing pressure at a rate of 100 MPa/min. Measurements were taken at the Corvinus University of Budapest, Faculty of Food Science, Dept. of Refrigeration and Livestock Products Technology RESATO FPU 100-2000 equipment. Denaturation was determined with calorimetric (DSC) tests. From our results, it appears that even at 250 MPa pressure treatment, the viable cell count decreases. Further, it can be said that microbe count went down in the egg white samples at 300-350 MPa, below the impact level. Significant denaturation was not detected during our examinations. In summary, we state that the most HHP-sensitive liquid egg type, egg white, can be pressure treated to reduce microbe count at a level less than that which causes denaturation. Microbe reduction was smaller in yolk and melange, so higher pressure values are appropriate for these products.

White sturgeon spawning areas in the lower Snake River

USGS Publications Warehouse

Parsley, M.J.; Kappenman, K.M.

2000-01-01

We documented 17 white sturgeon Acipenser transmontanus spawning locations in the Snake River from the mouth to Lower Granite Dam (river km 0 to 173). Spawning locations were determined by the collection of fertilized eggs on artificial substrates or in plankton nets. We collected 245 eggs at seven locations in McNary Reservoir, 22 eggs at three locations in Ice Harbor Reservoir, 30 eggs from two locations in Lower Monumental Reservoir, and 464 eggs at five locations in Little Goose Reservoir. All 17 locations were in high water velocity areas and between 1.0 and 7.0 km downstream from a hydroelectric dam. The documentation of spawning areas is important because this habitat is necessary to maintain natural and viable populations.

Unravelling proteome changes of chicken egg whites under carbon dioxide modified atmosphere packaging.

PubMed

Xu, Lei; Jia, Fei; Luo, Changyao; Yu, Qianqian; Dai, Ruitong; Li, Xingmin

2018-01-15

Unfertilized chicken eggs within 24h of laying were chosen and stored at 25°C and 45% humidity for 0, 20, and 40days. The experimental group (EG) was the carbon dioxide-modified atmosphere packaging (CDMAP) group, whereas the control group (CG) contained eggs without special handling. Egg freshness indexes were measured. The proteome of the egg whites was determined by LC-MS/MS using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 87 proteins were detected. The results indicated that CDMAP can control the change in protein abundance. Using a correlation analysis between the protein abundance and freshness indexes of the EG, Beta-hexosaminidase, Trypsin inhibitor ClTI-1 and Apolipoprotein D were determined to be potential predictors of egg freshness. In comparing the proteomes of the EG and CG, it was concluded that CDMAP could affect the proteins related to egg vitelline membranes, eggshell matrix and metabolic intensity to maintain egg freshness. Copyright © 2017. Published by Elsevier Ltd.

Food-cooking processes modulate allergenic properties of hen's egg white proteins.

PubMed

Liu, Xiaoyu; Feng, Bai-Sui; Kong, Xiaoli; Xu, Hong; Li, Xiumin; Yang, Ping-Chang; Liu, Zhigang

2013-01-01

Reducing the allergenicity of food allergens can suppress the clinical symptoms of food allergy. The objective of the present study was to investigate the effects of processing on the allergenic properties of hen's egg white proteins. Eggs were processed by traditional Chinese cooking, including steaming, water boiling, frying, spicing and tea boiling. The contents of processed egg protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; the allergenicity was evaluated by Western blotting, enzyme-linked immunosorbent assay and enzyme allergosorbent test inhibition. Circular dichroism spectrum analysis of four major egg allergens from various egg products was performed as well. A mouse model of food allergy was developed to test the allergenicity of processed egg protein in vivo. Protein degradation was significant following tea boiling and spiced-tea boiling. The total allergenic potential of water-boiled egg and fried egg was relatively higher than that of steamed egg, spiced egg and tea-boiled egg. Challenge with proteins from raw egg, water-boiled egg and fried egg induced skewed T-helper 2 pattern responses (Th2 responses) in the intestine of mice sensitized to egg proteins; however, when the mice sensitized to egg proteins were challenged with proteins from steamed egg, spiced egg and tea-boiled egg, respectively, only weak Th2 responses were induced in their intestine. Processing by steaming, spicing, or tea boiling can weaken the allergenicity of egg proteins. Copyright © 2012 S. Karger AG, Basel.

Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

PubMed Central

Mechaly, Ariel E.; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M.; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general. PMID:24823716

Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

PubMed

Correa, Agustín; Pacheco, Sabino; Mechaly, Ariel E; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Magnetic Fluids Have Ability to Decrease Amyloid Aggregation Associated with Amyloid-Related Diseases

NASA Astrophysics Data System (ADS)

Antosova, Andrea; Koneracka, Martina; Siposova, Katarina; Zavisova, Vlasta; Daxnerova, Zuzana; Vavra, Ivo; Fabian, Martin; Kopcansky, Peter; Gazova, Zuzana

2010-12-01

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several amyloid-related diseases. We have investigated the effect of four magnetic fluids (MFs)—electrostatically stabilized Fe3O4 magnetic nanoparticles (MF1) and sterically stabilized Fe3O4 magnetic nanoparticles by sodium oleate (MF2, MF3 and MF4) with adsorbed BSA (MF2) or dextran (MF4)—on amyloid aggregation of two proteins, human insulin and chicken egg lysozyme. The morphology, particle size and size distribution of the prepared magnetic fluids were characterized. We have found that MFs are able to decrease amyloid aggregation of both studied proteins and the extent of depolymerization depended on the MF properties. The most effective reduction was observed for MF4 as 90% decrease of amyloids was detected for insulin and lysozyme amyloid aggregates. Our findings indicate that MFs have potential to be used for treatment of amyloid diseases.

In situ observation of containerless protein crystallization by magnetically levitating crystal growth

NASA Astrophysics Data System (ADS)

Maki, Syou; Tanimoto, Yoshifumi; Udagawa, Chikako; Morimoto, Shotaro; Hagiwara, Masayuki

2016-03-01

We report on the results of the crystal growth of hen-egg lysozyme by magnetically levitating crystals in a small amount of buffer solution. The concentrations of lysozyme and the precipitating agent (gadolinium chloride) were 6.53 wt % and 0.362 mol/kg, respectively. Gadolinium chloride, which induces the magneto-Archimedes effect, was utilized to levitate the crystals with Bz · (dBz/dz) = 22.46 T2/m, where Bz is the vertical (z) component of the magnetic flux density vector. Although the collected crystals were small, we succeeded in maintaining the levitation of the crystals into a specific place in the liquid phase from the beginning of nucleation. In situ observation revealed that a state of pseudo-weightlessness was generated in the vicinity of the magnet bore edge, and small crystals were concentrated inside the domain moving along an hourglass-shaped surface. We found by numerical computations that the formation of the hourglass-shaped domain is attributable to the radial component of the magnetic force.

Keeping Your Heart Healthy: What You Should Know about Lipids When You Have Chronic Kidney Disease (Stages 1-4)

MedlinePlus

... come from animals. Cholesterol-rich foods include: n Egg yolks (egg whites have no fat or cholesterol) n Any ... kidney.org 23 Diet Suggestions Food Choose Decrease Eggs Meat, poultry Limit to 2 egg yolks a ...

Occurrence of Campylobacter in commercially broken liquid egg in Japan.

PubMed

Sato, Miki; Sashihara, Nobuhiro

2010-03-01

Samples of unpasteurized liquid egg (whole egg, egg yolk, and egg white) and pasteurized liquid whole egg were collected from egg-breaking facilities in Japan and were tested for the presence of Campylobacter. Isolates recovered were tested for freeze-thaw response, sensitivity to antimicrobials, and heat resistance. Campylobacter was isolated from 27.9% of unpasteurized liquid whole egg samples and 36.0% of unpasteurized liquid egg yolk samples. No Campylobacter was isolated from unpasteurized liquid egg white or pasteurized liquid whole egg samples. The contamination levels of Campylobacter ranged from <3 to 240/100 ml. Freeze-thaw response was tested by freezing and thawing liquid whole egg and egg yolk to examine whether those conditions influenced the survival rate. It was shown that freezing and thawing reduced Campylobacter counts. Sensitivity to all antimicrobials used in this study was observed in 47.6% of the isolates. The most predominant antibiotic resistance profile was similar to that of isolates from chicken. D(55 degrees C)-values of 0.16 to 0.38 min and 0.47 to 0.84 min were determined for Campylobacter isolates in liquid whole egg and egg yolk, respectively. These values were lower than those reported for Salmonella. The very weak heat tolerance of Campylobacter indicated that it could be eliminated at the current legal pasteurization condition. There is no safety concern for commercially broken pasteurized liquid egg pertaining to Campylobacter contamination.

Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella

USDA-ARS?s Scientific Manuscript database

We report detection of <13 CFU of Salmonella per 25 g egg white within 7 h by concentrating the bacteria using microfiltration through 0.2-lm cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross-flow on both sides of the hollow fibers, and media selecti...

Liquid egg white pasteurization using a centrifugal UV irradiator

USDA-ARS?s Scientific Manuscript database

Studies are lacking on UV nonthermal pasteurization of liquid egg white (LEW). The objective of this study was to inactivate Escherichia coli using a UV irradiator that centrifugally formed a thin film of LEW on the inside of a rotating cylinder. The LEW was inoculated with E. coli K12 to approximat...

Thermal inactivation of H5N2 high-pathogenicity avian influenza virus in dried egg white with 7.5% moisture.

PubMed

Thomas, Colleen; Swayne, David E

2009-09-01

High-pathogenicity avian influenza viruses (HPAIV) cause severe systemic disease with high mortality in chickens. Isolation of HPAIV from the internal contents of chicken eggs has been reported, and this is cause for concern because HPAIV can be spread by movement of poultry products during marketing and trade activity. This study presents thermal inactivation data for the HPAIV strain A/chicken/PA/1370/83 (H5N2) (PA/83) in dried egg white with a moisture content (7.5%) similar to that found in commercially available spray-dried egg white products. The 95% upper confidence limits for D-values calculated from linear regression of the survival curves at 54.4, 60.0, 65.5, and 71.1 degrees C were 475.4, 192.2, 141.0, and 50.1 min, respectively. The line equation y = [0.05494 x degrees C] + 5.5693 (root mean square error = 0.0711) was obtained by linear regression of experimental D-values versus temperature. Conservative predictions based on the thermal inactivation data suggest that standard industry pasteurization protocols would be very effective for HPAIV inactivation in dried egg white. For example, these calculations predict that a 7-log reduction would take only 2.6 days at 54.4 degrees C.

Proteomic comparison by iTRAQ combined with mass spectrometry of egg white proteins in laying hens (Gallus gallus) fed with soybean meal and cottonseed meal

PubMed Central

He, Tao; Zhang, Haijun; Wang, Jing; Wu, Shugeng; Yue, Hongyuan; Qi, Guanghai

2017-01-01

Cottonseed meal (CSM) is commonly used in hens’ diets to replace soybean meal (SBM). However, the molecular consequences of this substitution remains unclear. To investigate the impact of this substitution at the molecular level, iTRAQ combined with biochemical analysis was performed in Hy-Line W-36 hens supplemented with a mixed diet of CSM and SBM. Egg weight, albumen height, and Haugh unit were significantly reduced in the CSM100 group (100% crude protein of SBM replaced by CSM) compared with the SBM group (P<0.05). A total of 15 proteins, accounting for 75% of egg white proteins with various biological functions of egg whites, were found to be reduced. This finding may relate to the decrease of albumen quality in the CSM100 group. Oviduct magnum morphology and hormone analysis indicated that a reduced level of plasma progesterone caused reduced growth of the tubular gland and epithelial cells in the magnum, further decreasing egg white protein synthesis in the magnum. These findings help demonstrate the molecular mechanisms of a CSM diet that cause adverse effects on albumen quality, while also showing that SBM should not be totally replaced with CSM in a hen diet. PMID:28813468

Prevalence of IgE mediated cow's milk and egg allergy in children under 2 years of age in Sanliurfa, Turkey: the city that isn't almost allergic to cow's milk.

PubMed

Zeyrek, D; Koruk, I; Kara, B; Demir, C; Cakmak, A

2015-12-01

Food allergy is a common problem in children and adults. The aim of this study was to determine the frequency of cow's milk and egg allergy based on skin prick test and oral food challenge results We recruited 614 children aged 1 month-2 years in from different areas of Sanliurfa. An interview was conducted with each parents and family demographic, and prenatal-antenatal-postnatal information was recorded, then skin prick test was applied to the children for egg white, egg yolk and cow's milk. Cases that are required, sIgE levels for cow's milk, egg white and egg yolk were measured and food challenge tests were applied. Cow's milk and egg allergy was determined in 1 case (0.16%) and 8 cases (1.30%) respectively from the results of the tests applied. Six cases (0.97%) were accepted as sensitive to egg white and egg yolk. Within the factors which were questioned, (only the egg allergy evaluation was made without the cow's milk allergy case) of those with parental consanguinity was determined at a statistically significantly high level (OR=5.8, 95% CI: 1.2-27.2, P=0.01). The prevalence of cow's milk allergy in Sanliurfa is the lowest reported prevalence rate to date in children under 2 years of age. When infants develop a food allergy, this takes the form of egg allergy. Within the several factors evaluated to explain this situation, a significant relationship was determined only with parental consanguinity. However, as reported above, to date there is no genetic or epidemiological study to explain this situation.

Immunologic changes in children with egg allergy ingesting extensively heated egg.

PubMed

Lemon-Mulé, Heather; Sampson, Hugh A; Sicherer, Scott H; Shreffler, Wayne G; Noone, Sally; Nowak-Wegrzyn, Anna

2008-11-01

Prior studies have suggested that heated egg might be tolerated by some children with egg allergy. We sought to confirm tolerance of heated egg in a subset of children with egg allergy, to evaluate clinical and immunologic predictors of heated egg tolerance, to characterize immunologic changes associated with continued ingestion of heated egg, and to determine whether a diet incorporating heated egg is well tolerated. Subjects with documented IgE-mediated egg allergy underwent physician-supervised oral food challenges to extensively heated egg (in the form of a muffin and a waffle), with tolerant subjects also undergoing regular egg challenges (in a form of scrambled egg or French toast). Heated egg-tolerant subjects incorporated heated egg into their diets. Skin prick test wheal diameters and egg white, ovalbumin, and ovomucoid IgE levels, as well as ovalbumin and ovomucoid IgG4 levels, were measured at baseline for all subjects and at 3, 6, and 12 months for those tolerant of heated egg. Sixty-four of 117 subjects tolerated heated egg, 23 tolerated regular egg, and 27 reacted to heated egg. Heated egg-reactive subjects had larger skin test wheals and greater egg white-specific, ovalbumin-specific, and ovomucoid-specific IgE levels compared with heated egg- and egg-tolerant subjects. Continued ingestion of heated egg was associated with decreased skin test wheal diameters and ovalbumin-specific IgE levels and increased ovalbumin-specific and ovomucoid-specific IgG4 levels. The majority of subjects with egg allergy were tolerant of heated egg. Continued ingestion of heated egg was well tolerated and associated with immunologic changes that paralleled the changes observed with the development of clinical tolerance to regular egg.

Genetic loci inherited from hens lacking maternal behaviour both inhibit and paradoxically promote this behaviour.

PubMed

Basheer, Atia; Haley, Chris S; Law, Andy; Windsor, Dawn; Morrice, David; Talbot, Richard; Wilson, Peter W; Sharp, Peter J; Dunn, Ian C

2015-12-30

A major step towards the success of chickens as a domesticated species was the separation between maternal care and reproduction. Artificial incubation replaced the natural maternal behaviour of incubation and, thus, in certain breeds, it became possible to breed chickens with persistent egg production and no incubation behaviour; a typical example is the White Leghorn strain. Conversely, some strains, such as the Silkie breed, are prized for their maternal behaviour and their willingness to incubate eggs. This is often colloquially known as broodiness. Using an F2 linkage mapping approach and a cross between White Leghorn and Silkie chicken breeds, we have mapped, for the first time, genetic loci that affect maternal behaviour on chromosomes 1, 5, 8, 13, 18 and 19 and linkage group E22C19W28. Paradoxically, heterozygous and White Leghorn homozygous genotypes were associated with an increased incidence of incubation behaviour, which exceeded that of the Silkie homozygotes for most loci. In such cases, it is likely that the loci involved are associated with increased egg production. Increased egg production increases the probability of incubation behaviour occurring because egg laying must precede incubation. For the loci on chromosomes 8 and 1, alleles from the Silkie breed promote incubation behaviour and influence maternal behaviour (these explain 12 and 26% of the phenotypic difference between the two founder breeds, respectively). The over-dominant locus on chromosome 5 coincides with the strongest selective sweep reported in chickens and together with the loci on chromosomes 1 and 8, they include genes of the thyrotrophic axis. This suggests that thyroid hormones may play a critical role in the loss of incubation behaviour and the improved egg laying behaviour of the White Leghorn breed. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of selection for fertility and egg laying persistency and against maternal incubation behaviour.

White-faced ibis DDE-related reproductive problems continue at Carson Lake, Nevada

USGS Publications Warehouse

Henny, C.J.; Herron, G.B.

1988-01-01

Organochlorine, mercury, and selenium contamination was studied in White-faced Ibis (Plegadis chihi) nesting at Carson Lake, Nevada in 1985 and 1986. DDE was detected in 138 of 140 eggs sampled. Eggshell thickness was negatively correlated with residues of DDE. DDE residues in ibis eggs, unlike residues in most other wading bird eggs from the Great Basin have not declined during the last decade. At DDE levels in eggs above 4 ppm (wet weight), clutch size and productivity decreased, and the incidence of cracked eggs increased. Assuming that 4 ppm DOE is the critical residue level, 40% of the nesting population in 1985 and 1986 was adversely impacted by DDE, with a net loss of 20% of the population' s expected .production. Most eggs containing high levels (up to 29 ppm) also contained DDT, which implies the source was recently-used DDT. No evidence of breeding ground DDE-DDT contamination was found.

Poultry products enriched with nutricines have beneficial effects on human health.

PubMed

Kralik, Gordana; Kralik, Zlata

2017-02-01

The paper presents nutritive value of broiler meat and table eggs, as these animal products are used for human nutrition on a daily basis. In the Republic of Croatia, average consumption of poultry meat amounts to 18.3 kg and to 160 eggs per capita.The most quality parts of broiler carcass are breasts and drumsticks with thighs. Breast muscles contain 21-23% protein, 1.90-1.97% fat, 75.28-76.01% water and 0.74-0.77% collagen. Thigh muscles contain 4.70-6.05% fat, 19.03-19.93% protein and 0.91-1.13% collagen. White meat contains more potassium and magnesium and less zinc and iron than dark meat. In 100 g of edible egg part there is 12.5-13.5 g protein, 10.7-11.6 g fat and 1.0-1.1 g minerals. Caloric valueof eggs is 167 Kcal. Eggs contain high amount of essential amino acids, especially leucine, isoleucine, lysine, arginine, valine and phenylalanine. Furthermore, eggs contain many vitamins, especially A, D, E, K and B-complex, as well as various macroelements and microelements. Eggs and meat enriched with one or more functional ingredients - nutricines (polyunsaturated fatty acids n-3), eicosapentaenoic acid, docosahexaenoic acid, selenium and lutein) meet the criteria of functional food because of their added value and benefits for human health. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

The potency of chicken egg yolk immunoglobulin (IgY) specific as immunotherapy to Mycobacterium tuberculosis infection

PubMed Central

Sudjarwo, Sri Agus; Eraiko, Koerniasari; Sudjarwo, Giftania Wardani; Koerniasari

2017-01-01

The aim of this study was to characterize of chicken egg yolk immunoglobulins (IgYs) specific as immunotherapy to Mycobacterium tuberculosis complex (MTBC) infection. Lohmann laying hens were immunized intramuscularly with antigenic of MTBC. Egg yolk was separated from egg white, and IgY antibody was then purified by multiple polyethylene glycols 6000 extraction and ammonium sulfate purification steps. The IgY anti-MTBC concentration in egg yolk increased at 2 weeks and it reached a maximum at 4 weeks after immunization. After 6 weeks, the levels of IgY anti-MTBC decreased gradually. The antibody of MTBC was detected and produces a specific line of precipitation in agar gel precipitation test beginning the week 2 after the first immunization. Analysis of results obtained with ELISA showed a significant increase in the MTBC specific antibodies after 2 weeks and reached a plateau at 4 weeks from the booster immunization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the IgY preparation to be pure and dissociated into protein bands with molecular weights of 112, 78, 69, 49, and 28 kDa and Western blot analysis shown the presence of anti-MTBC IgY in egg yolks, with molecular weights of approximately 78 kDa. These results suggested that egg yolk could be a practical strategy in large-scale production of specific anti-MTBC IgY for immunotherapy of TBC. PMID:28795022

The potency of chicken egg yolk immunoglobulin (IgY) specific as immunotherapy to Mycobacterium tuberculosis infection.

PubMed

Sudjarwo, Sri Agus; Eraiko, Koerniasari; Sudjarwo, Giftania Wardani; Koerniasari

2017-01-01

The aim of this study was to characterize of chicken egg yolk immunoglobulins (IgYs) specific as immunotherapy to Mycobacterium tuberculosis complex (MTBC) infection. Lohmann laying hens were immunized intramuscularly with antigenic of MTBC. Egg yolk was separated from egg white, and IgY antibody was then purified by multiple polyethylene glycols 6000 extraction and ammonium sulfate purification steps. The IgY anti-MTBC concentration in egg yolk increased at 2 weeks and it reached a maximum at 4 weeks after immunization. After 6 weeks, the levels of IgY anti-MTBC decreased gradually. The antibody of MTBC was detected and produces a specific line of precipitation in agar gel precipitation test beginning the week 2 after the first immunization. Analysis of results obtained with ELISA showed a significant increase in the MTBC specific antibodies after 2 weeks and reached a plateau at 4 weeks from the booster immunization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the IgY preparation to be pure and dissociated into protein bands with molecular weights of 112, 78, 69, 49, and 28 kDa and Western blot analysis shown the presence of anti-MTBC IgY in egg yolks, with molecular weights of approximately 78 kDa. These results suggested that egg yolk could be a practical strategy in large-scale production of specific anti-MTBC IgY for immunotherapy of TBC.

Long range Debye-Hückel correction for computation of grid-based electrostatic forces between biomacromolecules

PubMed Central

2014-01-01

Background Brownian dynamics (BD) simulations can be used to study very large molecular systems, such as models of the intracellular environment, using atomic-detail structures. Such simulations require strategies to contain the computational costs, especially for the computation of interaction forces and energies. A common approach is to compute interaction forces between macromolecules by precomputing their interaction potentials on three-dimensional discretized grids. For long-range interactions, such as electrostatics, grid-based methods are subject to finite size errors. We describe here the implementation of a Debye-Hückel correction to the grid-based electrostatic potential used in the SDA BD simulation software that was applied to simulate solutions of bovine serum albumin and of hen egg white lysozyme. Results We found that the inclusion of the long-range electrostatic correction increased the accuracy of both the protein-protein interaction profiles and the protein diffusion coefficients at low ionic strength. Conclusions An advantage of this method is the low additional computational cost required to treat long-range electrostatic interactions in large biomacromolecular systems. Moreover, the implementation described here for BD simulations of protein solutions can also be applied in implicit solvent molecular dynamics simulations that make use of gridded interaction potentials. PMID:25045516

Effect of fibrillation conditions on the anti-amyloidogenic properties of polyphenols and their involved mechanisms.

PubMed

Mahdavimehr, Mohsen; Katebi, Bentolhoda; Meratan, Ali Akbar

2018-06-24

In the present study, we have investigated the effects of protein concentration and stirring on the in vitro assembly of Hen Egg White Lysozyme (HEWL), particularly with regard to the aggregate morphology and anti-amyloidogenic properties of two naturally occurring polyphenols, taxifolin and silibinin. The results obtained clearly demonstrated that applying stirring and concentration enhancement alter the amount as well as morphology of amyloid fibrils formed. Additionally, latter aggregates exhibited higher affinity for amyloid-specific dyes. The second part of the present investigation was devoted to studies involving anti-amyloidogenic properties of selected polyphenols. Importantly, we found that the potency of polyphenols to inhibit HEWL amyloid fibrillation and related toxicity is strongly dependent on the amyloidogenic conditions in which amyloid fibrils are produced. Based on obtained data, under condition where the rate of protein assembly is high (higher protein concentration and stirring), the capacity of polyphenols to inhibit HEWL fibrillogenesis and related cytotoxicity may dramatically decrease. Similar results were obtained when we used taxifolin to inhibit bovine insulin amyloid fibrillation. Additionally, amyloidogenic conditions may also affect the mechanism by which these molecules inhibit HEWL fibrillation. The possible mechanism by which selected polyphenols exert their inhibitory effects, under various experimental conditions, is also discussed. Copyright © 2018. Published by Elsevier B.V.

X-Ray Structure determination of the Glycine Cleavage System Protein H of Mycobacterium tuberculosis Using An Inverse Compton Synchrotron X-Ray Source

PubMed Central

Abendroth, Jan; McCormick, Michael S.; Edwards, Thomas E.; Staker, Bart; Loewen, Roderick; Gifford, Martin; Rifkin, Jeff; Mayer, Chad; Guo, Wenjin; Zhang, Yang; Myler, Peter; Kelley, Angela; Analau, Erwin; Hewitt, Stephen Nakazawa; Napuli, Alberto J.; Kuhn, Peter; Ruth, Ronald D.; Stewart, Lance J.

2010-01-01

Structural genomics discovery projects require ready access to both X-ray and NMR instrumentation which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive frequent use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large government-run synchrotron facilities for data collection. In an effort to eliminate the need to ship crystals for data collection, we have developed the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam. PMID:20364333

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

PubMed Central

Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

2017-01-01

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (~100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day−1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. PMID:28722205

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

PubMed

Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

2017-11-01

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day -1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. © 2017 Wiley Periodicals, Inc.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

PubMed

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

2014-01-01

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Idiotypic analysis of antibodies to hen egg-white lysozyme (HEL). II. Distribution and frequency of occurrence of idiotypes shared by A/J anti-HEL antibody.

PubMed Central

Semma, M; Sakato, N; Fujio, H; Amano, T

1981-01-01

Anti-HEL Ab from one A/J mouse (No. a-4),Ida-4(HEL), was s.c. inoculated into rabbits to induce anti-idiotypic (anti-Id) sera. After absorption with normal A/J Ig, two anti-Id serra (R101 and R102) were obtained. The interaction between 125I-Id(a-4)(HEL) and anti-Id sera was completely inhibited by unlabelled Ida-4(HEL), but not by non-specific Ig. The anti-Id sera were used to investigate the distribution of A/J (No. a-4) anti-HEL idiotypes in sera obtained from HEL-immunized animals. Idiotypes shared by Ida-4(HEL) were also detected in the sera of five examined mouse strains, but not in any of the examined rat, guinea-pig, goat, and sheep sear. Our experiments suggest the presence of inter- as well as intrastrain idiotype in mice. However, cross-reactivity appeared to be weak. When R101 and R102, respectively, were the anti-Id sera used, the frequency of occurrence of Ida-4(HEL) in stain A wa 1/106 and 1/53; in other mouse strains it was 1/277 and 1/85. PMID:7461724

Assessment study of ion-exchange chromatography combined with solution X-ray scattering measurement for protein characterization.

PubMed

Watanabe, Yasushi

2018-03-02

The performance of ion-exchange chromatography combined with small-angle X-ray scattering measurement was evaluated by characterization of the hen egg white lysozyme as a model protein. The X-ray transmittance was estimated using a micro-ionization chamber equipped with a sample cell holder for the real-time monitoring of the X-ray beam strength through the salt gradient elution. The radius of gyration of the eluted protein was estimated to be 1.50 ± 0.06 (n = 3) nm and 1.4 ± 0.1 nm as the value at the zero protein concentration. By using the X-ray transmittance values for the scattering intensity correction, the molecular weight of the eluted protein was estimated to be 15,200 ± 500 (n = 3) and 14,400 ± 200 as the value at the zero protein concentration. These values are close to those of the monomer of this protein. The ion-exchange chromatography combined with the small-angle X-ray scattering measurement system equipped with the X-ray transmittance monitor is a reliable method for protein characterization in solution. Copyright © 2018 Elsevier B.V. All rights reserved.

Adsorption and separation of proteins by collagen fiber adsorbent.

PubMed

Li, Juan; Liao, Xue-pin; Zhang, Qi-xian; Shi, Bi

2013-06-01

The separation of proteins is a key step in biomedical and pharmaceutical industries. In the present investigation, the collagen fiber adsorbent (CFA) was exploited as column packing material to separate proteins. Bovine serum albumin (BSA), bovine hemoglobin (Hb) and lysozyme (LYS) that have different isoelectric points (pIs) were selected as model proteins to investigate the separation ability of CFA to proteins. In batch adsorption, the adsorption behaviors of these proteins on CFA under different pHs and ionic strengths indicated that the electrostatic interaction plays a predominant role in the adsorption of proteins on CFA. CFA exhibited high adsorption capacity to Hb and LYS. In column separation, the proteins were completely separated by adjusting pH and ionic strength of the eluent. The increase of flow rate could reduce the separation time with no influence on the recovery of protein in the experimental range. The protein recovery was higher than 90% even when the CFA column was re-used for 4 times in separation of BSA and LYS, and the retention time of BSA or LYS was almost constant during the repeated applications. In addition, as a practical application, LYS was successfully separated from chicken egg white powder by CFA column. Copyright © 2013 Elsevier B.V. All rights reserved.

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

PubMed

Primo, Emiliano D; Otero, Lisandro H; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor.

PubMed

Thomas, Ekelijn; Bouma, Annemarie; van Eerden, Ellen; Landman, Wil J M; van Knapen, Frans; Stegeman, Arjan; Bergwerff, Aldert A

2006-08-31

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.

Influence of processing factors on the stability of model mayonnaise with whole egg during long-term storage.

PubMed

Ariizumi, Masahiro; Kubo, Megumi; Handa, Akihiro; Hayakawa, Takashi; Matsumiya, Kentaro; Matsumura, Yasuki

2017-04-01

Mayonnaise-like oil-in-water emulsions with different stabilities-evaluated from the degree of macroscopic defects, e.g., syneresis-were prepared by different formulations and processing conditions (egg yolk weight, homogenizer speed, and vegetable oil temperature). Emulsions prepared with lower egg yolk content were destabilized for shorter periods. The long-term stability of emulsions was weakly related to initial properties, e.g., oil droplet distribution and protein coverage at the interface. Protein aggregation between oil droplets was observed and would be responsible for the instability of emulsions exhibited by the appearance defects. SDS-PAGE results for adsorbed and unadsorbed proteins at the O/W interface suggested that predominant constituents adsorbed onto the interface were egg white proteins as compared with egg yolk components when the amount of added egg yolk was low. In present condition, egg white proteins adsorbed at the O/W interface could be a bridge of neighboring oil droplets thereby causing flocculation in emulsions.

A photographic technique for estimating egg density of the white pine weevil, Pissodes strobi (Peck)

Treesearch

Roger T. Zerillo

1975-01-01

Compares a photographic technique with visual and dissection techniques for estimating egg density of the white pine weevil, Pissodes strobi (Peck). The relatively high correlations (.67 and .79) between counts from photographs and those obtained by dissection indicate that the non-destructive photographic technique could be a useful tool for...

Verification of Egg Farming Systems from The Netherlands and New Zealand Using Stable Isotopes.

PubMed

Rogers, Karyne M; van Ruth, Saskia; Alewijn, Martin; Philips, Andy; Rogers, Pam

2015-09-30

Stable isotopes were used to develop authentication criteria of eggs laid under cage, barn, free range, and organic farming regimens from The Netherlands and New Zealand. A training set of commercial poultry feeds and egg albumen from 49 poultry farms across The Netherlands was used to determine the isotopic variability of organic and conventional feeds and to assess trophic effects of these corresponding feeds and barn, free range, and organic farming regimens on corresponding egg albumen. A further 52 brands of New Zealand eggs were sampled from supermarket shelves in 2008 (18), 2010 (30), and 2014 (4) to characterize and monitor changes in caged, barn, free range, and organic egg farming regimens. Stable carbon (δ(13)C) and nitrogen (δ(15)N) isotopes of 49 commercial poultry feeds and their corresponding egg albumens reveals that Dutch poultry are fed exclusively on a plant-based feed and that it is possible to discriminate between conventional and organic egg farming regimens in The Netherlands. Similarly, it is possible to discriminate between New Zealand organic and conventional egg farming regimens, although in the initial screening in 2008, results showed that some organic eggs had isotope values similar to those of conventional eggs, suggesting hens were not exclusively receiving an organic diet. Dutch and New Zealand egg regimens were shown to have a low isotopic correlation between both countries, because of different poultry feed compositions. In New Zealand, both conventional and organic egg whites have higher δ(15)N values than corresponding Dutch egg whites, due to the use of fishmeal or meat and bone meal (MBM), which is banned in European countries. This study suggests that stable isotopes (specifically nitrogen) show particular promise as a screening and authentication tool for organically farmed eggs. Criteria to assess truthfulness in labeling of organic eggs were developed, and we propose that Dutch organic egg whites should have a minimum δ(15)N value of 4.8‰ to account for an organic plant derived diet. Monitoring of New Zealand egg isotopes over the past 7 years suggests that organic eggs should have a minimum δ(15)N value of 6.0‰, and eggs falling below this value should be investigated further by certification authorities.

Natural course of sensitization to hen's egg in children not previously exposed to egg ingestion.

PubMed

de Boissieu, D; Dupont, C

2006-04-01

Clinical adverse reactions to egg may occur in infants or children who have never eaten egg. They may be sensitized or even react at first egg ingestion. Few studies are available concerning the reality of egg white allergy in such sensitized children, the natural evolution of this condition and the appropriate decisions to make. To analyze the actuality and natural course of egg allergy in children sensitized without previous of hen's egg ingestion. We set up a clinical decision tree based on clinical history and specific egg white IgE to manage patients who had never ingested egg but were sensitized as demonstrated by a positive SPT and report a cohort of 30 such children The mean level of egg white specific IgE at first analysis, i.e. before 12 months, was high, 28.3 KU(A) /L, with a large range, from 0.6 to >100 KU(A) /L, below 6 KU(A) /L in only 8 patients. In 6 children ("no challenge" group), IgE values remained >8 KU(A) /L by the end of the survey and the oral challenge with egg was always denied. Their mean + SD IgE level was at 51.7 + 38 KU(A) /L at 1 year and 19.7 + 13 KU(A) /L at a mean age of 34 + 5 months. All had an associated anaphylactic reaction with milk and 5 were still allergic to milk by the end of the survey. In the remaining 24 infants, egg was given for the first time at a mean age of 30 + 9 months, by error in 4 cases, all exhibiting an immediate reaction, and in a hospital setting in 20, among whom 14 reacted. Among those 18, with a specific IgE level at 9.1 + 10 KU(A) /L at 28 + 9 months, 4 became tolerant between 3 and 4 years, with specific IgE levels below 1.3 KU(A) /L and a 5th one with specific IgE >100 KU(A) /L at 6 months tolerated scrambled eggs at age 7 year, with specific IgE at 2.6 KU(A) /L. In the 6 others, labeled "non allergic", egg white specific IgE levels were significantly lower, whatever the age, than in the "no challenge" group. The age at challenge was 35 + 8 months, with a mean specific IgE level at 1.0 + 0.9 KU(A) /L. In children sensitized to egg without previous ingestion of that food, egg tolerance appears probably in some by the age of 3 but may reveal much more prolonged in a limited number.

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei.

PubMed

Sirirustananun, Nuttarin; Chen, Jiann-Chu; Lin, Yong-Chin; Yeh, Su-Tuen; Liou, Chyng-Hwa; Chen, Li-Li; Sim, Su Sing; Chiew, Siau Li

2011-12-01

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quantitative trait loci segregating in crosses between New Hampshire and White Leghorn chicken lines: I. egg production traits.

PubMed

Goraga, Z S; Nassar, M K; Brockmann, G A

2012-04-01

A genome scan was performed to detect chromosomal regions that affect egg production traits in reciprocal crosses between two genetically and phenotypically extreme chicken lines: the partially inbred line New Hampshire (NHI) and the inbred line White Leghorn (WL77). The NHI line had been selected for high growth and WL77 for low egg weight before inbreeding. The result showed a highly significant region on chromosome 4 with multiple QTL for egg production traits between 19.2 and 82.1 Mb. This QTL region explained 4.3 and 16.1% of the phenotypic variance for number of eggs and egg weight in the F(2) population, respectively. The egg weight QTL effects are dependent on the direction of the cross. In addition, genome-wide suggestive QTL for egg weight were found on chromosomes 1, 5, and 9, and for number of eggs on chromosomes 5 and 7. A genome-wide significant QTL affecting age at first egg was mapped on chromosome 1. The difference between the parental lines and the highly significant QTL effects on chromosome 4 will further support fine mapping and candidate gene identification for egg production traits in chicken. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

Hypersensitivity to total parenteral nutrition fat-emulsion component in an egg-allergic child.

PubMed

Lunn, Michael; Fausnight, Tracy

2011-10-01

Immunoglobulin E (IgE)-mediated food allergies affect 6% to 8% of children in the United States with symptoms ranging from localized hives to life-threatening anaphylaxis. Intravenous fat emulsions (IFEs) are a vital component of total parental nutrition, because they provide essential fatty acids. IFE is a sterile fat emulsion that contains egg-yolk phospholipids. Although egg allergy is listed as a contraindication, adverse reactions are uncommon. We report here the case of a hypersensitivity to IFE in a 2-year-old patient with previously undocumented egg allergy. Our patient was placed on total parental nutrition and a 20% IFE postoperatively and developed diffuse pruritus 14 days after initiation of therapy. She showed transient improvement with intravenous antihistamine, but her symptoms did not resolve until the IFE was stopped. On the basis of clinical history, including aversion to egg, we performed skin-prick testing, the results of which were positive for egg white allergy. Serum testing confirmed allergy to both egg yolk and egg white. To our knowledge, this is the first reported case of a pediatric patient with a history suggestive of egg allergy, positive skin-prick and serum testing to egg, and reaction to IFE infusion. Although ingestion of egg lecithin in cooked food is generally tolerated by egg-allergic people, administration of intravenous egg-containing lipid emulsions may cause significant adverse reactions.

Effect of tributyltin on reproduction in Japanese whiting, Sillago japonica.

PubMed

Shimasaki, Yohei; Oshima, Yuji; Inoue, Suguru; Inoue, Yoshiyuki; Kang, Ik Joon; Nakayama, Kei; Imoto, Hisaya; Honjo, Tsuneo

2006-07-01

We examined the effect of tributyltin (TBT) on reproduction in the Japanese whiting, Sillago japonica. Mature fish were placed in indoor 500-L polyethylene tanks (five males and three females per tank) with a flow-through system and received dietary exposure to tributyltin oxide at concentrations of 2, 20, or 200 microg/g for 30 days during the active spawning period. Eggs spawned from the fish were collected daily, and the floating egg rate, larval deformity, hatchability, and viable hatch were monitored. TBT concentration in eggs of 200-microg/g group ranged from 85.0 to 159.6 ng/g in the evaluation period (days 5-30). In this period, the floating egg rate (83.2%), viable hatchability (82.2%), and total number of viable larvae (422,000 larvae per 100 g of female) were all significantly decreased in the 200-microg/g group compared with the control group (93.0%, 91.9%, and 709,000 larvae, respectively). The rate of deformity (2.6%) in the 200-microg/g group was about three times that in the control group (0.8%), although this difference was not significant. From these results, the lowest observed effect concentration of TBT in eggs on reproduction in Japanese whiting was estimated to be less than 159.6 ng/g-eggs.

Withdrawal times of oxytetracycline and tylosin in eggs of laying hens after oral administration.

PubMed

Muñoz, Rubén; Cornejo, Javiera; Maddaleno, Aldo; Araya-Jordán, Carolina; Iragüen, Daniela; Pizarro, Nicolás; San Martín, Betty

2014-06-01

Antimicrobials administered to laying hens may be distributed into egg white or yolk, indicating the importance of evaluating withdrawal times (WDTs) of the pharmaceutical formulations. In the present study, oxytetracycline and tylosin's WDTs were estimated. The concentration and depletion of these molecules in eggs were linked to their pharmacokinetic and physicochemical properties. Twenty-seven Leghorn hens were used: 12 treated with oxytetracycline, 12 treated with tylosin, and 3 remained as an untreated control group. After completion of therapies, eggs were collected daily and drug concentrations in egg white and yolk were assessed. The yolk was used as the target tissue to evaluate the WDT; the results were 9 and 3 days for oxytetracycline and tylosin, respectively. In particular, oxytetracycline has a good oral bioavailability, a moderate apparent volume of distribution, a molecular weight of 460 g/mol, and is lightly liposoluble. Tylosin, a hydrosoluble compound, with a molecular weight of 916 g/mol, has a low oral bioavailability and a low apparent volume of distribution, too. Present results suggest that the WDTs of the studied antimicrobials are strongly influenced by their oral bioavailability, the distribution, and the molecular weight and solubility, and that these properties also influence the distribution between the egg yolk and white.

Protein precipitation behavior of condensed tannins from Lotus pedunculatus and Trifolium repens with different mean degrees of polymerization

USDA-ARS?s Scientific Manuscript database

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited...

Food allergy in infants and children: clinical evaluation and management.

PubMed

Levy, Y; Kornbroth, B; Ofer, I; Garty, B Z; Danon, Y L

1994-12-01

A total of 122 infants and children up to age 17 (69 males and 53 females) who were referred for food allergy to the Pediatric Allergy and Clinical Immunology Unit were evaluated by complete history, emphasizing the implicated foods, clinical presentation and involvement of various organ systems, physical examination, and prick skin tests to food allergens. Fourteen infants with a history of egg white allergy and positive skin tests to egg white also underwent skin tests (prick and intradermal in 1:100 dilution) to measles-mumps-rubella (MMR) vaccine; 35 children under 3 years old had 41 oral challenges with the suspected foods; and 9 children over 3 years old had 12 oral challenges with the suspected foods. We found that cow milk/humanized milk formula, egg white, soybean, and peanut are the main allergenic foods in the pediatric population. Thirteen children had 13 positive oral challenges: 12 to cow milk/humanized milk formula and one to egg white. Symptoms reproduced by oral challenges included urticarial and erythematous rash, conjunctival itching, angioedema, abdominal pain, vomiting, diarrhea, and rhinorrhea. No anaphylactic shock was reported. Negative skin test has an excellent predictive accuracy for negative oral challenge with the suspected food in children > 3 years old. The negative predictive accuracy of cow milk skin test in children < 3 years was 73%. Positive skin test is not a good predictor of a clinical reaction to food. Oral food challenge performed cautiously in a medical setting is the "gold standard" for diagnosis. MMR vaccine can be safely administered to infants with egg white allergy after skin tests with the vaccine are performed.

Analysis of the low-molecular weight protein profile of egg-white and its changes during early chicken embryological development.

PubMed

Fang, Jun; Ma, Mei H; Qiu, Ning; Wu, Xiao; Jin, Yong G

2012-01-01

Many low-molecular weight (LMW) proteins in egg-white are potentially bioactive, but the mass range and number of these are not yet fully characterized. The aim of the present study was to map the LMW protein profile in egg-white and provide the basis for further understanding of the physiological function of these proteins. For this purpose, six time points (days 0, 1, 2, 3, 4, 5 of incubation) were selected in an attempt to delineate the LMW proteomic profile in egg-white and its changes during early chicken embryological development. Samples were pretreated using gel chromatography techniques prior to analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Protein search focused on the mass range m/z 1,000 to 8,000. One hundred and fourteen mass signal peaks of LMW proteins ranging from m/z 1,035.88 to 7,112.91 were detected at all six time points. The observed changes in the LMW protein profile during development were highly dynamic. Eighty six novel mass signal peaks of LMW proteins were generated during incubation, the origin of which could be assigned to the high-molecular weight protein fractions.The list of egg-white LMW proteins provided in this paper is by far the most comprehensive and is intended to serve as a starting point for the isolation and functional characterization of interesting LMW proteins which may play a crucial role in early embryo nutrition and immunity.

Sanitation of chicken eggs by ionizing radiation: functional and nutritional assessment

NASA Astrophysics Data System (ADS)

Pinto, P.; Ribeiro, R.; Sousa, L.; Verde, S. Cabo; Lima, M. G.; Dinis, M.; Santana, A.; Botelho, M. L.

2004-09-01

Alternative technologies must be developed and implemented considering sanitation and preservation of eggs mainly for risk population. Food irradiation is an alternative to free Salmonella spp. and Campylobacter spp. eggs, as a low dose point to a safety assurance. This study presents the correlation between irradiation doses (0.5 kGy up to 5 kGy at dose rate of 1.0 kGy/h) and some of functional and nutritional egg properties. Viscosimetry of non-irradiated and irradiated eggs was evaluated by means of VT550 Haake with an NV sensor and co-axes cylinders. After irradiation at 5 kGy, the yolk colour die (pale yellow) and the white egg was modified to a turbid yellow. The Cross equation was utilized to viscosimetry curves. Based on the assymptotics viscosimetry data indicated that increase of doses corresponds to an increase of the yolk and a decrease of white egg viscosimetry as such a Newton behaviour. Irradiation effects on nutritional properties were evaluated by means of egg protein patterns that were assessed by polyacrylamide gel electrophoresis. Lipids were identified by TLC. Based on results the sanitation dose is lower than the limit dose for the decrease of the main eggs properties.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

NASA Astrophysics Data System (ADS)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

Dirt detection on brown eggs by means of color computer vision.

PubMed

Mertens, K; De Ketelaere, B; Kamers, B; Bamelis, F R; Kemps, B J; Verhoelst, E M; De Baerdemaeker, J G; Decuypere, E M

2005-10-01

In the last 20 yr, different methods for detecting defects in eggs were developed. Until now, no satisfying technique existed to sort and quantify dirt on eggshells. The work presented here focuses on the design of an off-line computer vision system to differentiate and quantify the presence of different dirt stains on brown eggs: dark (feces), white (uric acid), blood, and yolk stains. A system that provides uniform light exposure around the egg was designed. In this uniform light, pictures of dirty and clean eggs were taken, stored, and analyzed. The classification was based on a few standard logical operators, allowing for a quick implementation in an online set-up. In an experiment, 100 clean and 100 dirty eggs were used to validate the classification algorithm. The designed vision system showed an accuracy of 99% for the detection of dirt stains. Two percent of the clean eggs had a light-colored eggshell and were subsequently mistaken for showing large white stains. The accuracy of differentiation of the different kinds of dirt stains was 91%. Of the eggs with dark stains, 10.81% were mistaken for having bloodstains, and 33.33% of eggs with bloodstains were mistaken for having dark stains. The developed system is possibly a first step toward an on line dirt evaluation technique for brown eggs.

Microwave-Assisted Synthesis of Red-Light Emitting Au Nanoclusters with the Use of Egg White

ERIC Educational Resources Information Center

Tian, Jinghan; Yan, Lei; Sang, Aohua; Yuan, Hongyan; Zheng, Baozhan; Xiao, Dan

2014-01-01

We developed a simple, cost-effective, and eco-friendly method to synthesize gold nanoclusters (AuNCs) with red fluorescence. The experiment was performed using HAuCl[subscript 4], egg white, Na[subscript 2]CO[subscript 3] (known as soda ash or washing soda), and a microwave oven. In our experiment, fluorescent AuNCs were prepared within a…

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

PubMed

Harrison, L C; Honeyman, M C; Trembleau, S; Gregori, S; Gallazzi, F; Augstein, P; Brusic, V; Hammer, J; Adorini, L

1997-03-17

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Current understanding of egg allergy

PubMed Central

Caubet, Jean-Christoph; Wang, Julie

2011-01-01

SYNOPSIS Egg is one of the most important allergens in childhood feeding, and egg allergy can pose quality of life concerns. A clear clinical history and the detection of egg white specific IgE will confirm the diagnosis of IgE-mediated reactions. Non-IgE-mediated symptoms such as in eosinophilic diseases of the gut might also be observed. Egg avoidance and education regarding the treatment of allergic reactions are the cornerstones of management of egg allergy. In this review, we discuss epidemiology, risk factors, diagnosis, treatment and natural history of egg allergy. PMID:21453811

77 FR 47816 - Certain Pasta from Italy: Notice of Initiation of Antidumping Duty Changed Circumstances Review

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-10

... certain non-egg dry pasta in packages of five pounds four ounces or less, whether or not enriched or..., milk, gluten, diastasis, vitamins, coloring and flavorings, and up to two percent egg white. The pasta... refrigerated, frozen, or canned pastas, as well as all forms of egg pasta, with the exception of non-egg dry...

76 FR 71311 - Certain Pasta From Italy: Notice of Partial Rescission of Antidumping Duty Administrative Review

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... certain non-egg dry pasta in packages of five pounds four ounces or less, whether or not enriched or..., milk, gluten, diastasis, vitamins, coloring and flavorings, and up to two percent egg white. The pasta... refrigerated, frozen, or canned pastas, as well as all forms of egg pasta, with the exception of non-egg dry...

Dietary administration of Gynura bicolor (Roxb. Willd.) DC water extract enhances immune response and survival rate against Vibrio alginolyticus and white spot syndrome virus in white shrimp Litopeneaus vannamei.

PubMed

Wu, Chih-Chung; Chang, Yueh-Ping; Wang, Jyh-Jye; Liu, Chun-Hung; Wong, Saou-Lien; Jiang, Chii-Ming; Hsieh, Shu-Ling

2015-01-01

Gynura bicolor (Roxb. & Willd.) DC., a perennial plant belonging to the Asteraceae family, is originated from the tropical area of Asia. The total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity were examined after white shrimp Litopenaeus vannamei had been fed diets containing the water extract of G. bicolor at 0 (control), 0.5, 1.0, and 2.0 g (kg diet)(-1) for 7-28 days. The results indicated that these parameters increased accordingly with the amount of extract and time. THCs of the shrimp fed the G. bicolor diets at 1.0 and 2.0 g (kg diet)(-1) were significantly higher than that fed the control diet for 14-28 days. For the shrimp fed the G. bicolor diets at 0.5, 1.0, and 2.0 g (kg diet)(-1), the PO, RBs, and lysozyme activities reached the highest levels after 7 days, whereas SOD activity reached the highest levels after 14 days. In a separate experiment, white shrimp L. vannamei fed the diets containing the G. bicolor extract for 28 days were challenged with Vibrio alginolyticus at 3 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1). The survival rate of the shrimp fed the G. bicolor diets was significantly higher than that of the shrimp fed the control diet at 48-144 h post challenge V. alginolyticus and WSSV. For the shrimp fed the G. bicolor diets at 0.5, 1 and 2 g (kg diet)(-1) under challenges of V. alginolyticus and WSSV, their LPS- and β-1,3-glucan-binding protein (LGBP) and peroxinectin (PE) mRNA expressions were significantly higher than those of the challenged control shrimp at 12-96 and 24-144 h post-challenge, respectively. We concluded that dietary administration of a G. bicolor extract could enhance the innate immunity within 28 days as evidenced by the increases in immune parameters (PO, RBs, and lysozyme) and antioxidant enzyme (SOD) activities of shrimp to against V. alginolyticus and WSSV infections. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kootenai River White Sturgeon Investigations; White Sturgeon Spawning and Recruitment Evaluation, 1996 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paragamian, Vaughn L.

1997-09-01

Test flows for Kootenai River white sturgeon Acipenser transmontanus spawning, scheduled for June 1996, were postponed until July. However, an estimated 126% snow pack and unusually heavy precipitation created conditions for sturgeon spawning that were similar to those occurring before construction of Libby Dam. Discharge in the Kootenai River at Bonners Ferry rose to nearly 1,204 m{sup 3}/s (42,500 cfs) during May and water temperature ranged from 5.8 C to 8.4 C (42 F to 47 F). Migration of adult white sturgeon into spawning areas occurred in late May during a rising hydrograph. Discharge and water temperature were rising andmore » had reached approximately 1,077 m{sup 3}/s (38,000 cfs) and 8 C (46 F). Discharge at Bonners Ferry peaked at about 1,397 m{sup 3}/s (49,300 cfs) on June 5. A total of 348 eggs (and one egg shell) were collected with 106,787 h of mat effort during the flow events. The first white sturgeon eggs were collected on June 8 and continued through June 30. Staging of eggs and back-calculating to spawning dates indicated there were at least 18 spawning episodes between June 6 and June 25. Discharge on June 6 was 1,196 m{sup 3}/s (42,200 cfs) and decreased steadily to 850 m{sup 3}/s (30,000 cfs) by June 26. Although sturgeon spawned in the same reach of river that they had during 1994 and 1995, the majority of eggs were found significantly (P = 0.0001) farther upstream than 1994 and 1995 and this in turn may be related to elevation of Kootenay Lake.« less

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

PubMed

Yang, Chih-Chiu; Lu, Chung-Lun; Chen, Sherwin; Liao, Wen-Liang; Chen, Shiu-Nan

2015-05-01

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

PubMed

Nishio, Shunsuke; Kohno, Yoshinori; Iwata, Yuki; Arai, Mayumi; Okumura, Hiroki; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2014-11-01

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs. © 2014 by the Society for the Study of Reproduction, Inc.

Vitamin D₃ and 25-Hydroxyvitamin D₃ Content of Retail White Fish and Eggs in Australia.

PubMed

Dunlop, Eleanor; Cunningham, Judy; Sherriff, Jill L; Lucas, Robyn M; Greenfield, Heather; Arcot, Jayashree; Strobel, Norbert; Black, Lucinda J

2017-06-22

Dietary vitamin D may compensate for inadequate sun exposure; however, there have been few investigations into the vitamin D content of Australian foods. We measured vitamin D₃ and 25-hydroxyvitamin D₃ (25(OH)D₃) in four species of white fish (barramundi, basa, hoki and king dory), and chicken eggs (cage and free-range), purchased from five Australian cities. Samples included local, imported and wild-caught fish, and eggs of varying size from producers with a range of hen stocking densities. Raw and cooked samples were analysed using high performance liquid chromatography with photodiode array. Limits of reporting were 0.2 and 0.1 μg/100 g for vitamin D₃ and 25(OH)D₃, respectively. The vitamin D₃ content of cooked white fish ranged from <0.1 to 2.3 μg/100 g, and the 25(OH)D₃ content ranged from 0.3 to 0.7 μg/100 g. The vitamin D₃ content of cooked cage eggs ranged from 0.4 to 0.8 μg/100 g, and the 25(OH)D₃ content ranged from 0.4 to 1.2 μg/100 g. The vitamin D₃ content of cooked free-range eggs ranged from 0.3 to 2.2 μg/100 g, and the 25(OH)D₃ content ranged from 0.5 to 0.8 μg/100 g. If, as has been suggested, 25(OH)D₃ has five times greater bioactivity than vitamin D₃, one cooked serve (100 g) of white fish, and one cooked serve of cage or free-range eggs (120 g) may provide 50% or 100%, respectively, of the current guidelines for the adequate intake of vitamin D (5 µg) for Australians aged 1-50 years.

EndoU is a novel regulator of AICD during peripheral B cell selection

PubMed Central

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.

2014-01-01

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22−/−[B6]) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag–specific B cells in IgTg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. PMID:24344237

EndoU is a novel regulator of AICD during peripheral B cell selection.

PubMed

Poe, Jonathan C; Kountikov, Evgueni I; Lykken, Jacquelyn M; Natarajan, Abirami; Marchuk, Douglas A; Tedder, Thomas F

2014-01-13

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

An LED-based UV-B irradiation system for tiny organisms: System description and demonstration experiment to determine the hatchability of eggs from four Tetranychus spider mite species from Okinawa.

PubMed

Suzuki, Takeshi; Yoshioka, Yoshio; Tsarsitalidou, Olga; Ntalia, Vivi; Ohno, Suguru; Ohyama, Katsumi; Kitashima, Yasuki; Gotoh, Tetsuo; Takeda, Makio; Koveos, Dimitris S

2014-03-01

We developed a computer-based system for controlling the photoperiod and irradiance of UV-B and white light from a 5×5 light-emitting diode (LED) matrix (100×100mm). In this system, the LED matrix was installed in each of four irradiation boxes and controlled by pulse-width modulators so that each box can independently emit UV-B and white light at irradiances of up to 1.5 and 4.0Wm(-2), respectively, or a combination of both light types. We used this system to examine the hatchabilities of the eggs of four Tetranychus spider mite species (T. urticae, T. kanzawai, T. piercei and T. okinawanus) collected from Okinawa Island under UV-B irradiation alone or simultaneous irradiation with white light for 12hd(-1) at 25°C. Although no eggs of any species hatched under the UV-B irradiation, even when the irradiance was as low as 0.02Wm(-2), the hatchabilities increased to >90% under simultaneous irradiation with 4.0Wm(-2) white light. At 0.06Wm(-2) UV-B, T. okinawanus eggs hatched (15% hatchability) under simultaneous irradiation with white light, whereas other species showed hatchabilities <1%. These results suggest that photolyases activated by white light may reduce UV-B-induced DNA damage in spider mite eggs and that the greater UV-B tolerance of T. okinawanus may explain its dominance on plants in seashore environments, which have a higher risk of exposure to reflected UV-B even on the undersurface of leaves. Our system will be useful for further examination of photophysiological responses of tiny organisms because of its ability to precisely control radiation conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Song, Xiaoling; Zhang, Yue; Wei, Song; Huang, Jie

2013-03-01

The effects of different hydrolysis methods on peptidoglycan (PG) were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp, Litop enaeus vannamei. PG derived from Bifidobacterium thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations. A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp. The composition, concentration, and molecular weight ranges of the soluble PG were analyzed. Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6—31 of the experiment. The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus (WSSV). Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h, and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme. SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods. Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes. In addition, the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV, whereas no increased resistance was observed for preparations containing less low-molecular-weight PG. These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.

Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study.

PubMed

Wizert, Alicja; Iskander, D Robert; Cwiklik, Lukasz

2017-12-01

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates. Copyright © 2017 Elsevier B.V. All rights reserved.

Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorption methods by kinetics of thermal inactivation

NASA Astrophysics Data System (ADS)

Puentes-Camacho, Daniel; Velázquez, Enrique F.; Rodríguez-Félix, Dora E.; Castillo-Ortega, Mónica; Sotelo-Mundo, Rogerio R.; del Castillo-Castro, Teresa

2017-12-01

Proteins suffer changes in their tertiary structure when they are immobilized, and enzymatic activity is affected due to the low biocompatibility of some supporting materials. In this work immobilization of lysozyme on carbon nanotubes previously functionalized by microwave irradiation was studied. The effectiveness of the microwave-assisted acid treatment of carbon nanotubes was evaluated by XPS, TEM, Raman and FTIR spectroscopy. The carboxylic modification of nanotube surfaces by this fast, simple and feasible method allowed the physical adsorption and covalent linking of active lysozyme onto the carbonaceous material. Thermal inactivation kinetics, thermodynamic parameters and storage stability were studied for adsorbed and covalent enzyme complexes. A major stability was found for lysozyme immobilized by the covalent method, the activation energy for inactivation of the enzyme was higher for the covalent method and it was stable after 50 d of storage at 4 °C. The current study highlights the effect of protein immobilization method on the biotechnological potential of nanostructured biocatalysts.

Can fatty acid and mineral compositions of sturgeon eggs distinguish between farm-raised versus wild white (Acipenser transmontanus) sturgeon origins in California? Preliminary report.

PubMed

DePeters, Edward J; Puschner, Birgit; Taylor, Scott J; Rodzen, Jeff A

2013-06-10

The objective was to investigate the potential of using fatty acid and mineral compositions of sturgeon eggs to distinguish their source, either farm-raised or wild fish. Trafficking of illegally obtained wild white sturgeon eggs is a major concern to the California Department of Fish and Game, but there is no forensic method to separate wild and farm-raised white sturgeon eggs. The extension of these findings in future work will be to use the fatty acid and mineral compositions as forensic indicators of caviar produced legally from farm raised sturgeon compared with illegal caviar produced from sturgeon poached from the wild. Samples (10) of sturgeon eggs were collected from a commercial aquaculture facility in the Sacramento Valley. Eggs from wild sturgeon (9) were obtained primarily from confiscations of illegally caught sturgeon by fish and game law enforcement personnel. The total lipid content of sturgeon eggs was analyzed for fatty acid composition. The most notable difference was the higher concentration (P<0.001) of C18:2n6 in farm raised eggs (6.5 mg/100g total lipid) than wild eggs (0.6 mg/100g total lipid) while other differences between fatty acids were smaller. Eicosapentaenoic acid (C20:5n3) was higher (P<0.02) in farm-raised (5.56 mg/100g) than wild (4.49 mg/100g). Docosahexaenoic acid (C22:6n3), C18:1 cis 9&10, and C20:4n6 were not different for origin of the eggs. Concentration of selenium was markedly higher (P<0.001) in eggs from wild sturgeon (10.0 mg/kg dry weight) than farm-raised sturgeon (2.7 mg/kg dry weight). Concentrations of iron, zinc, copper, phosphorus, sulfur, calcium, and potassium did not differ between farm-raised and wild eggs. Arsenic concentration in wild eggs was 3.3mg/kg dry weight whereas arsenic was not detected in the farm-raised eggs. Fatty acid and mineral compositions of eggs differed significantly between farm-raised and wild sturgeon and these should be investigated further as biological markers for forensic identification of caviar origin. Copyright © 2013. Published by Elsevier Ireland Ltd.

Effects of yolk contamination, shearing, and heating on foaming properties of fresh egg white.

PubMed

Wang, G; Wang, T

2009-03-01

A series of experiments were conducted to evaluate effects of yolk contamination, shearing, and thermal treatment on foaming properties of liquid egg white. Samples obtained from industrial processing were also evaluated. Whipping and purging methods were both used to assess their effectiveness and sensitivity in evaluating foaming. A concentration as low as 0.022% (as-is basis) of yolk contamination caused significant reductions in foaming capacity and foaming speed. The neutral lipid fraction of egg yolk caused the major detrimental effect on foaming, and phospholipids fraction did not give significant foaming reduction at a concentration as high as 0.1%. High-speed and short-time shearing caused no apparent damage but longer shearing time significantly impaired foaming. Heat-induced foaming change is a function of temperature and holding time. Foaming was significantly reduced at a temperature of 55 degrees C for 10 min, whereas it did not change up to 3 min at a heating temperature of 62 to 64 degrees C. Industrial processing steps (pumping, pipe transfer, and storage) did not produce negative effects on foaming of the final products and the controlled pasteurization was actually beneficial for good foaming performance. Therefore, yolk contamination of the egg white was the major factor in reducing foaming properties of the white protein.

In vitro tests of substitute lubricants for wear testing orthopaedic biomaterials.

PubMed

Scholes, Susan C; Joyce, Thomas J

2013-06-01

Bovine serum is the lubricant recommended by several international standards for the wear testing of orthopaedic biomaterials; however, there are issues over its use due to batch variation, degradation, cost and safety. For these reasons, alternative lubricants were investigated. A 50-station Super-CTPOD (circularly translating pin-on-disc) wear test rig was used, which applied multidirectional motion to ultra-high-molecular-weight polyethylene test pins rubbing against cobalt chromium discs. Thirteen possible alternative lubricants were tested. The use of soy protein as a lubricant gave statistically higher wear, while soya oil, olive oil, Channel Island milk, whole milk, whey, wheatgerm oil, 11 mg/mL egg white, albumin/globulin mix and albumin/globulin/chondroitin sulphate mix all gave statistically lower wear than bovine serum. The lubricants giving the closest wear results to bovine serum were 20 and 40 mg/mL egg white solutions. A light absorbance assay found that these egg white solutions suffered from a high degradation rate that increased with increasing protein content. While egg white solutions offer the best alternative lubricant to bovine serum due to the wear volumes produced, cost-effectiveness and safety of handling, protein degradation will still occur, leading to the need for regular lubricant replacement. Of the lubricants tested in this study, none were found to be superior to bovine serum.

Consumer Shell Egg Consumption and Handling Practices: Results from a National Survey.

PubMed

Kosa, Katherine M; Cates, Sheryl C; Bradley, Samantha; Godwin, Sandria; Chambers, Delores

2015-07-01

Numerous cases and outbreaks of Salmonella infection are attributable to shell eggs each year in the United States. Safe handling and consumption of shell eggs at home can help reduce foodborne illness attributable to shell eggs. A nationally representative Web survey of 1,504 U.S. adult grocery shoppers was conducted to describe consumer handling practices and consumption of shell eggs at home. Based on self-reported survey data, most respondents purchase shell eggs from a grocery store (89.5%), and these eggs were kept refrigerated (not at room temperature; 98.5%). As recommended, most consumers stored shell eggs in the refrigerator (99%) for no more than 3 to 5 weeks (97.6%). After cracking eggs, 48.1% of respondents washed their hands with soap and water. More than half of respondents who fry and/or poach eggs cooked them so that the whites and/or the yolks were still soft or runny, a potentially unsafe practice. Among respondents who owned a food thermometer (62.0%), only 5.2% used it to check the doneness of baked egg dishes when they prepared such a dish. Consumers generally followed two of the four core "Safe Food Families" food safety messages ("separate" and "chill") when handling shell eggs at home. To prevent Salmonella infection associated with shell eggs, consumers should improve their practices related to the messages "clean" (i.e., wash hands after cracking eggs) and "cook" (i.e., cook until yolks and whites are firm and use a food thermometer to check doneness of baked egg dishes) when preparing shell eggs at home. These findings will be used to inform the development of science-based consumer education materials that can help reduce foodborne illness from Salmonella infection.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

PubMed Central

Osanai-Futahashi, M; Tatematsu, K-i; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

2016-01-01

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects. PMID:26328757

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

PubMed

Osanai-Futahashi, M; Tatematsu, K-I; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

2016-02-01

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

77 FR 69793 - Certain Pasta From Italy; Final Results of Countervailing Duty Administrative Review; 2010

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-21

... the Order Imports covered by the order are shipments of certain non-egg dry pasta in packages of five... flavorings, and up to two percent egg white. The pasta covered by the scope of the order is typically sold in... well as all forms of egg pasta, with the exception of non-egg dry pasta containing up to two percent...

Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability.

PubMed

Goliomytis, M; Orfanou, H; Petrou, E; Charismiadou, M A; Simitzis, P E; Deligeorgis, S G

2014-02-01

1. The purpose of this study was to evaluate the effects of dietary supplementation with hesperidin (one or 3 g/kg of feed) for 31 d on the performance, egg quality and yolk oxidative stability of brown and white laying hens (26-wk old). 2. Supplementation with hesperidin did not affect egg production, egg weight and egg quality traits. 3. No hesperidin effect on yolk and plasma cholesterol was observed. A strain effect was found with lower total and per g yolk cholesterol of brown hens in comparison to the white ones. 4. Oxidative stability of egg yolk, expressed as ng MDA/g yolk, was significantly improved in the hesperidin groups even from the first week of supplementation. At the same time, a significant improvement in the oxidative stability of egg yolk due to the incorporation of hesperidin in hens' diet was observed after 30 and 90 d of storage at 20°C and 4°C, respectively. 5. No hesperidin by strain interaction was detected for any of the traits measured. 6. In conclusion, incorporation of hesperidin to laying hens' feed did not affect productive and egg qualitative traits. On the other hand, dietary hesperidin supplementation significantly improved oxidative stability of both fresh and stored eggs. Antioxidant properties of hesperidin seem to make it a promising natural agent for improving the shelf life of eggs.

Showing Food Foams Properties with Common Dairy Foods

NASA Astrophysics Data System (ADS)

Bravo-Diaz, Carlos; Gonzalez-Romero, Elisa

1997-09-01

Most writers on food either ignore scientific principles that underlie cooking or disparage the value of such information on the grounds that can not be easily reduced to the test tube. However, people who have not yet logged years preparing food might require some explanation about what is going on or just simple may be curious about what foods are and how cooking works. In this work we show some easy to carry, inexpensive and safe experiments developed using familiar kitchen materials related with egg foams. Eggs's properties are not only limited to prepare excellent and delicious emulsions like mayonnaise but also makes excellent foams, increasing their volume significantly in two primary ways: the first one may be attributed to the albumen (a major component of egg white) because it is a thick viscous solution and it drains more slowly out of bubble walls than does a thin liquid and the second one is because egg white introduces a kind of reinforcement into the bubble walls. As the egg white is beaten and air bubbles are incorporated into it, the proteins in the bubble wall are subjected to an imbalance of forces due to the air-liquid interface which makes them to unfold and bond each other forming a delicate but definitely reinforcing network.

Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure.

PubMed

Tanley, Simon W M; Schreurs, Antoine M M; Kroon-Batenburg, Loes M J; Helliwell, John R

2012-11-01

The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδ and the Nℇ atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin and N-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδ and Nℇ atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the Nℇ binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check with Fo-Fc OMIT electron-density maps. The occupancies of each binding site were calculated using SHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.

αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin

PubMed Central

2005-01-01

αB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of αB-crystallin on the fibril growth of the Aβ (amyloid β)-peptides Aβ-(1–40) and Aβ-(1–42). αB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Aβ-(1–40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric α-crystallins in preventing Aβ-(1–40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on α-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Aβ-(1–40). αB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Aβ-(1–42), as well as the fibril growth of Aβ-(1–40) when seeded with the Aβ-(1–42) fibril seed. Sedimentation velocity measurements show that αB-crystallin does not form a stable complex with Aβ-(1–40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. αB-crystallin binds to the amyloid fibrils of Aβ-(1–40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that αB-crystallin prevents the fibril growth of β2-microglobulin under acidic conditions. It also retards the depolymerization of β2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease. PMID:16053447

77 FR 471 - Emergency Food Assistance Program; Availability of Foods for Fiscal Year 2012

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-05

..., poultry, fish, vegetables, dry beans, juices, and fruits. Approximately $37.5 million in surplus foods..., frozen ham, frozen turkey roast, blackeye beans, garbanzo beans, great northern beans, light red kidney beans, lentils, lima beans, pinto beans, egg mix, shell eggs, lowfat bakery mix, egg noodles, white and...

75 FR 78674 - Emergency Food Assistance Program; Availability of Foods for Fiscal Year 2011

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-16

... meat, poultry, fish, vegetables, dry beans, juices, and fruits. Approximately $155.9 million in surplus... turkey roast, blackeye beans, garbanzo beans, great northern beans, light red kidney beans, lentils, lima beans, pinto beans, egg mix, shell eggs, lowfat bakery mix, egg noodles, white and yellow corn grits...

Theoretical and functional complexity of white variegation of unripe fleshy fruits

PubMed Central

Lev-Yadun, Simcha

2013-01-01

In many plant species, the bright colors of ripe fruit serve to attract frugivores to enable efficient seed dispersal. Here I show that the fleshy fruit of several dozens of species originating from Asia (southeastern, eastern and central), the Middle East, Africa, America (South, Central and North), Australia, Polynesia and Micronesia, with fruit usually larger than 1 cm, have white or light green spots while they are still unripe. In many of these species, while the spots are conspicuous, the unripe fruit is known to be poisonous, bitter or sour. I propose that this fruit syndrome may signal frugivores that the fruit is still unripe. Similarly to the succulent leaves of window-plants, these spots form windows that enable light to penetrate deeper into the photosynthetic layers in the developing fruit. This seems to be a solution to overcome the limitations of light harvest because of the high volume to surface ratio of developing fleshy fruits. The white or whitish variegation in these unripe fleshy fruits may serve at least five functions: 1) Windows for photosynthesis, 2) camouflage, 3) signaling to frugivores that they are not ripe (possibly sometimes a type of mutualism with frugivores), 4) signaling to frugivores that they are poisonous - aposematism, and 5) mimicking insect eggs to reduce egg laying. All these functions may be partly or fully simultaneous. Because these white spots appear in plants of diverse geographical and taxonomic origin, it is probably an old adaptation, and such a syndrome has appeared and been selected for many times. PMID:23921545

A high precision method for length-based separation of carbon nanotubes using bio-conjugation, SDS-PAGE and silver staining.

PubMed

Borzooeian, Zahra; Taslim, Mohammad E; Ghasemi, Omid; Rezvani, Saina; Borzooeian, Giti; Nourbakhsh, Amirhasan

2018-01-01

Parametric separation of carbon nanotubes, especially based on their length is a challenge for a number of nano-tech researchers. We demonstrate a method to combine bio-conjugation, SDS-PAGE, and silver staining in order to separate carbon nanotubes on the basis of length. Egg-white lysozyme, conjugated covalently onto the single-walled carbon nanotubes surfaces using carbodiimide method. The proposed conjugation of a biomolecule onto the carbon nanotubes surfaces is a novel idea and a significant step forward for creating an indicator for length-based carbon nanotubes separation. The conjugation step was followed by SDS-PAGE and the nanotube fragments were precisely visualized using silver staining. This high precision, inexpensive, rapid and simple separation method obviates the need for centrifugation, additional chemical analyses, and expensive spectroscopic techniques such as Raman spectroscopy to visualize carbon nanotube bands. In this method, we measured the length of nanotubes using different image analysis techniques which is based on a simplified hydrodynamic model. The method has high precision and resolution and is effective in separating the nanotubes by length which would be a valuable quality control tool for the manufacture of carbon nanotubes of specific lengths in bulk quantities. To this end, we were also able to measure the carbon nanotubes of different length, produced from different sonication time intervals.

Multi-Conformation Monte Carlo: A Method for Introducing Flexibility in Efficient Simulations of Many-Protein Systems.

PubMed

Prytkova, Vera; Heyden, Matthias; Khago, Domarin; Freites, J Alfredo; Butts, Carter T; Martin, Rachel W; Tobias, Douglas J

2016-08-25

We present a novel multi-conformation Monte Carlo simulation method that enables the modeling of protein-protein interactions and aggregation in crowded protein solutions. This approach is relevant to a molecular-scale description of realistic biological environments, including the cytoplasm and the extracellular matrix, which are characterized by high concentrations of biomolecular solutes (e.g., 300-400 mg/mL for proteins and nucleic acids in the cytoplasm of Escherichia coli). Simulation of such environments necessitates the inclusion of a large number of protein molecules. Therefore, computationally inexpensive methods, such as rigid-body Brownian dynamics (BD) or Monte Carlo simulations, can be particularly useful. However, as we demonstrate herein, the rigid-body representation typically employed in simulations of many-protein systems gives rise to certain artifacts in protein-protein interactions. Our approach allows us to incorporate molecular flexibility in Monte Carlo simulations at low computational cost, thereby eliminating ambiguities arising from structure selection in rigid-body simulations. We benchmark and validate the methodology using simulations of hen egg white lysozyme in solution, a well-studied system for which extensive experimental data, including osmotic second virial coefficients, small-angle scattering structure factors, and multiple structures determined by X-ray and neutron crystallography and solution NMR, as well as rigid-body BD simulation results, are available for comparison.

Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

DOE PAGES

Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; ...

2015-03-27

X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat formore » conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.« less

Pure-phase selective excitation in fast-relaxing systems.

PubMed

Zangger, K; Oberer, M; Sterk, H

2001-09-01

Selective pulses have been used frequently for small molecules. However, their application to proteins and other macromolecules has been limited. The long duration of shaped-selective pulses and the short T(2) relaxation times in proteins often prohibited the use of highly selective pulses especially on larger biomolecules. A very selective excitation can be obtained within a short time by using the selective excitation sequence presented in this paper. Instead of using a shaped low-intensity radiofrequency pulse, a cluster of hard 90 degrees pulses, delays of free precession, and pulsed field gradients can be used to selectively excite a narrow chemical shift range within a relatively short time. Thereby, off-resonance magnetization, which is allowed to evolve freely during the free precession intervals, is destroyed by the gradient pulses. Off-resonance excitation artifacts can be removed by random variation of the interpulse delays. This leads to an excitation profile with selectivity as well as phase and relaxation behavior superior to that of commonly used shaped-selective pulses. Since the evolution of scalar coupling is inherently suppressed during the double-selective excitation of two different scalar-coupled nuclei, the presented pulse cluster is especially suited for simultaneous highly selective excitation of N-H and C-H fragments. Experimental examples are demonstrated on hen egg white lysozyme (14 kD) and the bacterial antidote ParD (19 kD). Copyright 2001 Academic Press.

Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

NASA Astrophysics Data System (ADS)

Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

2013-03-01

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

Salts employed in hydrophobic interaction chromatography can change protein structure - insights from protein-ligand interaction thermodynamics, circular dichroism spectroscopy and small angle X-ray scattering.

PubMed

Komaromy, Andras Z; Kulsing, Chadin; Boysen, Reinhard I; Hearn, Milton T W

2015-03-01

Key requirements of protein purification by hydrophobic interaction chromatography (HIC) are preservation of the tertiary/quaternary structure, maintenance of biological function, and separation of the correctly folded protein from its unfolded forms or aggregates. This study examines the relationship between the HIC retention behavior of hen egg white lysozyme (HEWL) in high concentrations of several kosmotropic salts and its conformation, assessed by circular dichroism (CD) spectroscopy. Further, the physicochemical properties of HEWL in the presence of high concentrations of ammonium sulfate, sodium chloride and magnesium chloride were investigated by small angle X-ray scattering (SAXS) at different temperatures. Radii of gyration were extrapolated from Guinier approximations and the indirect transform program GNOM with protein-protein interaction and contrast variation taken into account. A bead model simulation provided information on protein structural changes using ab initio reconstruction with GASBOR. These results correlated to the secondary structure content obtained from CD spectroscopy of HEWL. These changes in SAXS and CD data were consistent with heat capacity ΔCp -values obtained from van't Hoff plot analyses of the retention data. Collectively, these insights enable informed decisions to be made on the choice of chromatographic conditions, leading to improved separation selectivity and opportunities for innovative column-assisted protein refolding methods. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine

X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat formore » conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.« less

Multicomponent T2 relaxation studies of the avian egg.

PubMed

Mitsouras, Dimitris; Mulkern, Robert V; Maier, Stephan E

2016-05-01

To investigate the tissue-like multiexponential T2 signal decays in avian eggs. Transverse relaxation studies of raw, soft-boiled and hard-boiled eggs were performed at 3 Tesla using a three-dimensional Carr-Purcell-Meiboom-Gill imaging sequence. Signal decays over a TE range of 11 to 354 ms were fitted assuming single- and multicomponent signal decays with up to three separately decaying components. Fat saturation was used to facilitate spectral assignment of observed decay components. Egg white, yolk and the centrally located latebra all demonstrate nonmonoexponential T2 decays. Specifically, egg white exhibits two-component decays with intermediate and long T2 times. Meanwhile, yolk and latebra are generally best characterized with triexponential decays, with short, intermediate and very long T2 decay times. Fat saturation revealed that the intermediate component of yolk could be attributed to lipids. Cooking of the egg profoundly altered the decay curves. Avian egg T2 decay curves cover a wide range of decay times. Observed T2 components in yolk and latebra as short as 10 ms, may prove valuable for testing clinical sequences designed to measure short T2 components, such as myelin-associated water in the brain. Thus we propose that the egg can be a versatile and widely available MR transverse relaxation phantom. © 2015 Wiley Periodicals, Inc.

Coevolution in action: disruptive selection on egg colour in an avian brood parasite and its host.

PubMed

Yang, Canchao; Liang, Wei; Cai, Yan; Shi, Suhua; Takasu, Fugo; Møller, Anders P; Antonov, Anton; Fossøy, Frode; Moksnes, Arne; Røskaft, Eivin; Stokke, Bård G

2010-05-26

Trait polymorphism can evolve as a consequence of frequency-dependent selection. Coevolutionary interactions between hosts and parasites may lead to selection on both to evolve extreme phenotypes deviating from the norm, through disruptive selection. Here, we show through detailed field studies and experimental procedures that the ashy-throated parrotbill (Paradoxornis alphonsianus) and its avian brood parasite, the common cuckoo (Cuculus canorus), have both evolved egg polymorphism manifested in discrete immaculate white, pale blue, and blue egg phenotypes within a single population. In this host-parasite system the most common egg colours were white and blue, with no significant difference in parasitism rates between hosts laying eggs of either colour. Furthermore, selection on parasites for countering the evolution of host egg types appears to be strong, since ashy-throated parrotbills have evolved rejection abilities for even partially mimetic eggs. The parrotbill-cuckoo system constitutes a clear outcome of disruptive selection on both host and parasite egg phenotypes driven by coevolution, due to the cost of parasitism in the host and by host defences in the parasite. The present study is to our knowledge the first to report the influence of disruptive selection on evolution of discrete phenotypes in both parasite and host traits in an avian brood parasitism system.

Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

PubMed Central

Kim, Dohyang; Nam, Yu Hwa; Cui, Xiang-Shun; Kim, Nam-Hyung

2018-01-01

The transgenic chicken has been considered as a prospective bioreactor for large-scale production of costly pharmaceutical proteins. In the present study, we report successful generation of transgenic hens that lay eggs containing a high concentration of human erythropoietin (hEPO) in the ovalbumin. Using a feline immunodeficiency virus (FIV)-based pseudotyped lentivirus vector enveloped with G glycoproteins of the vesicular stomatitis virus, the replication-defective vector virus carrying the hEPO gene under the control of the chicken ovalbumin promoter was microinjected to the subgerminal cavity of freshly laid chicken eggs (stage X). Stable germline transmission of the hEPO transgene to the G1 progeny, which were non-mosaic and hemizygous for the hEPO gene under the ovalbumin promoter, was confirmed by mating of a G0 rooster with non-transgenic hens. Quantitative analysis of hEPO in the egg whites and in the blood samples taken from G1 transgenic chickens showed 4,810 ~ 6,600 IU/ml (40.1 ~ 55.0 μg/ml) and almost no detectable concentration, respectively, indicating tightly regulated oviduct-specific expression of the hEPO transgene. In terms of biological activity, there was no difference between the recombinant hEPO contained in the transgenic egg white and the commercially available counterpart, in vitro. We suggest that these results imply an important step toward efficient production of human cytokines from a transgenic animal bioreactor. PMID:29847554

Kootenai River White Sturgeon Investigations : White Sturgeon Spawning and Recruitment Evaluation, 1998 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paragamian, Vaughn L.; Kruse, Gretchen L.; Wakkinen, Virginia

2001-03-01

Flows in the Kootenai River for white sturgeon Acipenser transmontanus spawning in 1998 were expected to be at a minimum because the snow pack in the basin was only about 79% normal, and local inflow was expected to be very low, <142 m{sup 3}/s (5,000 cfs). Flows in the Kootenai River at Bonners Ferry from late April through early May were at about 425 m{sup 3}/s (15,000 cfs) while water temperature ranged from about 8 to 10 C (45 to 50 F). Spawning and incubation flows from Libby Dam began on May 18 when flow at the dam was broughtmore » up to 765 m{sup 3}/s (27,000 cfs). Unusually frequent rains and several enormous storms brought peak flows at Bonners Ferry to over 1,175 m{sup 3}/s (41,500 cfs) on May 27, temperature ranged between 8 and 10.6 C (45 to 51 F). Flow gradually subsided at Bonners Ferry during June and was steady at 708 to 765 m{sup 3}/s (25,000 to 27,000 cfs) while temperature gradually rose to 14.4 C (58 F). Forty-seven adult white sturgeon were captured with 4,220 hours of angling and setlining effort between March 1 and April 15, 1998 by the Idaho Department of Fish and Game (IDFG). Sonic and radio tags were attached to four female and five male sturgeon during this effort. From April 1 through July 31, 1998, a total of 17 fish were monitored specifically for pre-spawn and spawning activities. White sturgeon spawning location, timing, frequency, and habitat were evaluated by sampling for eggs with artificial substrate mats. Four hundred and eighty-four eggs were collected, 393 eggs (81%) were collected on 60 standard mats, and 91 eggs (19%) were collected on seven experimental mats with drift nets. Ten eggs collected with experimental mats were found mixed with sand, suggesting eggs are moving in the lower water column with sand. The middle Shorty's Island reach (rkm 229.6-231.5) produced the most eggs (173) while the Deep Creek section (rkm 237.6-240.5) produced 112 eggs. No eggs were collected above the Deep Creek section (>rkm 240.5). Four hundred and twenty (87%) of the 484 white sturgeon eggs collected in 1998 were viable. Development ranged from stage 12 to 28 (1 h to 12 d old), with 95% of the viable eggs at stage 21 (about 2.4 days) or earlier. The oldest egg was estimated at 293 hours old or about 12 days. Based on ages of viable eggs and the dates of egg collection, we estimated that white sturgeon spawned during at least 20 days in 1998. The first spawning episode was estimated to have occurred on May 6. The next episode was estimated to have occurred May 7 with a gap in spawning until May 10. Thereafter, spawning occurred for the next three days with a second break. From May 22, spawning occurred nearly every day through June 6. Peak spawning appeared to occur between from May 23 through May 28. Juvenile sampling yielded 163 individual fish (several fish were recaptured) of which 160 were hatchery and three wild sturgeon recruited from flow test years. Food habit studies of hatchery age-3 sturgeon indicated Chironomids comprised about 36% of the total food items by number while the Ephemeropterans Ephemerellidae and Baetidae contributed 22%. Continuous seismic profiling of a 12-km (7.45 mi) reach of the Kootenai River (rkm 228-240) indicated the riverbed was comprised primarily of fine and coarse sand. There was no evidence to suggest pre-dam gravels were overlain with sand. Recommendations for the 1999 spawning season include coordinating the flow test with sturgeon behavior and river temperatures of 8-10 C (46-50 F), and discharge should be in increments of 57 m{sup 3}/s (2,000 cfs) per day to a minimum of 1,130 m{sup 3}/s (40,000 cfs) at Bonners Ferry. We also recommend no load following.« less

Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt-water systems.

PubMed

Wijaya, Emmy C; Separovic, Frances; Drummond, Calum J; Greaves, Tamar L

2016-09-21

Improving protein stabilisation is important for the further development of many applications in the pharmaceutical, specialty chemical, consumer product and agricultural sectors. However, protein stabilization is highly dependent on the solvent environment and, hence, it is very complex to tailor protein-solvent combinations for stable protein maintenance. Understanding solvent features that govern protein stabilization will enable selection or design of suitable media with favourable solution environments to retain protein native conformation. In this work the structural conformation and activity of lysozyme in 29 solvent systems were investigated to determine the role of various solvent features on the stability of the enzyme. The solvent systems consisted of 19 low molecular weight polar solvents and 4 protic ionic liquids (PILs), both at different water content levels, and 6 aqueous salt solutions. Small angle X-ray scattering, Fourier transform infrared spectroscopy and UV-vis spectroscopy were used to investigate the tertiary and secondary structure of lysozyme along with the corresponding activity in various solvation systems. At low non-aqueous solvent concentrations (high water content), the presence of solvents and salts generally maintained lysozyme in its native structure and enhanced its activity. Due to the presence of a net surface charge on lysozyme, electrostatic interactions in PIL-water systems and salt solutions enhanced lysozyme activity more than the specific hydrogen-bond interactions present in non-ionic molecular solvents. At higher solvent concentrations (lower water content), solvents with a propensity to exhibit the solvophobic effect, analogous to the hydrophobic effect in water, retained lysozyme native conformation and activity. This solvophobic effect was observed particularly for solvents which contained hydroxyl moieties. Preferential solvophobic effects along with bulky chemical structures were postulated to result in less competition with water at the specific hydration layer around the protein, thus reducing protein-solvent interactions and retaining lysozyme's native conformation. The structure-property links established in this study are considered to be applicable to other proteins.

Wintering area DDE source to migratory white-faced ibis revealed by satellite telemetry and prey sampling.

PubMed

Yates, Michael A; Fuller, Mark R; Henny, Charles J; Seegar, William S; Garcia, Jaqueline

2010-01-01

Locations of contaminant exposure for nesting migratory species are difficult to fully understand because of possible additional sources encountered during migration or on the wintering grounds. A portion of the migratory white-faced ibis (Plegadis chihi) nesting at Carson Lake, Nevada continues to be exposed to dichloro-diphenyldichloro-ethylene (DDE) with no change, which is unusual, observed in egg concentrations between 1985 and 2000. About 45-63% of the earliest nesting segment shows reduced reproductive success correlated with elevated egg concentrations of >4 microg/g wet weight (ww). Local prey (primarily earthworms) near nests contained little DDE so we tracked the migration and wintering movements of 20 adult males during 2000-2004 to determine the possible source. At various wintering sites, we found a correlation (r (2) = 0.518, P = 0.0125, N = 11) between DDE in earthworm composites and DDE in blood plasma of white-faced ibis wintering there, although the plasma was collected on their breeding grounds soon after arrival. The main source of DDE was wintering areas in the Mexicali Valley of Baja California Norte, Mexico, and probably the adjacent Imperial Valley, California, USA. This unusual continuing DDE problem for white-faced ibis is associated with: the long-term persistence in soil of DDE; the earthworms' ability to bioconcentrate DDE from soil; the proclivity of white-faced ibis to feed on earthworms in agricultural fields; the species's extreme sensitivity to DDE in their eggs; and perhaps its life history strategy of being a "capital breeder". We suggest surveying and sampling white-faced ibis eggs at nesting colonies, especially at Carson Lake, to monitor the continuing influence of DDE.

Wintering area DDE source to migratory white-faced ibis revealed by satellite telemetry and prey sampling

USGS Publications Warehouse

Yates, M.A.; Fuller, M.R.; Henny, C.J.; Seegar, W.S.; Garcia, Jorge H.

2010-01-01

Locations of contaminant exposure for nesting migratory species are difficult to fully understand because of possible additional sources encountered during migration or on the wintering grounds. A portion of the migratory white-faced ibis (Plegadis chihi) nesting at Carson Lake, Nevada continues to be exposed to dichloro-diphenyldichloro-ethylene (DDE) with no change, which is unusual, observed in egg concentrations between 1985 and 2000. About 45-63% of the earliest nesting segment shows reduced reproductive success correlated with elevated egg concentrations of >4 ??g/g wet weight (ww). Local prey (primarily earthworms) near nests contained little DDE so we tracked the migration and wintering movements of 20 adult males during 2000-2004 to determine the possible source. At various wintering sites, we found a correlation (r 2 = 0.518, P = 0.0125, N = 11) between DDE in earthworm composites and DDE in blood plasma of white-faced ibis wintering there, although the plasma was collected on their breeding grounds soon after arrival. The main source of DDE was wintering areas in the Mexicali Valley of Baja California Norte, Mexico, and probably the adjacent Imperial Valley, California, USA. This unusual continuing DDE problem for white-faced ibis is associated with: the long-term persistence in soil of DDE; the earthworms' ability to bioconcentrate DDE from soil; the proclivity of white-faced ibis to feed on earthworms in agricultural fields; the species's extreme sensitivity to DDE in their eggs; and perhaps its life history strategy of being a "capital breeder". We suggest surveying and sampling white-faced ibis eggs at nesting colonies, especially at Carson Lake, to monitor the continuing influence of DDE. ?? 2009 Springer Science+Business Media, LLC.

Interactions between canola meal and flaxseed oil in the diets of White Lohmann hens on fatty acid profile and sensory characteristics of table eggs.

PubMed

Goldberg, Erin M; Ryland, Donna; Aliani, Michel; House, James D

2016-08-01

The current study was designed to assess the fatty acid composition and sensory attributes of eggs procured from hens consuming diets containing canola meal (CM) and/or flax oil (FO). A total of 96 group-caged White Lohmann hens received 1 of 4 isonitrogenous and isoenergetic diets for a period of 4 weeks. Diets were arranged in a 2 × 2 factorial design, containing 24% canola meal, 7.5% flax oil, both, or neither (control). All yolk fatty acids were affected by flax oil inclusion, with the exception of stearic acid (SA) and docosapentaenoic acid (DPA). Only SA was affected by CM inclusion. Additionally, significant interactions between CM and FO were observed for linoleic acid (LA) and total omega-6 polyunsaturated fatty acids (PUFA), with DPA approaching significance (P = 0.069). Trained panelists (n = 8) evaluated 7 aroma ('egg', 'creamy', 'buttery', 'salty', 'sweet', 'barny', and 'oceanic') and 6 flavor ('egg', 'creamy', 'buttery', 'salty', 'brothy', and 'oceanic') attributes of cooked egg product. No significant differences (P > 0.05) in aroma attributes were found between eggs from different dietary treatments. However, egg, creamy, buttery, and oceanic flavors were significantly different between the dietary treatments (P < 0.05). While oceanic flavor significantly increased with inclusion of FO, egg and creamy flavors showed a significant decrease (P < 0.05). Although CM addition alone did not result in significant sensory changes, the pairing of CM and FO resulted in even greater sensory changes than using FO alone, specifically with regard to egg flavor. Results from partial least squares analyses showed a strong association between oceanic flavor and omega-3 PUFA. Oppositely, egg, creamy, and buttery flavors were more correlated with the presence of omega-6 PUFA and palmitic acid. This experiment provides evidence that the interaction between CM and FO in the White Lohmann hen diet results in sensory changes of cooked eggs associated in part with changes in yolk fatty acid content. © 2016 Poultry Science Association Inc.

Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum

NASA Astrophysics Data System (ADS)

Sanders, Lori; Slimmer, Scott; Angelini, Thomas; Wong, Gerard C. L.

2003-03-01

Cystic fibrosis sputum is a complex fluid consisting of mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems containing actin, lysozyme, and KCl were created to simulate CF sputum in vitro. These model systems were studied using small angle x-ray scattering and confocal fluorescence microscopy. Preliminary results will be presented. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

Effects of inbreeding and temperature stress on life history and immune function in a butterfly.

PubMed

Franke, K; Fischer, K

2013-03-01

Theory predicts that inbreeding depression should be more pronounced under environmental stress due to an increase in the expression of recessive deleterious alleles. If so, inbred populations may be especially vulnerable to environmental change. Against this background, we here investigate effects of inbreeding, temperature stress and its interactions with inbreeding in the tropical butterfly Bicyclus anynana. We use a full-factorial design with three levels of inbreeding (F = 0/0.25/0.38) and three temperature treatments (2 h exposure to 1, 27 or 39 °C). Despite using relatively low levels of inbreeding significant inbreeding depression was found in pupal mass, pupal time, thorax mass, abdomen fat content, egg hatching success and fecundity. However, stress resistance traits (heat tolerance, immune function) were not affected by inbreeding and interactions with temperature treatments were virtually absent. We thus found no support for an increased sensitivity of inbred individuals to environmental stress, and suspect that such patterns are restricted to harsher conditions. Our temperature treatments evidently imposed stress, significantly reducing longevity, fecundity, egg hatching success and haemocyte numbers, while fat content, protein content and lysozyme activity remained unaffected. Males and females differed in all traits measured except pupal time, protein content and phenoloxidase (PO) activity. Correlation analyses revealed, among others, a trade-off between PO and lysozyme activity, and negative correlations between fat content and several other traits. We stress that more data are needed on the effects of inbreeding, temperature variation and sexual differences on insect immune function before more general conclusions can be drawn. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Erythrocyte Saturation with IgG Is Required for Inducing Antibody-Mediated Immune Suppression and Impacts Both Erythrocyte Clearance and Antigen-Modulation Mechanisms.

PubMed

Cruz-Leal, Yoelys; Marjoram, Danielle; Lazarus, Alan H

2018-02-15

Anti-D prevents hemolytic disease of the fetus and newborn, and this mechanism has been referred to as Ab-mediated immune suppression (AMIS). Anti-D, as well as other polyclonal AMIS-inducing Abs, most often induce both epitope masking and erythrocyte clearance mechanisms. We have previously observed that some Abs that successfully induce AMIS effects could be split into those that mediate epitope masking versus those that induce erythrocyte clearance, allowing the ability to analyze these mechanisms separately. In addition, AMIS-inducing activity has recently been shown to induce Ag modulation (Ag loss from the erythrocyte surface). To assess these mechanisms, we immunized mice with transgenic murine RBCs expressing a single Ag protein comprising a recombinant Ag composed of hen egg lysozyme, OVA sequences comprising aa 251-349, and the human Duffy transmembrane protein (HOD-Ag) with serial doses of polyclonal anti-OVA IgG as the AMIS-inducing Ab. The anti-OVA Ab induced AMIS in the absence of apparent epitope masking. AMIS occurred only when the erythrocytes appeared saturated with IgG. This Ab was capable of inducing HOD-RBC clearance, as well as loss of the OVA epitope at doses of Ab that caused AMIS effects. HOD-RBCs also lost reactivity with Abs specific for the hen egg lysozyme and Duffy portions of the Ag consistent with the initiation of Ag modulation and/or trogocytosis mechanisms. These data support the concept that an AMIS-inducing Ab that does not cause epitope masking can induce AMIS effects in a manner consistent with RBC clearance and/or Ag modulation. Copyright © 2018 by The American Association of Immunologists, Inc.

Kootenai River White Sturgeon Investigations; White Sturgeon Spawning and Recruitment Evaluation, 1999 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paragamian, Vaughn L.; Kruse, Gretchen L.; Wakkinen, Virginia

2001-11-01

Sampling for adult Kootenai River white sturgeon Acipenser transmontanus began in March and continued through April 1999. Forty-six adult sturgeon were captured with 4,091 hours of angling and set-lining effort, while an additional three adult sturgeon were captured during gillnetting for juveniles. Flows for Kootenai River white sturgeon spawning were expected to be high because the snow pack in the basin was estimated at 130% of normal, but runoff came very slowly. Discharge from Libby Dam from mid-March through mid-June was maintained at 113 m{sup 3}/s (4,000 cfs). Flows in the Kootenai River at Bonners Ferry during early April, includingmore » local inflow, were 227-255 m{sup 3}/s (8,000-9,000 cfs) but increased gradually in late April to a peak of 657 m{sup 3}/s (23,200 cfs). Flows subsided in early May to about 340 m{sup 3}/s (12,000 cfs), but rose to 1,031 m{sup 3}/s (36,370 cfs) by Mary 26 because of local runoff, and white sturgeon began spawning. However, flows subsided again to 373 m{sup 3}/s (13,200 cfs) June 11, 1999 and some female white sturgeon with transmitters began leaving the spawning reach. Water temperature ranged from about 8 C to 10 C (45 F to 50 F) during these two weeks. On June 13 (two weeks after sturgeon began spawning), spawning and incubation flows from Libby Dam began. The flow was brought up to 1,136 m{sup 3}/s (40,100 cfs) and temperature rose to about 11 C (52 F). They sampled for 3,387 mat days (one mat day is a single 24 h set) with artificial substrate mats and captured 184 white sturgeon eggs. The Middle Shorty's Island reach (river kilometer [rkm] 229.6-231.5) produced the most eggs (144), with 388 mat days of effort; the Refuge section (rkm 234.8 to 237.5) with 616 mat days of effort produced 23 eggs; and the Lower Shorty's section produced 19 eggs with 548 days of mat effort. No eggs were collected above the Refuge section (> rkm 240.5) with 988 mat days of effort. They do not believe flows for sturgeon spawning in 1999 were very time for adequate spawning. Most spawning is thought to have occurred before the spawning flows, when 66% of the spawning events and 87% of the eggs were collected. Recommendations for the 2000 spawning season are given.« less

The efficacy of two electrodes radiofrequency technique: comparison study using a cadaveric interspinous ligament and temperature measurement using egg white.

PubMed

Lee, Chang-Hyung; Derby, Richard; Choi, Hyun-Seok; Lee, Sang-Heon; Kim, Se Hoon; Kang, Yoon Kyu

2010-01-01

One technique in radiofrequency neurotomies uses 2 electrodes that are simultaneously placed to lie parallel to one another. Comparing lesions on cadaveric interspinous ligament tissue and measuring the temperature change in egg white allows us to accurately measure quantitatively the area of the lesion. Fresh cadaver spinal tissue and egg white tissue were used. A series of samples were prepared with the electrodes placed 1 to 7 mm apart. Using radiofrequency, the needle electrodes were heated in sequential or simultaneous order and the distance of the escaped lesion area and temperature were measured. Samples of cadaver interspinous ligament showed sequential heating of the needles limits the placement of the needle electrodes up to 2 mm apart from each other and up to 4 mm apart when heated simultaneously. The temperature at the escaped lesion area decreased according to the distance for egg white. There was a significant difference in temperature at the escaped lesion area up to 6 mm apart and the temperature was above 50 degrees celsius up to 5 mm in simultaneous lesion and 3 mm in the sequential lesion. The limitations of this study include cadaveric experimentation and use of intraspinous ligament rather than medial branch of the dorsal ramus which is difficult to identify. Heating the 2 electrodes simultaneously appears to coagulate a wider area and potentially produce better results in less time.

Safety and Efficacy of Low-Dose Oral Immunotherapy for Hen's Egg Allergy in Children.

PubMed

Yanagida, Noriyuki; Sato, Sakura; Asaumi, Tomoyuki; Nagakura, Kenichi; Ogura, Kiyotake; Ebisawa, Motohiro

2016-01-01

The minimal dose for oral immunotherapy (OIT) tolerance is unknown. We investigated the efficacy and safety of low-dose OIT with 1/32 of the volume of a whole egg. Thirty-three children (aged ≥5 years) with egg allergies confirmed by oral food challenge against 1/32 of a heated whole egg (194 mg of egg protein) were enrolled. The OIT group ingested a scrambled egg once a day. The volume was gradually increased up to a maximum of 1/32 of a heated whole egg. Egg consumption was completely absent in the control group. There were no significant differences in background between the OIT and control groups. Respectively, 71% (15/21) and 0% (0/12) of the patients in the OIT and control groups exhibited sustained unresponsiveness to 1/32 of a whole egg 2 weeks after stopping OIT after 12 months (p < 0.001); 33% (7/21) and 0% (0/12; p = 0.032), respectively, showed sustained unresponsiveness to 1/2 of a whole egg. Egg white- or ovomucoid-specific IgE levels in the OIT group were significantly lower than at baseline after 12 months. Egg white- or ovomucoid-specific IgG as well as IgG4 levels in the OIT group were significantly higher than baseline levels after 1, 3, 6, and 12 months. Adverse allergic reactions were rare, and most symptoms were mild. Low-dose OIT induced sustained unresponsiveness to 1/32 and 1/2 of a whole egg, with no severe symptoms. To improve food allergies, continuous intake of small amounts of these foods may be as effective as the consumption of larger quantities. © 2017 The Author(s) Published by S. Karger AG, Basel.

The evolution of egg colour and patterning in birds.

PubMed

Kilner, R M

2006-08-01

Avian eggs differ so much in their colour and patterning from species to species that any attempt to account for this diversity might initially seem doomed to failure. Here I present a critical review of the literature which, when combined with the results of some comparative analyses, suggests that just a few selective agents can explain much of the variation in egg appearance. Ancestrally, bird eggs were probably white and immaculate. Ancient diversification in nest location, and hence in the clutch's vulnerability to attack by predators, can explain basic differences between bird families in egg appearance. The ancestral white egg has been retained by species whose nests are safe from attack by predators, while those that have moved to a more vulnerable nest site are now more likely to lay brown eggs, covered in speckles, just as Wallace hypothesized more than a century ago. Even blue eggs might be cryptic in a subset of nests built in vegetation. It is possible that some species have subsequently turned these ancient adaptations to new functions, for example to signal female quality, to protect eggs from damaging solar radiation, or to add structural strength to shells when calcium is in short supply. The threat of predation, together with the use of varying nest sites, appears to have increased the diversity of egg colouring seen among species within families, and among clutches within species. Brood parasites and their hosts have probably secondarily influenced the diversity of egg appearance. Each drives the evolution of the other's egg colour and patterning, as hosts attempt to avoid exploitation by rejecting odd-looking eggs from their nests, and parasites attempt to outwit their hosts by laying eggs that will escape detection. This co-evolutionary arms race has increased variation in egg appearance both within and between species, in parasites and in hosts, sometimes resulting in the evolution of egg colour polymorphisms. It has also reduced variation in egg appearance within host clutches, although the benefit thus gained by hosts is not clear.

Lysozyme pattern formation in evaporating droplets

NASA Astrophysics Data System (ADS)

Gorr, Heather Meloy

Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ring-like structures due to the redistribution of solute during evaporation (the "coffee ring effect"). The forms of the deposited patterns depend on complex interactions between solute(s), solvent, and substrate in a rapidly changing, far from equilibrium system. Solute self-organization during evaporation of colloidal sessile droplets has attracted the attention of researchers over the past few decades due to a variety of technological applications. Recently, pattern formation during evaporation of various biofluids has been studied due to potential applications in medical screening and diagnosis. Due to the complexity of 'real' biological fluids and other multicomponent systems, a comprehensive understanding of pattern formation during droplet evaporation of these fluids is lacking. In this PhD dissertation, the morphology of the patterns remaining after evaporation of droplets of a simplified model biological fluid (aqueous lysozyme solutions + NaCl) are examined by atomic force microscopy (AFM) and optical microscopy. Lysozyme is a globular protein found in high concentration, for example, in human tears and saliva. The drop diameters, D, studied range from the micro- to the macro- scale (1 microm -- 2 mm). In this work, the effect of evaporation conditions, solution chemistry, and heat transfer within the droplet on pattern formation is examined. In micro-scale deposits of aqueous lysozyme solutions (1 microm < D < 50 microm), the protein motion and the resulting dried residue morphology are highly influenced by the decreased evaporation time of the drop. The effect of electrolytes on pattern formation is also investigated by adding varying concentrations NaCl to the lysozyme solutions. Finally, a novel pattern recognition program is described and implemented which classifies deposit images by their solution chemistries. The results presented in this PhD dissertation provide insight into the evaporative behavior and pattern formation in droplets of simplified model biological fluids (aqueous lysozyme + NaCl). The patterns that form depend sensitively on the evaporation conditions, characteristic time and length scales, and the physiochemical properties of the solutions. The patterns are unique, dependent on solution chemistry, and may therefore act as a "fingerprint" in identifying fluid properties.

Effects of white phosphorus on mallard reproduction

USGS Publications Warehouse

Vann, S.I.; Sparling, D.W.; Ottinger, M.A.

2000-01-01

Extensive waterfowl mortality involving thousands of ducks, geese, and swans has occurred annually at Eagle River Flats, Alaska since at least 1982. The primary agent for this mortality has been identified as white phosphorus. Although acute and subacute lethality have been described, sublethal effects are less well known. This study reports on the effects of white phosphorus on reproductive function in the mallard (Anas platyrhynchos) in captivity. Fertility, hatching success, teratogenicity, and egg laying frequency were examined in 70 adult female mallards who received up to 7 daily doses of 0, 0.5, 1.0, and 2.0 mg/kg of white phosphorus. Measurements of fertility and hatchability were reduced by the white phosphorus. Teratogenic effects were observed in embryos from hens dosed at all treatment levels. Egg laying frequency was reduced even at the lowest treatment level; treated hens required a greater number of days to lay a clutch of 12 eggs than control hens. After two doses at 2.0 mg/kg, all females stopped laying completely for a minimum of 10 days and laying frequency was depressed for at least 45 days. Fertility of 10 adult male mallards dosed with 1.0 mg/kg of white phosphorus did not differ from 10 controls, but plasma testosterone levels were significantly (p < 0.05) reduced in the treated males 1 day after dosing ended. These results provide evidence that productivity of free-ranging mallards may be impaired if they are exposed to white phosphorus at typical field levels.

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

USGS Publications Warehouse

Work, Thierry M.; Balazs, George H.; Rameyer, Robert; Chang, S.P.; Berestecky, J.

2000-01-01

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund’s complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

Magnetic Resonance Mediated Radio Frequency Coagulation for Vascular Repair

NASA Astrophysics Data System (ADS)

Zhao, Ming

Purpose. Magnetic Resonance Mediated Radiofrequency Coagulation employs the RF heating effect of MRI scanning to coagulate biomaterials for repair of vascular defects. Coagulation of a protein biomaterial by MR-induced RF heating is a novel means to effect repair of defects such as aneurysms or arteriovenous malformations. Our novel method is to coagulate a thermosetting material (such as egg white, which can be used for investigating heat coagulation behavior and MR relaxation properties) delivered endovascularly by catheter and coagulated by RF-induced heating of an intracatheter resonant wire antenna in the scanner. Methods. Experiments were performed on a Siemens 1.5 T MRI scanner and a Bruker 14T NMR spectrometer. Egg white was brought to equilibrium at seven temperatures (20, 30, 40, 50, 60, 70 and 37 °C) in sequence. Measurement of the water spin-lattice relaxation time Ti, spin-spin relaxation time T2, spin-lattice relaxation time in the rotating frame T1p, or full width at half maximum of the MT spectrum were performed at each temperature. Relaxation parameters of raw egg white and egg white after coagulation at 70 °C were measured in the scanner at 20 °C to determine optimum inversion time, echo time and offset frequency for good image contrast between coagulated and uncoagulated protein. Finally, coagulation of egg white within a glass aneurysm phantom by RF heating in the scanner was performed to demonstrate the MR coagulation methodology and the ability to achieve image contrast between coagulated and uncoagulated biomaterial. Results. Water T2, T1p and MT gave the most definitive indication of the change from uncoagulated at low temperature to fully coagulated at 60 °C, while water T1 showed only the expected gradual increase with temperature, and no response to coagulation. MT weighted imaging is expected to be the optimum method to establish the coagulation condition of the biomaterial.

Removal of Salmonella Enteritidis from commercial unpasteurized liquid egg white using pilot scale cross flow tangential microfiltration.

PubMed

Mukhopadhyay, Sudarsan; Tomasula, Peggy M; Luchansky, John B; Porto-Fett, Anna; Call, Jeffrey E

2010-09-01

Effectiveness of a cross flow microfiltration (MF) process for removal of a cocktail of Salmonella enterica serovar Enteritidis species from commercial unpasteurized liquid egg white (LEW) from a local egg breaking plant, while maintaining its functional properties was evaluated. To facilitate MF, LEW was wedge screened, homogenized and then diluted (1:2 w/w) with distilled water containing 0.5% sodium chloride. Diluted unpasteurized LEW was inoculated with five strains of S. Enteritidis (ATCC 4931, ATCC BAA-708, ATCC 49215, ATCC 49218, and ATCC BAA-1045) to a level of approximately 10(7)CFU/mL of LEW and microfiltered using a ceramic membrane. Process parameters influencing egg white functional properties and pathogen removal efficiency were evaluated. Average permeates flux increased by almost 126% when pH of LEW was adjusted from pH 8 to pH 7 at 25 degrees C. Microbial removal efficiency was at least, on average, 6.8Log(10)CFU/mL (limit of detection < or =0.5Log(10)CFU/mL). Functional property analysis indicated that the MF process did not alter the foaming power of LEW. Published by Elsevier B.V.

Caenorhabditis elegans as a model to study the impact of exposure to light emitting diode (LED) domestic lighting.

PubMed

Abdel-Rahman, Fawzia; Okeremgbo, Bethel; Alhamadah, Fatimah; Jamadar, Sakha; Anthony, Kevin; Saleh, Mahmoud A

2017-04-16

This study aimed to investigate the biological impact of exposure on domestic light emitting diodes (LED) lighting using the free-living nematode Caenorhabditis elegans as a model. Nematodes were separately exposed to white LED light covering the range of 380-750 nm, blue light at 450 nm and black light at 380-420 nm for one life cycle (egg to adult) with dark exposure as the control. Each light range induced stress to the nematode C. elegans such as reducing the number of the hatched eggs and/or delayed the maturation of the hatched eggs to the adult stage. In addition, it lowered or prevented the ability of adults to lay eggs and impaired the locomotion in the exposed worms. The observed type of biological stress was also associated with the production of reactive oxygen species (ROS) as compared to nematodes grown in the dark. It is concluded that the blue light component of white LED light may cause health problems, and further investigation is required to test commercial brands of white LEDs that emit different amounts of blue light.

Vitamin D3 and 25-Hydroxyvitamin D3 Content of Retail White Fish and Eggs in Australia

PubMed Central

Dunlop, Eleanor; Cunningham, Judy; Sherriff, Jill L.; Lucas, Robyn M.; Greenfield, Heather; Arcot, Jayashree; Strobel, Norbert; Black, Lucinda J.

2017-01-01

Dietary vitamin D may compensate for inadequate sun exposure; however, there have been few investigations into the vitamin D content of Australian foods. We measured vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in four species of white fish (barramundi, basa, hoki and king dory), and chicken eggs (cage and free-range), purchased from five Australian cities. Samples included local, imported and wild-caught fish, and eggs of varying size from producers with a range of hen stocking densities. Raw and cooked samples were analysed using high performance liquid chromatography with photodiode array. Limits of reporting were 0.2 and 0.1 μg/100 g for vitamin D3 and 25(OH)D3, respectively. The vitamin D3 content of cooked white fish ranged from <0.1 to 2.3 μg/100 g, and the 25(OH)D3 content ranged from 0.3 to 0.7 μg/100 g. The vitamin D3 content of cooked cage eggs ranged from 0.4 to 0.8 μg/100 g, and the 25(OH)D3 content ranged from 0.4 to 1.2 μg/100 g. The vitamin D3 content of cooked free-range eggs ranged from 0.3 to 2.2 μg/100 g, and the 25(OH)D3 content ranged from 0.5 to 0.8 μg/100 g. If, as has been suggested, 25(OH)D3 has five times greater bioactivity than vitamin D3, one cooked serve (100 g) of white fish, and one cooked serve of cage or free-range eggs (120 g) may provide 50% or 100%, respectively, of the current guidelines for the adequate intake of vitamin D (5 µg) for Australians aged 1–50 years. PMID:28640196

Cheese is a reliable alternative meal for solid-phase gastric emptying study.

PubMed

Drubach, Laura A; Kourmouzi, Vasiliki; Fahey, Frederic H

2010-05-01

We evaluated the labeling stability of several alternative meals that could be used to perform solid-phase gastric emptying study. Cooked egg whites labeled with technetium-99m sulfur colloid served as a control. Packaged instant oatmeal and instant mashed potatoes were prepared by adding hot water. Cheddar cheese was melted. Peanut butter was added to bread. The different meals were mixed with technetium-99m sulfur colloid (2.2-3.7 MBq), chopped into small pieces and placed in a glass tube containing gastric juice. Four samples of each meal were analyzed after 1 and 4 h of agitation with a 3-D rotator (two samples per time point). The meal samples were washed with 2 ml of saline and filtered using a blood transfusion filter. The activity in each sample before and after filtering was assayed in a dose calibrator. The percentage of initial radioactivity remaining with the meal of admixture with gastric juice was measured and the average of the two samples was taken. The percentage of activity bound to the solid phase was 98.2+/-1.9, 95.6+/-1.1, 62.1+/-1.7, 41.8+/-0.6, and 74.5+/-3.8% at 1 h and 98.5+/-1.0, 95.8+/-2.6, 77.2+/-6.8, 55.5+/-3.4 and 40.2+/-22.1 at 4 h for egg whites, cheese, oatmeal, mashed potatoes and peanut butter respectively. For egg whites and cheese, there was no significant difference between the values at 1 and 4 h (P>0.8). Cheddar cheese provides an alternative meal for assessing solid gastric emptying in children comparable to egg whites. Oatmeal and mashed potatoes had low and variable labeling stability and are not recommended. In view of the significant proportion of pediatric patients who refuse to eat scrambled eggs or have allergy to eggs, the availability of other meal choices is essential. The versatility of cheddar cheese, which can be added to macaroni or as a topping on pizza, makes it a useful alternative to labeled eggs.

Study on development of Vipera lebetina snake anti-venom in chicken egg yolk for passive immunization

PubMed Central

Zolfagharian, Hossein; Dounighi, Naser Mohammadpour

2015-01-01

Chicken egg yolk antibodies against Vipera lebetina venom were evaluated for their antivenom potential. White leghorn hens were immunized with detoxified V. lebetina venom (γ-irradiated venom). The detoxified venom (200 μg) was mixed with an equal volume of complete Freund's adjuvant and was injected intramuscularly into the hens. The antibodies showed high activity (1.6 LD50/mL) in egg yolks after 12 d of venom injection. The eggs were collected after 12 days, and the egg yolks were removed and washed with purified water to remove any contamination with egg whites. The purification was performed using a method described by Maya Devi et al., followed by gel filtration (Sephadex G-50). The purity and molecular weight of antivenom antibodies (IgY) were determined using electrophoresis, and the molecular weight was found to be approximately 185 kDa. The potency of IgY was 6 LD50/mL (mice), i.e., 1 mL of IgY could neutralize 43.8 μg of standard V. lebetina venom). Our results showed that chicken egg yolk antibodies were effective in neutralizing the lethality and several pharmacological effects of V. lebetina venom and could be used for developing effective antivenom. PMID:25700656

Multi-Component T2 Relaxation Studies of the Avian Egg

PubMed Central

Mitsouras, Dimitris; Mulkern, Robert V.; Maier, Stephan E.

2015-01-01

Purpose To investigate the tissue-like multiexponential T2 signal decays in avian eggs. Methods Transverse relaxation studies of raw, soft-boiled and hard-boiled eggs were performed at 3 Tesla using a 3D Carr-Purcell-Meiboom-Gill (CPMG) imaging sequence. Signal decays over a TE range of 11 to 354 ms were fitted assuming single- and multi-component signal decays with up to three separately decaying components. Fat saturation was used to facilitate spectral assignment of observed decay components. Results Egg white, yolk and the centrally located latebra all demonstrate nonmonoexponential T2 decays. Specifically, egg white exhibits two-component decays with intermediate and long T2 times. Meanwhile, yolk and latebra are generally best characterized with triexponential decays, with short, intermediate and very long T2 decay times. Fat saturation revealed that the intermediate component of yolk could be attributed to lipids. Cooking of the egg profoundly altered the decay curves. Conclusion Avian egg T2 decay curves cover a wide range of decay times. Observed T2 components in yolk and latebra as short as 10 ms, may prove valuable for testing clinical sequences designed to measure short T2 components, such as myelin-associated water in the brain. Thus we propose that the egg can be a versatile and widely available MR transverse relaxation phantom. PMID:26037128

Effects of temperature on the development and survival of eggs of four coastal California fishes

USGS Publications Warehouse

Gadomski, D.M.; Caddell, S.M.

1996-01-01

Laboratory experiments were conducted to determine the effects of temperature on egg development and survival of four fish species found off southern California. Our objectives were to further understanding ofnatural spawning patterns and to aid in identifying and ageing field-collected specimens. An egg-staging procedure was devised and eggs were observed every two hours during development at a range of temperatures (8-28°Cl. Barred sand bass, Parolabrax nebulifer, eggs survived to hatching and produced viable embryos at the highest temperature range (l6-28°C), reflecting this species'summerspawning season. Fantail sole, Xystreurys liolepis, eggs also hatched at a higher temperature range (l6-24°C). This species spawns primarily from summer through fall. Eggs of the two species with winter-spring spawning peaks, white croaker, Genyonemuslineatus, and California halibut, Paralichthys californicus, had lower temperature-tolerance ranges (12- 2O"C). Developmental rate at a specific temperature did not significantly differ between species, whereas within tolerance limits, temperature strongly affected rate of development for all species. Time to hatching for all species was inversely related to temperature, and the relationship was approximately exponential. Species-specific differences in egg-stage sequence were observed; embryonic organogenesisin relation to germ-ring migration and blastopore closure was faster for barred sand bass and fantail sole than for white croaker and California halibut.

Albumin-stabilized fluorescent silver nanodots

NASA Astrophysics Data System (ADS)

Sych, Tomash; Polyanichko, Alexander; Kononov, Alexei

2017-07-01

Ligand-stabilized Ag nanoclusters (NCs) possess many attractive features including high fluorescence quantum yield, large absorption cross-section, good photostability, large Stokes shift and two-photon absorption cross sections. While plenty of fluorescent clusters have been synthesized on various polymer templates, only a few studies have been reported on the fluorescent Ag clusters on peptides and proteins. We study silver NCs synthesized on different protein matrices, including bovine serum albumin, human serum albumin, egg albumin, equine serum albumin, and lysozyme. Our results show that red-emitting Ag NCs can effectively be stabilized by the disulfide bonds in proteins and that the looser structure of the denatured protein favors formation of the clusters.