Sample records for white lysozyme fibrillation

  1. Effects of Flow on Hen Egg White Lysozyme (HEWL) Fibril Formation: Lenght Distribution, Flexibility, and Kinetics

    NARCIS (Netherlands)

    Humblet-Hua, K.N.P.; Sagis, L.M.C.; Linden, van der E.


    The effect of steady shear and turbulent flow on the formation of amyloid fibrils from hen egg white lysozyme (HEWL) was studied. The conversion and size distribution of fibrils obtained by heating HEWL solutions at pH 2 were determined. The formation of fibrils was quantified using flow-induced

  2. Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils. (United States)

    Sasahara, Kenji; Yagi, Hisashi; Naiki, Hironobu; Goto, Yuji


    Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.

  3. Effect of nitrogen-doped graphene quantum dots on the fibrillation of hen egg-white lysozyme. (United States)

    Zeng, Hua-Jin; Miao, Min; Liu, Zhe; Yang, Ran; Qu, Ling-Bo


    In this study, the fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of different amount of nitrogen-doped graphene quantum dots (N-GQDs) was studied by Thioflavin T (ThT) spectroscopy, Congo red (CR) binding assays, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assay, circular dichroism (CD) and transmission electron microscopy (TEM). The experimental results indicated that not only the fibrillation of HEWL at high temperature (65°C) and low pH (pH=2.0) could be inhibited effectively by N-GQDs, but the inhibition of HEWL by N-GQDs followed a dose-dependent manner. The results of this work suggested that the N-GQDs had a great potential for designing new therapeutic agents and were promising for future treatment of amyloid-related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Interaction of magnetic nanoparticles with lysozyme amyloid fibrils (United States)

    Gdovinová, Veronika; Tomašovičová, Natália; Batko, Ivan; Batková, Marianna; Balejčíková, Lucia; Garamus, Vasyl M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Kopčanský, Peter


    This work is devoted to the structural study of complex solutions of magnetic nanoparticles with lysozyme amyloid fibrils due to possible ordering of such system by applying the external magnetic field. The interaction of magnetic nanoparticles with amyloid fibrils has been followed by atomic force microscopy and small-angle X-ray scattering. It has been observed that magnetic nanoparticles (MNPs) adsorb to lysozyme amyloid fibrils. It was found that MNPs alter amyloids structures, namely the diameter of lysozyme amyloid fibrils is increased whereas the length of fibrils is decreased. In the same time MNPs do not change the helical pitch significantly.

  5. Myricetin Prevents Fibrillogenesis of Hen Egg White Lysozyme


    He, Jianwei; Wang, Yu; Chang, Alan K.; Xu, Linan; Wang, Na; Chong, Xiaoying; Li, Hui; Zhang, Bing; Jones, Gary W.; Song, Youtao


    Myricetin is a natural flavonol found in many grapes, berries, fruits, vegetables, and herbs as well as other plants. Recent studies have identified potential antiamyloidogenic activity for this compound. In this study, the kinetics of amyloid fibril formation by hen egg white lysozyme (HEWL) and the antifibril-forming activity of myricetin were investigated. We demonstrate that myricetin significantly inhibits the fibrillation of HEWL and the inhibitory effect is dose-dependent. Int...

  6. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    Energy Technology Data Exchange (ETDEWEB)

    Kopcansky, Peter; Siposova, Katarina [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Melnikova, Lucia, E-mail: [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Bednarikova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Institute of Chemical Sciences, Faculty of Sciences, Safarik University, Kosice (Slovakia); Timko, Milan; Mitroova, Zuzana; Antosova, Andrea [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Garamus, Vasil M. [Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research, Max-Planck-Street 1, 21502 Geesthacht (Germany); Petrenko, Viktor I. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Kyiv Taras Shevchenko National University, Volodymyrska Street 64, Kyiv 01033 (Ukraine); Avdeev, Mikhail V. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Gazova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Department of Medical and Clinical Biochemistry and LABMED, Tr. SNP 1, 040 11 Kosice (Slovakia)


    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size. - Highlights: • The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. • Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  7. Different conditions of fibrillogenesis cause polymorphism of lysozyme amyloid fibrils (United States)

    Sulatskaya, Anna I.; Rodina, Natalia P.; Povarova, Olga I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.


    Structural differences of lysozyme amyloid fibrils prepared under different conditions were examined with the use of electron microscopy, CD spectroscopy together with a specially developed approach based on the absorption and fluorescence spectroscopy of solutions of amyloid fibrils with a specific fluorescent probe thioflavin T, prepared by equilibrium microdialysis. It was shown that the amyloid fibrils differ in their photophysical properties, morphology, parameters of thioflavin T binding. Furthermore, characteristic of the dye bound to fibrils obtained in various conditions are different. These results lead us to conclude that the conditions of fibrillogenesis can influence the rate of formation as well as the properties and structure of investigated amyloid fibrils.

  8. Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Shannon E Hill

    Full Text Available 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

  9. Myricetin prevents fibrillogenesis of hen egg white lysozyme. (United States)

    He, Jianwei; Wang, Yu; Chang, Alan K; Xu, Linan; Wang, Na; Chong, Xiaoying; Li, Hui; Zhang, Bing; Jones, Gary W; Song, Youtao


    Myricetin is a natural flavonol found in many grapes, berries, fruits, vegetables, and herbs as well as other plants. Recent studies have identified potential antiamyloidogenic activity for this compound. In this study, the kinetics of amyloid fibril formation by hen egg white lysozyme (HEWL) and the antifibril-forming activity of myricetin were investigated. We demonstrate that myricetin significantly inhibits the fibrillation of HEWL and the inhibitory effect is dose-dependent. Interestingly, the inhibitory effect toward HEWL fibrillation was stronger than that exerted by the previously characterized fibril-forming inhibitor quercetin, which has high structural similarity with myricetin. Spectrofluorometric and computational studies suggest that the mechanism underlying the inhibitory action of myricetin at a molecular level is to reduce the population of partially unfolded HEWL intermediates. This action is achieved by the tight binding of myricetin to the aggregation-prone region of the β-domain of HEWL and linking to the relatively stable α-domain, thus resulting in the inhibition of amyloid fibril formation.

  10. The effect of concentration, temperature and stirring on hen egg white lysozyme amyloid formation. (United States)

    Ow, Sian-Yang; Dunstan, Dave E


    Lysozyme is associated with hereditary systemic amyloidosis in humans. Hen egg white lysozyme (HEWL) has been extensively studied as an amyloid forming protein. In this study, we investigated HEWL amyloid formation over a range of temperatures at two stirring speeds and at low concentrations to avoid gel formation. The amyloid fibril formation was found to follow first order kinetics with the rate determining step being the unfolding of the lysozyme. Both the rate of formation and final amount of amyloid formed show maxima with temperature at approximately at 65 °C. CD measurements show that the lysozyme is unfolded by 55 °C. The decrease in amyloid formation at temperatures above 65 °C is attributed to competing amorphous aggregation. The majority of the non-fibrillar aggregates are small and uniform in size with a few larger amorphous aggregates observed in the AFM images.

  11. Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters. (United States)

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


    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.

  12. Quinopeptide formation associated with the disruptive effect of epigallocatechin-gallate on lysozyme fibrils. (United States)

    Cao, Na; Zhang, Yu-Jie; Feng, Shuang; Zeng, Cheng-Ming


    Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBT-staining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products. (United States)

    Meratan, Ali Akbar; Ghasemi, Atiyeh; Nemat-Gorgani, Mohsen


    Recent findings implicate that fibrillation products, the protein aggregates formed during the various steps leading to formation of mature fibrils, induce neurotoxicity predominantly in their intermediate oligomeric state. This has been shown to occur by increasing membrane permeability, eventually leading to cell death. Despite accumulating reports describing mechanisms of membrane permeabilization by oligomers in model membranes, studies directly targeted at characterizing the events occurring in biological membranes are rare. In the present report, we describe interaction of the original native structure, prefibrils and fibrils of hen egg white lysozyme (HEWL) with mitochondrial membranes, as an in vitro biological model, with the aim of gaining insight into possible mechanism of cytotoxicity at the membrane level. These structures were first characterized using a range of techniques, including fluorescence, size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were found to be flexible/hydrophobic structures with the capacity to interact with mitochondrial membranes. Possible permeabilization of mitochondria was explored utilizing sensitive fluorometric and luminometric assays. Results presented demonstrate release of mitochondrial enzymes upon exposure to HEWL oligomers, but not native enzyme monomer or mature fibrils, in a concentration-dependent manner. Release of cytochrome c was also observed, as reported earlier, and membrane stabilization promoted by addition of calcium prevented release. Moreover, the oligomer-membrane interaction was influenced by high concentrations of NaCl and spermine. The observed release of proteins from mitochondria is suggested to occur by a nonspecific perturbation mechanism. Copyright © 2011. Published by Elsevier Ltd.

  14. Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white. (United States)

    Wang, Xuejiao; Dong, Shaohua; Bai, Quan


    Molecular imprinting as a promising and facile separation technique has received much attention because of its high selectivity for target molecules. In this study, lysozyme molecularly imprinted polymers (Lys-MIPs) were successfully prepared by the entrapment method with lysozyme as the template molecule, acrylamide as the functional monomer and N,N-methylenebisacrylamide as the cross-linker. The removal of the template lysozyme from the molecularly imprinted polymers was investigated in detail by two methods. The synthesized Lys-MIPs were characterized by scanning electron microscopy and Fourier transform-infrared, and the adsorption capacity, selectivity and reproducibility of the Lys-MIPs were also evaluated. The maximum adsorption capacity reached 94.8 mg/g, which is twice that of nonmolecularly imprinted polymers, and satisfactory selectivity and reproducibility were achieved. Using the Lys-MIP column, lysozyme could be separated completely from egg white, with purity close to 100% and mass recovery of 98.2%. This illustrated that the synthesized Lys-MIPs had high specific recognition and selectivity to the template lysozyme when they were applied to a mixture of protein standards and a real sample. Copyright © 2014 John Wiley & Sons, Ltd.

  15. Antimicrobial peptides derived from goose egg white lysozyme. (United States)

    Thammasirirak, Sompong; Pukcothanung, Yuwatida; Preecharram, Sutthidech; Daduang, Sakda; Patramanon, Rina; Fukamizo, Tamo; Araki, Tomohiro


    Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS-MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20-28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.

  16. Amyloid fibril formation from a 9 amino acid peptide, 55th-63rd residues of human lysozyme. (United States)

    Tokunaga, Yuhei; Matsumoto, Mitsuharu; Sugimoto, Yasushi


    Hen egg white lysozyme (HEWL) readily forms amyloid fibrils in vitro. We have previously identified a core structure, termed HEWL K-peptide, involved in fibril formation. Two major peptides, peptide #3 (50th-102nd residues of human lysozyme (hLZ)) and peptide #5 (54th-102nd residues), were isolated from the hLZ amyloid fibrils that had been exposed to pH 2.0 and 58 °C, and precipitated by ultra-centrifugation. These peptides cover most of the beta-domain and C-helix of hLZ including a 9 residues sequence corresponds to a human counterpart of HEWL K-peptide, GIFQINSRY (55th-63rd residues of hLZ, thus named "human K-peptide"). Chemically synthesized human K-peptide readily formed amyloid fibrils, as in HEWL K-peptide. It was demonstrated that both at least 9 residue length and Phe residue at 3rd position of the human K-peptide were crucial for amyloidogenesis in vitro. Short peptides covering COOH-terminal region of peptides #3 and #5 did not form amyloid fibrils. These data suggested that human K-peptide region with high propensity of amyloidogenesis plays a key role as a fibril-forming core sequence of hLZ. Interestingly, human K-peptide region is flanked by two predicted semi-disordered regions (39th-52nd and 67th-75th residues). We discuss the possible role of these regions. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. The solubility of hen egg-white lysozyme (United States)

    Howard, Sandra B.; Twigg, Pamela J.; Baird, James K.; Meehan, Edward J.


    The equilibrium solubility of chicken egg-white lysozyme in the presence of crystalline solid state was determined as a function of NaCl concentration, pH, and temperature. The solubility curves obtained represent a region of the lysozyme phase diagram. This diagram makes it possible to determine the supersaturation of a given set of conditions or to achieve identical supersaturations by different combinations of parameters. The temperature dependence of the solubility permits the evaluation of Delta-H of crystallization. The data indicate a negative heat of crystallization for the tetragonal crystal form but a positive heat of crystallization for the high-temperature orthorhombic form.

  18. Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

    Directory of Open Access Journals (Sweden)

    Josephine W Wu

    Full Text Available Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

  19. Concentration dependent switch in the kinetic pathway of lysozyme fibrillation: Spectroscopic and microscopic analysis (United States)

    Kiran Kumar, E.; Prasad, Deepak Kumar; Prakash Prabhu, N.


    Formation of amyloid fibrils is found to be a general tendency of many proteins. Investigating the kinetic mechanisms and structural features of the intermediates and the final fibrillar state is essential to understand their role in amyloid diseases. Lysozyme, a notable model protein for amyloidogenic studies, readily formed fibrils in vitro at neutral pH in the presence of urea. It, however, showed two different kinetic pathways under varying urea concentrations when probed with thioflavin T (ThT) fluorescence. In 2 M urea, lysozyme followed a nucleation-dependent fibril formation pathway which was not altered by varying the protein concentration from 2 mg/ml to 8 mg/ml. In 4 M urea, the protein exhibited concentration dependent change in the mechanism. At lower protein concentrations, lysozyme formed fibrils without any detectable nuclei (nucleation-independent polymerization pathway). When the concentration of the protein was increased above 3 mg/ml, the protein followed nucleation-dependent polymerization pathway as observed in the case of 2 M urea condition. This was further verified using microscopic images of the fibrils. The kinetic parameters such as lag time, elongation rate, and fibrillation half-time, which were derived from ThT fluorescence changes, showed linear dependency against the initial protein concentration suggested that under the nucleation-dependent pathway conditions, the protein followed primary-nucleation mechanism without any significant secondary nucleation events. The results also suggested that the differences in the initial protein conformation might alter the mechanism of fibrillation; however, at the higher protein concentrations lysozyme shifted to nucleation-dependent pathway.

  20. Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation. (United States)

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


    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.

  1. Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme. (United States)

    Basu, Anirban; Suresh Kumar, Gopinatha


    Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

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


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


    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 o...

  3. Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis (United States)

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


    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.

  4. Isozymes of lysozyme in leukocytes and egg white: evidence for the species-specific control of egg-white lysozyme synthesis. (United States)

    Hindenburg, A; Spitznagel, J; Arnheim, N


    Two structurally distinct forms of eggwhite lysozyme (EC are known. The egg white of some species contains both of these forms, while the egg white of other species appears to contain only one or the other of them. We have immunological and electrophoretic evidence that the chicken, which has only one lysozyme type in its egg white, contains both types in its polymorphonuclear leukocytes. Experiments on Embden goose bone marrow showed that this tissue also contains both lysozymes, even though the egg white of this species contains only one of them. Our studies suggest that many avian species have the genetic loci that code for both forms of lysozyme, but that a species-specific regulatory mechanism controls whether one or the other or both of them are expressed during egg white production. The fact that two distinct lysozymes are present in chicken leukocytes may be of significance to the antibacterial mechanism of these cells, especially in light of the fact that they lack myeloperoxidase, an important leukocyte enzyme in mammals.

  5. Raman study of lysozyme amyloid fibrils suspended on super-hydrophobic surfaces by shear flow

    KAUST Repository

    Moretti, Manola


    The shear flow generated at the rim of a drop evaporating on a micro-fabricated super-hydrophobic surface has been used to suspend and orient single/few lysozyme amyloid fibrils between two pillars for substrate-free characterization. Micro Raman spectroscopy performed on extended fibers evidenced a shift of the Amide I band main peak to the value attributed to β-sheet secondary structure, characteristic of the amyloid fibers. In addition, given the orientation sensitivity of the anisotropic molecule, the Raman signal of the main secondary structure was nicely enhanced for a fiber alignment parallel to the polarization direction of the laser. The substrate-free sample generated by this suspending technique is suitable for other structural analysis methods, where fiber crystals are investigated. It could be further employed for generation of arrays and patterns in a controllable fashion, where bio-compatible material is needed.

  6. Thioflavin T derivatives for the characterization of insulin and lysozyme amyloid fibrils in vitro: Fluorescence and quantum-chemical studies

    Energy Technology Data Exchange (ETDEWEB)

    Vus, Kateryna, E-mail: [Department of Nuclear and Medical Physics, V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61022 (Ukraine); Trusova, Valeriya; Gorbenko, Galyna [Department of Nuclear and Medical Physics, V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61022 (Ukraine); Sood, Rohit; Kinnunen, Paavo [Department of Biomedical Engineering and Computational Science, School of Science and Technology, Aalto University, FI-00076 Espoo (Finland)


    Two charged Thioflavin T (ThT) derivatives, referred to here as ICT2 and ICT3, showed higher fluorescence response, association constants and the blue-shifted emission maxima in the presence of lysozyme fibrils compared to insulin aggregates. In turn, the other two ThT derivatives, ICT4 and ICT5, possessed much weaker sensitivity to amyloid fibrils. Furthermore, a direct correlation was found between the “light-up” ability of the fibril-bound fluorophores and those observed in concentrated dichlormethane or glycerol solutions. To explain this behavior, the ground and lowest non-relaxed excited state properties of the dyes were evaluated with the 6-31G(d,p) basis set, using DFT and the CIS method. The excited state energy dependences along the torsion angle between the benzothiazole and phenyl moieties of the ICT4, ICT5 turned out to have three directly observed minima, corresponding to the locally excited (LE) and twisted intramolecular charge transfer (TICT) states. Thus, stronger stabilization of the ICT4, ICT5 LE states resulted in significantly greater quantum yield of these dyes in buffer solution and the absence of the “light-up” feature in the presence of insulin amyloid fibrils, compared to ICT2 and ICT3. - Highlights: • The ThT derivatives–ICT{sub 2}, ICT{sub 3} had the sensitivity to lysozyme fibrillar aggregates. • The insulin fibril (InsF)-bound ICT{sub 2}, ICT{sub 3} showed higher fluorescence intensities. • The lysozyme fibril-bound dyes have blue-shifted emission maxima compared to InsF. • Fluorescence of the fibril-bound dyes strongly correlates with that in glycerol. • The propylamine/metoxy moieties → the stabilization of the LE state of ICT4/ICT5. • Such a stabilization → the weak dye sensitivity to the environmental factors.

  7. Human Interleukin-2 and Hen Egg White Lysozyme: Screening for Bacteriolytic Activity against Various Bacterial Cells (United States)

    Levashov, P. A.; Ovchinnikova, E. D.; Morozova, O. A.; Matolygina, D. A.; Osipova, H. E.; Cherdyntseva, T. A.; Savin, S. S.; Zakharova, G. S.; Alekseeva, A. A.; Belogurova, N. G.; Smirnov, S. A.; Tishkov, V. I.; Levashov, A. V.


    The bacteriolytic activity of interleukin-2 and hen egg white lysozyme against 34 different species of microorganisms has been studied. It was found that 6 species of microorganisms are lysed in the presence of interleukin-2. All interleukin-2-sensitive microorganisms belong either to the Enterobacteriaceae, Bacillaceae, or the Lactobacillaceae family. It was also found that 12 species of microorganisms are lysed in the presence of lysozyme, and 16 species of microorganisms are lysed in the presence of sodium dodecyl sulfate (SDS). The bacteriolytic activity of interleukin-2 and lysozyme was studied at various pH values. PMID:27099789

  8. Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme. (United States)

    Bai, P; Peng, Z


    Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently. Copyright 2001 Academic Press.

  9. Functionalisation of Polyvinylpyrrolidone on Gold Nanoparticles Enhances Its Anti-Amyloidogenic Propensity towards Hen Egg White Lysozyme

    National Research Council Canada - National Science Library

    Tulika Das; Vidyalatha Kolli; Srijeeb Karmakar; Nandini Sarkar


    ...)-conjugated gold nanoparticles (PVP-AuNps) on hen egg white lysozyme (HEWL) amyloids. The synthesized nanoparticles have been characterized using various biophysical techniques like Ultraviolet-Visible (UV-Vis...

  10. Effect of guar gum conjugation on functional, antioxidant and antimicrobial activity of egg white lysozyme. (United States)

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


    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.

  11. NMR-based localization of ions involved in salting out of hen egg white lysozyme. (United States)

    Poznański, Jarosław


    NaCl-induced aggregation of hen egg white lysozyme (HEWL) was monitored by NMR spectroscopy. Small, but significant, changes induced by salt addition in TOCSY spectra were attributed to the effect of local reorganization of protein backbone upon ion binding. Salt-induced variations in HN and H alpha chemical shifts were mapped on the HEWL 3D structure which allowed the construction of a scheme of the spatial localization of potential ion binding sites. It was found that in a 0.5 M NaCl solution six chloride anions and at least one sodium cation are bound to preferred sites on the HEWL surface.

  12. Kinetic study on thermal denaturation of hen egg-white lysozyme involving precipitation. (United States)

    Nohara, D; Mizutani, A; Sakai, T


    A kinetic study on the thermal denaturation accompanying precipitation of hen egg-white lysozyme was performed at temperatures between 50 and 90 degrees C. Visible precipitation occurred at lysozyme concentrations higher than 10(-5)M. Even at the concentration of 10(-6)M where no visible precipitation was observed, irreversible and reversible denaturation could be clearly discriminated. The former involves two different reactions with activation energies of approximately 93 and 50 kJ x mol(-1). On the other hand, enthalpy and entropy changes in the latter are 443 kJ x mol(-1) and 1280 J x K(-1) x mol(-1), respectively, indicating a large conformational change. The contradiction that the denaturation or deactivation reaction fitted first-order reaction kinetics while its rate constant depended on the protein concentration, was resolved by newly proposed schemes. The apparent first-order rate constant obtained experimentally depended on the initial protein concentration being on the order of almost unity. Moreover, it was revealed that the apparent first-order reaction involved a second-order reaction that characterized the aggregation of denatured protein molecules. The theory developed here explained reasonably the thermal denaturation accompanying precipitation that occurs at high protein concentration and at high temperature, and was also successfully applied to the lower concentration range with no accompanying precipitation.

  13. Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. (United States)

    Maachupalli-Reddy, J; Kelley, B D; De Bernardez Clark, E


    The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated. Separate experiments were conducted in which known amounts of individual contaminants were added to test their effect on renaturation kinetics. On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding reaction, but some of them significantly affected the rate of the aggregation reaction and, thus, the overall renaturation yield. While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a decrease of about 10% in the overall renaturation yield. Phospholipids were found to improve refolding yields by about 15% by decreasing the overall rate of the aggregation reaction without affecting the rate of the folding reaction. Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were found to significantly decrease renaturation yields by promoting aggregation. This effect was strongly dependent on the concentration of the proteinaceous impurity. On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.

  14. Re-refinement of 4xan: hen egg-white lysozyme with carboplatin in sodium bromide solution

    NARCIS (Netherlands)

    Tanley, Simon W. M.; Schreurs, Antoine M. M.; Kroon - Batenburg, Louise; Helliwell, John R.


    A re-refinement of 4xan, hen egg white lysozyme (HEWL) with carboplatin crystallised in NaBr solution, has been made (Tanley et al 2016). This follows our Response article (Tanley et al 2015) to the Critique article of Shabalin et al 2015, suggesting the need for corrections to some solute molecule

  15. Hydrophobic interaction adsorption of hen egg white proteins albumin, conalbumin, and lysozyme. (United States)

    Rojas, Edwin E Garcia; dos Reis Coimbra, Jane S; Minim, Luis A; Saraiva, Sérgio H; da Silva, César A Sodré


    Hydrophobic adsorption equilibrium data of the hen egg white proteins albumin, conalbumin, and lysozyme were obtained in batch systems, at 25 degrees C, using the Streamline Phenyl resin as adsorbent. The influence of three types of salt, NaCl, Na(2)SO(4), or (NH(4))(2)SO(4), and their concentration on the equilibrium data were evaluated. The salt Na(2)SO(4) showed the higher interaction with the studied proteins, thus favoring the adsorption of proteins by the adsorbent, even though each type of salt interacted in a distinct manner with each protein. The isotherm models of Langmuir, Langmuir exponential, and Chen and Sun were well fitted to the equilibrium data, with no significant difference being observed at the 5% level of significance. The mass transfer model applied simulated correctly adsorption kinetics of the proteins under the studied conditions.

  16. Local pH at the surface of hen egg white lysozyme (United States)

    Otosu, Takuhiro; Kobayashi, Kaito; Yamaguchi, Shoichi


    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.

  17. An experimental model of affinity filtration for the isolation of egg white Lysozyme using Cibacron Blue immobilized to yeast cells. (United States)

    Ferraris, María del Pilar; Gonzalez, Ulises A; Aguilar, Carlos F; Rodríguez, Jorge A


    An experimental model of affinity filtration process was designed using a macroligand composed by Cibacron Blue F3GA immobilized to yeast cells. Its performance was evaluated, at bench scale, through the recovery of egg white Lysozyme. The selective and reversible binding between the Cibacron ligand molecule and the enzyme is described. The separation of Lysozyme from the protein mixture included the application of stages such as affinity adsorption, concentration, diafiltration and elution. A tangential microfiltration system with an inorganic membrane was designed. The main finding was the development of the diafiltration operation, key stage in the enzyme isolation. The macroligand particle kept its integrity along the whole process and the degree of purity of the isolated Lysozyme was significant. Copyright © 2016. Published by Elsevier B.V.

  18. Inhibition of lysozyme amyloidogenesis by phospholipids. Focus on long-chain dimyristoylphosphocholine. (United States)

    Ponikova, Slavomira; Kubackova, Jana; Bednarikova, Zuzana; Marek, Jozef; Demjen, Erna; Antosova, Andrea; Musatov, Andrey; Gazova, Zuzana


    Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro. Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis. Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount. Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-β structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in β-sheets which are required for destroying of amyloid fibrils. The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Denaturation of hen egg white lysozyme in electromagnetic fields: a molecular dynamics study. (United States)

    English, Niall J; Mooney, Damian A


    Nonequilibrium molecular dynamics simulations of hen egg white lysozyme have been performed in the canonical ensemble at 298 K in the presence of external electromagnetic fields of varying intensity in the microwave to far-infrared frequency range. Significant nonthermal field effects were noted, such as marked changes in the protein's secondary structure which led to accelerated incipient local denaturation relative to zero-field conditions. This occurred primarily as a consequence of alignment of the protein's total dipole moment with the external field, although the enhanced molecular mobility and dipolar alignment of water molecules is influential on sidechain motion in solvent-exposed regions. The applied field intensity was found to be highly influential on the extent of denaturation in the frequency range studied, and 0.25-0.5 V Arms-1 fields were found to induce initial denaturation to a comparable extent to thermal denaturation in the 400 to 500 K range. In subsequent zero-field simulations following exposure to the e/m field, the extent of perturbation from the native fold and the degree of residual dipolar alignment were found to be influential on incipient folding.

  20. Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites. (United States)

    Asperger, Arndt; Marx, Kristina; Albers, Christian; Molin, Laura; Pinato, Odra


    Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

  1. Novel polydopamine imprinting layers coated magnetic carbon nanotubes for specific separation of lysozyme from egg white. (United States)

    Gao, Ruixia; Zhang, Lili; Hao, Yi; Cui, Xihui; Liu, Dechun; Zhang, Min; Tang, Yuhai


    Novel core-shell nanocomposites, consisting of magnetic carbon nanotubes (MCNTs) core surrounded by a thin polydopamine (PDA) imprinting shell for specific recognition of lysozyme (Lyz), were fabricated for the first time. The obtained products were characterized and the results showed that the PDA layer was successfully attached onto the surface of MCNTs and the corresponding thickness of imprinting layer was just about 10nm which could enable the template access the recognition cavities easily. The polymerization conditions and adsorption performance of the resultant nanomaterials were investigated in detail. The results indicated that the obtained imprinted polymers showed fast kinetic and high affinity towards Lyz and could be used to specifically separate Lyz from real egg white. In addition, the prepared materials had excellent stability and no obvious deterioration after five adsorption-regeneration cycles. Easy preparation, rapid separation, high binding capacity, and satisfactory selectivity for the template protein make this polymer attractive in biotechnology and biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Use of lysozyme from chicken egg white as a nitrite replacer in an Italian-type chicken sausage

    Directory of Open Access Journals (Sweden)

    Nalaka Sandun Abeyrathne


    Full Text Available Background: Sodium or potassium nitrite is widely used as a curing agent in sausages and other cured meat products. Nitrite has strong antimicrobial and antioxidant effects and generates cured meat color. Nitrite, however, can react with secondary or tertiary amines in meat to form carcinogenic, teratogenic and mutagenic N-nitroso compounds. Several findings have been suggested that high consumption of processed meat may increase the risk of cancer, and emphasized that dietary nitrosamines are positively associated with cancer. Lysozyme is one of the major egg proteins that have antimicrobial and antioxidant characteristics. Therefore, lysozyme can be used in meat processing to prevent microbial growth and oxidative degradation in meat products during storage. This study is focused on evaluating the antimicrobial and antioxidant effects of lysozyme extracted from egg white as a replacer of nitrite in a cooked Italian-type chicken sausage. Methods: Four curing treatments including 100% nitrite (control, 100% lysozyme (treatment 1, 25% nitrite + 75% lysozyme (treatment 2 and 50% nitrite + 50% lysozyme (treatment 3 were used to prepare Italian-type chicken sausage samples. Recipe was developed with 64% (w/w meat, 17% (w/w binder (bread crumble, 12% (w/w ice, 4% (w/w vegetable oil, 2% (w/w salt, 1% (w/w spices (chili, black pepper, cardamom. Prepared samples were cooked in an 80 °C smoke house to a core temperature of 65 °C and cooled in cold water to 20-25 °C subsequently packed in polyethylene and stored in a freezer (-18 °C. The antimicrobial effect lysozyme was tested using Escherichia coli and Salmonella. The growth of these pathogens at 0, 3 and 5 days of storage of spore inoculation was determined. The antioxidant activity of lysozyme was determined using the TBARS value during the 25 d storage period. The redness (a*, lightness (L*, and yellowness (b* of sausages were analyzed using a Minolta color meter (CR 410, Konica Minolta Inc

  3. A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. (United States)

    Goldberg, M E; Rudolph, R; Jaenicke, R


    The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.

  4. Hen egg white lysozyme permeabilizes Escherichia coli outer and inner membranes. (United States)

    Derde, Melanie; Lechevalier, Valérie; Guérin-Dubiard, Catherine; Cochet, Marie-Françoise; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique; Nau, Françoise


    Natural preservatives answer the consumer demand for long shelf life foods, synthetic molecules being perceived as a health risk. Lysozyme is already used because of its muramidase activity against Gram-positive bacteria. It is also described as active against some Gram-negative bacteria; membrane disruption would be involved, but the mechanism remains unknown. In this study, a spectrophotometric method using the mutant Escherichia coli ML-35p has been adapted to investigate membrane disruption by lysozyme for long durations. Lysozyme rapidly increases the permeability of the outer membrane of E. coli due to large size pore formation. A direct delayed activity of lysozyme against the inner membrane is also demonstrated, but without evidence of perforations.

  5. Renaturation of reduced hen egg white lysozyme containing two blocked sulfhydryl groups

    Energy Technology Data Exchange (ETDEWEB)

    Acharya, A.S.; Taniuchi, H.


    Formation of native lysozyme from the reduced form involves many pathways in two processes: incorrect pairing of half-cystine residues by oxidation and rearrangement of disulfide (SS) bonds. The energy barrier against suflhydryl (SH)-disulfide interchange of the native or nativelike species thus formed causes accumulation of these species. For example, the enzymatically active isomers containing three presumably native SS bonds and one open SS bond may be thermodynamically favorable over the nonnative isomers and can be formed from reduced lysozyme or lysozyme containing scrambled SS bonds by nonobligatory and flexible pathways. As an extension of these observations formation of nativelike species from reduced lysozyme containing the average of two carboxymethyl (CM)-cysteine was investigated.

  6. Robust identification of binding hot spots using continuum electrostatics: application to hen egg-white lysozyme. (United States)

    Hall, David H; Grove, Laurie E; Yueh, Christine; Ngan, Chi Ho; Kozakov, Dima; Vajda, Sandor


    Binding hot spots, protein regions with high binding affinity, can be identified by using X-ray crystallography or NMR spectroscopy to screen libraries of small organic molecules that tend to cluster at such hot spots. FTMap, a direct computational analogue of the experimental screening approaches, uses 16 different probe molecules for global sampling of the surface of a target protein on a dense grid and evaluates the energy of interaction using an empirical energy function that includes a continuum electrostatic term. Energy evaluation is based on the fast Fourier transform correlation approach, which allows for the sampling of billions of probe positions. The grid sampling is followed by off-grid minimization that uses a more detailed energy expression with a continuum electrostatics term. FTMap identifies the hot spots as consensus clusters formed by overlapping clusters of several probes. The hot spots are ranked on the basis of the number of probe clusters, which predicts their binding propensity. We applied FTMap to nine structures of hen egg-white lysozyme (HEWL), whose hot spots have been extensively studied by both experimental and computational methods. FTMap found the primary hot spot in site C of all nine structures, in spite of conformational differences. In addition, secondary hot spots in sites B and D that are known to be important for the binding of polysaccharide substrates were found. The predicted probe-protein interactions agree well with those seen in the complexes of HEWL with various ligands and also agree with an NMR-based study of HEWL in aqueous solutions of eight organic solvents. We argue that FTMap provides more complete information on the HEWL binding site than previous computational methods and yields fewer false-positive binding locations than the X-ray structures of HEWL from crystals soaked in organic solvents. © 2011 American Chemical Society

  7. Kinetics of secondary structure recovery during the refolding of reduced hen egg white lysozyme. (United States)

    Roux, P; Delepierre, M; Goldberg, M E; Chaffotte, A F


    We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 microM oxidized glutathione, and 20 microM reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.

  8. Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk. (United States)

    Deckers, Daphne; Vanlint, Dietrich; Callewaert, Lien; Aertsen, Abram; Michiels, Chris W


    Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.

  9. Effect of natural biopolymers on amyloid fibril formation and morphology. (United States)

    Ow, Sian-Yang; Bekard, Innocent; Dunstan, Dave E


    Amyloid fibrils are associated with the pathogenesis of protein misfolding diseases such as Alzheimer's disease. These fibrils typically exhibit different morphologies when grown in vitro, and this has been known to affect their biological properties and cytotoxicity. The formation kinetics and resultant morphology of fibrils formed from the model proteins Bovine Insulin and Hen Egg White Lysozyme have been measured. We show that the presence of gum arabic and pectin during fibril formation cause the amyloid fibrils formed to associate into higher order fibrillar aggregates. It is postulated that the carbohydrates act as a template to promote inter-fibril association, resulting in larger, thicker fibrils. This observation provides some insight into the differences in growing amyloid fibrils in vitro in the absence or presence of other high molecular weight compounds. Furthermore, these findings suggest a method of tailoring fibril structure for applications in nanotechnology and bio-template applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Interaction between Sorbitol and Hen Egg White Lysozyme: From Dynamics to Structure

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Ramezani


    Full Text Available Background: The study of the molecular dynamics of proteins based on internal motions is crucial for understanding the mechanism of protein function. Internal movements of proteins play an important role in both protein folding processes and the mechanism of action of enzyme. Knowledge about these movements will help us in understanding these processes correctly. Osmolites are small organic molecules which are utilized by the cells of all organisms, except hallobacteria. Osmolites are produced under extreme stressful conditions in order to help in stabilizing macro-molecules and retaining their biological function. They interact directly with macromolecules, but they exert their effect only when the characteristics or conditions of the solvent present around the cell change. Therefore, their presence affects the stability of the protein indirectly. Materials and Methods: In this work we studied the effect(s of the osmolite Sorbitol on the dynamics of Hen Egg White Lysozyme, and through our study we used NMR Hydrogen-Deuterium Exchange to characterize the effect(s. We performed spectrometery in two different conditions, in the absence and presence of sorbitol. The extent of peptide hydrogen exchange was assigned as a function time and in a decreasing manner in the intensity of the peak corresponding to amino acids. The resulting TOCSY spectra were assigned using SPARKY software, as well as we integrated the peaks using this software. Results: The speed of hydrogen exchange, hydrogen atoms of the peptide bond, will give us information about the local structural oscillations where the exchange is taking place. The rate of hydrogen exchange varies from one amino acid to another, and the intensity of the peak of these hydrogen decreases as time passes by. Conclusion: We found that the presence of sorbitol causes a decrease in proton exchange rate, and since there is no noticeable chemical shifts in the peaks of the spectra, in the presence or absence

  11. Lysozyme expression in Lactococcus lactis

    NARCIS (Netherlands)

    Guchte, Maarten van de; Wal, Fimme Jan van der; Kok, Jan; Venema, Gerhardus

    Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence

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

    National Research Council Canada - National Science Library

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


    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...

  13. ACE inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme. (United States)

    Rao, Shengqi; Sun, Jun; Liu, Yuntao; Zeng, Huawei; Su, Yujie; Yang, Yanjun


    Lysozyme from hen egg white is a well-known antimicrobial protein with high ratio of hydrophobic and positively charged amino acid residues. In order to explore functional bioactivities of enzymatic hydrolysates of lysozyme, the protein was subjected to a simulated gastrointestinal digestion and the resulting hydrolysate (LPH2) showed a strong competitive angiotensin I-converting enzyme (ACE) inhibitory activity (IC(50)=12.6μg/ml) and a remarkable antioxidant activity. The LPH2 was fractionated using a 3kDa cut-off membrane and the obtained permeate LPH2-3kDa was analysed by MALDI-TOF-TOF MS. Using this technology, 38 different peptides were identified and some of these peptides were well fit with structure requirements of ACE inhibitory peptides and/or antioxidant peptides. The findings from this study suggest that the protein containing high proportion of hydrophobic and positively charged residues have the potential to generate multifunctional peptides, and these peptides would be beneficial ingredient to be used in functional foods. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  14. Nonequilibrium molecular dynamics study of electric and low-frequency microwave fields on hen egg white lysozyme. (United States)

    English, Niall J; Solomentsev, Gleb Y; O'Brien, Paul


    Nonequilibrium molecular dynamics simulations of various mutants of hen egg white lysozyme have been performed at 300 K and 1 bar in the presence of both external static electric and low-frequency microwave (2.45 GHz) fields of varying intensity. Significant nonthermal field effects were noted, such as marked changes in the protein's secondary structure relative to the zero-field state, depending on the field conditions, mutation, and orientation with respect to the applied field. This occurred primarily as a consequence of alignment of the protein's total dipole moment with the external field, although the dipolar alignment of water molecules in both the solvation layer and the bulk was also found to be influential. Substantial differences in behavior were found for proteins with and without overall net charges, particularly with respect to translational motion. Localized motion and perturbation of hydrogen bonds were also found to be evident for charged residues.

  15. Self-assembly dynamics for the transition of a globular aggregate to a fibril network of lysozyme proteins via a coarse-grained Monte Carlo simulation

    Directory of Open Access Journals (Sweden)

    R. B. Pandey


    Full Text Available The self-organizing dynamics of lysozymes (an amyloid protein with 148 residues with different numbers of protein chains, Nc = 1,5,10, and 15 (concentration 0.004 – 0.063 is studied by a coarse-grained Monte Carlo simulation with knowledge-based residue-residue interactions. The dynamics of an isolated lysozyme (Nc = 1 is ultra-slow (quasi-static at low temperatures and becomes diffusive asymptotically on raising the temperature. In contrast, the presence of interacting proteins leads to concentration induced protein diffusion at low temperatures and concentration-tempering sub-diffusion at high temperatures. Variation of the radius of gyration of the protein with temperature shows a systematic transition from a globular structure (at low T to a random coil (high T conformation when the proteins are isolated. The crossover from globular to random coil becomes sharper upon increasing the protein concentration (i.e. with Nc = 5,10, with larger Rg at higher temperatures and concentration; Rg becomes smaller on adding more protein chains (e.g. Nc = 15 a non-monotonic response to protein concentration. Analysis of the structure factor (S(q provides an estimate of the effective dimension (D ≥ 3, globular conformation at low temperature, and D ∼ 1.7, random coil, at high temperatures of the isolated protein. With many interacting proteins, the morphology of the self-assembly varies with scale, i.e. at the low temperature (T = 0.015, D ∼ 2.9 on the scale comparable to the radius of gyration of the protein, and D ∼ 2.3 at the large scale over the entire sample. The global network of fibrils appears at high temperature (T = 0.021 with D ∼ 1.7 (i.e. a random coil morphology at large scale involving tenuous distribution of micro-globules (at small scales.

  16. Preliminary Work in Obtaining Site-Directed Mutants of Hen Egg White Lysozyme (United States)

    Holmes, Leonard D.


    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

  17. Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

    Directory of Open Access Journals (Sweden)

    Haruhiko Koizumi


    Full Text Available Dislocation-free tetragonal hen egg white (HEW lysozyme crystals were grown from a seed crystal in a cell. The rates of tetragonal HEW lysozyme crystal growth normal to the (110 and (101 faces with and without a 1-MHz external electric field were measured. A decrease in the typical growth rates of the crystal measured under an applied field at 1 MHz was observed, although the overall driving force increased. Assuming that the birth and spread mechanism of two-dimensional nucleation occurs, an increase in the effective surface energy of the step ends was realized in the presence of the electric field, which led to an improvement in the crystal quality of the tetragonal HEW lysozyme crystals. This article also discusses the increase in the effective surface energy of the step ends with respect to the change in the entropy of the solid.

  18. Amyloid protofibrils of lysozyme nucleate and grow via oligomer fusion. (United States)

    Hill, Shannon E; Robinson, Joshua; Matthews, Garrett; Muschol, Martin


    The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.

  19. Molecular cloning, inducible expression and antibacterial analysis of a novel i-type lysozyme (lyz-i2) in Pacific white shrimp, Litopenaeus vannamei. (United States)

    Chen, Ting; Ren, Chunhua; Wang, Yanhong; Luo, Peng; Jiang, Xiao; Huang, Wen; Chen, Chang; Hu, Chaoqun


    The full-length cDNA coding for a novel invertebrate (i-type) lysozyme was identified in Pacific white shrimp (Litopenaeus vannamei). The newly obtained L. vannamei lysozyme is similar to the Penaeus monodon i-type lysozyme 2, but it is distant from the known L. vannamei c-type lysozyme and i-type lysozyme 1 in protein sequence; therefore, it was defined as L. vannamei i-type lysozyme 2 (lyz-i2). Expression of L. vannamei lyz-i2 transcripts were ubiquitously detected in all tissues we selected, with the highest abundance observed in the hemolymph. Challenge with Vibrio harveyi might elicit L. vannamei lyz-i2 mRNA expression in the hepatopancreas, intestine, muscle, gill and hemolymph. In the themolymph, specifically, the stimulatory effects of Vibrio and lipopolysaccharide (LPS) on lyz-i2 transcript levels were durable and transient, respectively; while Polyinosinic:polycytidylic acid [Poly (I:C)] treatment did not affect lyz-i2 expression. L. vannamei lyz-i2 recombinant protein was generated in an Escherichia coli system. By lysoplate and turbidimetric assays, the L. vannamei lyz-i2 recombinant protein showed a broad spectrum of antimicrobial properties with high activities against Micrococcaceae lysodeikticus and various Vibrio species and relatively low activity against E. coli. In conclusion, L. vannamei lyz-i2 might be a potent antibacterial protein with a role in innate immunity in Penaeid shrimp. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ali Chaari

    Full Text Available 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.

  2. Occupational asthma induced by inhaled egg lysozyme. (United States)

    Bernstein, J A; Kraut, A; Bernstein, D I; Warrington, R; Bolin, T; Warren, C P; Bernstein, I L


    A 26-year-old man employed in a company which manufactured hen egg white derived lysozyme for use in the pharmaceutical industry was evaluated for occupational asthma. The worker began to experience immediate-onset asthmatic symptoms two months after starting to work with egg lysozyme powder. The work process involved the production of approximately 1,000 kg of purified dried lysozyme powder per week. Prick skin testing was positive to egg lysozyme (50 mg/ml) and other egg protein components, but negative to whole egg white and egg yolk reagents. Serum specific IgE to egg lysozyme was documented. Decrements in serial peak expiratory flow rates were associated with lysozyme exposure at work. A specific bronchoprovocation challenge to lysozyme powder was positive demonstrating an isolated immediate asthmatic response (48 percent decrease from baseline FEV1). This is the first reported case of lysozyme-induced asthma specifically caused by inhalational exposure to egg lysozyme.

  3. Measurements of thermal conductivity and thermal diffusivity of hen egg-white lysozyme crystals using a short hot wire method (United States)

    Fujiwara, Seiji; Maki, Syou; Tanaka, Seiichi; Maekawa, Ryunosuke; Masuda, Tomoki; Hagiwara, Masayuki


    Thermal conductivity and thermal diffusivity of hen egg-white lysozyme (HEWL) crystals were examined by using the transient short hot wire method. This method is based on the conventional hot wire method, but improved by using a wire that is much shorter than conventional ones. The magneto-Archimedes levitation technique was utilized to attach the HEWL crystals onto the wire. Owing to the upward magnetic force, the HEWL crystals were deposited at the air-liquid interface of the protein buffer solution where the short hot wire was preliminarily fixed. In situ observation clarified that the wire was completely buried into the HEWL crystals. By means of these techniques, the measurement of thermal conductivity and thermal diffusivity of HEWL crystals was realized for the first time. Gadolinium chloride (a paramagnetic subject) was used as a precipitant agent of crystallization. Crystal growth was carried out over 20 h at 17.2 °C. The applied magnetic field was 4 T. Measurements were conducted during the crystal growth at two different times. The thermal conductivity and diffusivity of the HEWL crystals were determined to be 0.410 W/(m.K) and 3.77×10-8 m2/s at 14 h after, and 0.438 W/(m.K) and 5.18×10-8 m2/s at 20 h after, respectively. We emphasize that this method is versatile and applicable for other protein crystals.

  4. Lysozyme fractionation from egg white at pilot scale by means of tangential flow membrane adsorbers: Investigation of the flow conditions. (United States)

    Brand, Janina; Voigt, Katharina; Zochowski, Bianca; Kulozik, Ulrich


    The application of membrane adsorbers instead of classical packed bed columns for protein fractionation is still a growing field. In the case of egg white protein fractionation, the application of classical chromatography is additionally limited due to its high viscosity that impairs filtration. By using tangential flow membrane adsorbers as stationary phase this limiting factor can be left out, as they can be loaded with particle containing substrates. The flow conditions existing in tangential flow membrane adsorbers are not fully understood yet. Thus, the aim of the present study was to gain a deeper understanding of the transport mechanisms in tangential flow membrane adsorbers. It was found that loading in recirculation mode instead of single pass mode increased the binding capacity (0.39 vs. 0.52 mg cm(-2)). Further, it was shown that either higher flow rates (0.39 mg cm(-2) vs. 0.57 mg cm(-2) at 1 CV min(-1) or 20 CV min(-1), respectively) or higher amounts of the target protein in the feed (0.24 mg cm(-2) vs. 0.85 mg cm(-2) for 2.5 or 39.0 g lysozyme, respectively) led to more protein binding. These results show that, in contrast to radial flow or flat sheet membrane adsorbers, the transport in tangential flow membrane adsorbers is not purely based on convection, but on a mix of convection and diffusion. Additionally, investigations concerning the influence of fouling formation were performed that can lead to transport limitations. It was found that this impact is neglectable. It can be concluded that the usage of tangential flow membrane adsorbers is very recommendable for egg white protein fractionations, although the transport is partly diffusion-limited. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Effect of human serum albumin on the kinetics of 4-methylumbelliferyl-β-D-N-N'-N″ Triacetylchitotrioside hydrolysis catalyzed by hen egg white lysozyme. (United States)

    Calderon, Cristian; Abuin, Elsa; Lissi, Eduardo; Montecinos, Rodrigo


    The effect of human serum albumin (HSA) addition on the rate of hydrolysis of the synthetic substrate 4-methylumbelliferyl-β-D-N-N'-N″ triacetylchitotrioside ((NAG)(3)-MUF) catalyzed by hen egg white lysozyme has been measured in aqueous solution (citrate buffer 50 mM pH = 5.2 at 37 °C). The presence of HSA leads to a decrease in the rate of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. The catalytic rate constant decreases tenfold when the albumin concentration increases, while the Michaelis constant remains almost constant in the albumin concentration range employed. Ultracentrifugation experiments indicate that the main origin of the observed variation in the kinetic behavior is related to the existence of an HSA-lysozyme interaction. Interestingly, the dependence of the catalytic rate constant with albumin concentration parallels the decrease of the free enzyme concentration. We interpret these results in terms of the presence in the system of two enzyme populations; namely, the HSA associated enzyme which does not react and the free enzyme reacting as in the absence of albumin. Other factors such as association of the substrate to albumin or macromolecular crowding effects due to the presence of albumin are discarded. Theoretical modeling of the structure of the HSA-lysozyme complex shows that the Glu35 and Asp52 residues located in the active site of lysozyme are oriented toward the HSA surface. This conformation will inactivate lysozyme molecules bound to HSA.

  6. Synergistic contributions of asparagine 46 and aspartate 52 to the catalytic mechanism of chicken egg white lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Ichiro; Kirsch, J.F. [Univ. of California, Berkeley, CA (United States)


    The X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and Asp52 with the D-subsite N-acetylmuramic acid due help to distort that pyranose ring into the reactive half-chair conformation and that a hydrogen bond is formed between Asn46 and Asp52. These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL. The Michaelis constants of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type K{sub M}; the corresponding k{sub cat} values are 25- and 50-fold lower, respectively, than the wild-type k{sub cat}. These results support the proposal of Strynadka and James. The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do not vary. The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues. The mutations do not change the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived substrates differ in conformation from complexes with bacterial peptidoglycans. 23 refs., 7 figs., 2 tabs.

  7. Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Ichiro; Kirsch, J.F. [Univ. of California, Berkeley, CA (United States)


    The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity. ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans. The active-site aspartate 52 residue of ChEWL, which is postulated to stabilize the oxocarbenium ion intermediate, has no counterpart in GoEWL. The substrate specificity of the D52A ChEWL mutant was compared with those of wild-type ChEWL and GoEWL. D52A ChEWL retains approximately 4% of the wild-type catalytic activity in reactions with three different bacterial cell suspensions. Asp52 therefore is not essential to the catalytic mechanism, accounting for only a 2 kcal/mol decrease in AG. The function of Asp52 in D52A ChEWL- and GoEWL-catalyzed cleavage of (carboxymethyl)chitin may be partially fulfilled by an appropriately positioned carboxyl group on the substrate (substrate-assisted catalysis). D52A ChEWL and GoEWL, unlike wild-type ChEWL, exhibit biphasic kinetics in the clearing of Micrococcus luteus cell suspensions, suggesting preferences for subsets of the linkages in the M. luteus peptidoglycan. These subsets do not exist in the peptidoglycans of Escherichia coli or Sarcina lutea, since neither D52A ChEWL nor GoEWL exhibits initial bursts in reactions with suspensions of these bacteria. We propose that substrate-assisted catalysis occurs in reactions of D52A ChEWL and GoEWL with M. luteus peptidoglycans, with the glycine carboxyl group of uncross-linked peptides attached to N-acetylmuramic acid partially substituting the function of the missing Asp52. 52 refs., 6 figs., 1 tab.

  8. Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study (United States)

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


    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.

  9. Comparative insight into surfactants mediated amyloidogenesis of lysozyme. (United States)

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


    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.

  10. Heavy-atom derivatives in lipidic cubic phases: results on hen egg-white lysozyme tetragonal derivative crystals with Gd-HPDO3A complex. (United States)

    Girard, Eric; Pebay-Peyroula, Eva; Vicat, Jean; Kahn, Richard


    Gd-HPDO3A, a neutral gadolinium complex, is a good candidate for obtaining heavy-atom-derivative crystals by the lipidic cubic phase crystallization method known to be effective for membrane proteins. Gadolinium-derivative crystals of hen egg-white lysozyme were obtained by co-crystallizing the protein with 100 mM Gd-HPDO3A in a monoolein cubic phase. Diffraction data were collected to a resolution of 1.7 A using Cu Kalpha radiation from a rotating-anode generator. Two binding sites of the gadolinium complex were located from the strong gadolinium anomalous signal. The Gd-atom positions and their refined occupancies were found to be identical to those found in derivative crystals of hen egg-white lysozyme obtained by co-crystallizing the protein with 100 mM Gd-HPDO3A using the hanging-drop technique. Moreover, the refined structures are isomorphous. The lipidic cubic phase is not disturbed by the high concentration of Gd-HPDO3A. This experiment demonstrates that a gadolinium complex, Gd-HPDO3A, can be used to obtain derivative crystals by the lipidic cubic phase crystallization method. Further studies with membrane proteins that are known to crystallize in lipidic cubic phases will be undertaken with Gd-HPDO3A and other Gd complexes to test whether derivative crystals with high Gd-site occupancies can be obtained.

  11. The contrasting effect of macromolecular crowding on amyloid fibril formation.

    Directory of Open Access Journals (Sweden)

    Qian Ma

    Full Text Available Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1 have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins has been

  12. Stable self-assembled nanostructured hen egg white lysozyme exhibits strong anti-proliferative activity against breast cancer cells. (United States)

    Mahanta, Sailendra; Paul, Subhankar; Srivastava, Ankit; Pastor, Ashutosh; Kundu, Bishwajit; Chaudhuri, Tapan K


    Chemotherapy side effects have long been a matter of great concern. Here we describe a structurally stable self-assembled nanostructured lysozyme (snLYZ) synthesized using a simple desolvation technique that exhibited anticancer activity, as well as excellent hemocompatibility. Field emission scanning electron microscopy; atomic force microscopy and dynamic particle size analyzer were used for analyzing the synthesized snLYZ. The analysis revealed spherical shape with an average size of 300 nm. Circular dichroism and tryptophan fluorescence spectroscopic analysis revealed its gross change in secondary as well as the tertiary level of the structure. snLYZ also demonstrated excellent structural as well as the functional stability of LYZ in a wide range of pH and temperature with a fair level of protection against proteinase K digestion. When applied to MCF-7 breast cancer cells, it exhibited approximately 95% cell death within 24h, involving a reactive oxygen species (ROS) based mechanism, and showed excellent hemocompatibility. Fluorescence microscopy imaging revealed distinct cellular internalization of snLYZ and the formation of cytoplasmic granules, which initiated a cell-killing process through membrane damage. In order to mimic targeted therapy, we tagged folic acid with snLYZ, which further enhanced cytotoxicity against MCF-7 cells. Therefore, this is the first report of its kind where we demonstrated the preparation of a highly stable self-assembled nanostructured lysozyme with a strong anti-proliferative activity against breast cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Emadi, Saeed, E-mail:; 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 incubation 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.

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

    Vogtt, K; Winter, R


    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.

  15. Quasi-Laue neutron-diffraction study of the water arrangement in crystals of triclinic hen egg-white lysozyme. (United States)

    Bon, C; Lehmann, M S; Wilkinson, C


    Triclinic crystals of lysozyme, hydrogen-deuterium exchanged in deuterated solvent, have been studied using neutron quasi-Laue techniques and a newly developed cylinder image-plate detector. The wavelength range employed was from 2.7 to 3.5 A, which gave 9426 significant reflections [F >/= 2sigma(F)] to a resolution limit of 1. 7 A. The deuteration states of the H atoms in the protein molecule were identified, followed by an extensive analysis of the water structure surrounding the protein. The final R factor was 20.4% (Rfree = 22.1%). In total, the 244 observed water molecules form approximately one layer of water around the protein with far fewer water molecules located further away. Water molecules covering the apolar patches make tangential layers at 4-5 A from the surface or form C-H...O contacts, and several water-molecule sites can be identified in the apolar cavities. Many of the water molecules are apparently orientationally disordered, and only 115 out of the 244 water molecules sit in mean single orientations. Comparison of these results with quasi-elastic neutron scattering observations of the water dynamics leads to a picture of the water molecules forming an extended constantly fluctuating network covering the protein surface.

  16. Lysozyme, intradermal diffusion of india ink and peritoneal capillary permeability in mice. (United States)

    Bianchi, C; Goi, A; Ronchini, A


    Hen egg white lysozyme injected intracutaneously into mice inhibits the intradermal diffusion of India Ink. The effect of lysozyme persists in presence of hyaluronidase. Lysozyme injected i.p. into mice inhibits acetic acid (i.p.) induced leakage of Pontamine Sky Blue into the abdominal cavity. The data suggest that lysozyme might be tried for applications outside the microbiological field.

  17. Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system


    Gourbatsi, Evdoxia; Povey, Jane; Uddin, Shahid; Smales, C. Mark


    Objectives The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Results Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic pepti...

  18. Measurements of Thermal Conductivity and Thermal Diffusivity of Hen Egg-White Lysozyme Crystals and Its Solution Using the Transient Short Hot Wire Method (United States)

    Fujiwara, Seiji; Maki, Syou; Maekawa, Ryunosuke; Tanaka, Seiichi; Hagiwara, Masayuki


    Protein crystals are an essentially important biological sample to advance the analysis of X-ray structure, but their thermophysical properties, especially thermal conductivity and thermal diffusivity, have not been studied sufficiently. This current situation can be attributed to various kinds of technical problems; e.g., the fragility of protein crystals and the difficulty of nucleation control. Ideally speaking, protein crystallization should be carried out under a " containerless condition" to eliminate any mechanical distortion of the crystals from the walls. To realize the condition, we have developed an original crystallization method by means of the magneto-Archimedes effect. In this paper, a transient short hot wire method was combined with the technique of magneto-Archimedes effect to realize simultaneous measurement of thermal conductivity and thermal diffusivity of hen egg-white lysozyme (HEWL) crystals. As the results, thermal conductivity and thermal diffusivity of HEWL crystals were found to be 0.410-0.438 \\hbox {W}\\cdot \\hbox {m}^{-1}\\cdot \\hbox {K}^{-1} and 3.77-5.18× 10^{-8} \\hbox {m}2\\cdot \\hbox {s}^{-1}, respectively. We clarified by the crystallizing process of HEWL that the crystals were magnetically levitated at the air-liquid interface and the short hot wire was completely buried into them as the crystals grew. We also measured the HEWL solution by the same methods. The thermal conductivity of the solution had almost the same value as that of water and had little dependency on the concentration of HEWL, but the thermal diffusivity was unclear.

  19. Polymorphism of Lysozyme Condensates. (United States)

    Safari, Mohammad S; Byington, Michael C; Conrad, Jacinta C; Vekilov, Peter G


    Protein condensates play essential roles in physiological processes and pathological conditions. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Here we employ the antimicrobial enzyme lysozyme and examine the similarities between mesoscopic clusters, amyloid structures, and disordered aggregates consisting of chemically modified protein. We show that the mesoscopic clusters are distinct from the other two classes of aggregates. Whereas cluster formation and amyloid oligomerization are both reversible, aggregation triggered by reduction of the intramolecular S-S bonds is permanent. In contrast to the amyloid structures, protein molecules in the clusters retain their enzymatic activity. Furthermore, an essential feature of the mesoscopic clusters is their constant radius of less than 50 nm. The amyloid and disordered aggregates are significantly larger and rapidly grow. These findings demonstrate that the clusters are a product of limited protein structural flexibility. In view of the role of the clusters in the nucleation of ordered protein solids, our results suggest that fine-tuning the degree of protein conformational stability is a powerful tool to control and direct the pathways of protein condensation.

  20. Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system. (United States)

    Gourbatsi, Evdoxia; Povey, Jane; Uddin, Shahid; Smales, C Mark


    The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.

  1. Attenuation of lysozyme amyloid cytotoxicity by SPION-mediated modulation of amyloid aggregation. (United States)

    Naik, Aafreen; Kambli, Priyanka; Borana, Mohanish; Mohanpuria, Neha; Ahmad, Basir; Kelkar-Mane, Varsha; Ladiwala, Uma


    The formation and deposition of proteinaceous aggregates of amyloid fibrils characterize diverse degenerative diseases, such as Alzheimer's, Parkinson's, and systemic amyloidosis. The presence of these aggregates is associated with clinical manifestations, and various forms of amyloid aggregates have been identified to be cytotoxic. Although the exact mechanism of amyloid toxicity remains to be elucidated, prevention of amyloid fibril formation and aggregation forms a possible therapeutic approach. Nanomaterials possess the potential for such a strategy. Using hen egg white lysozyme (HEWL) as a prototypic amyloid-forming protein, we found a reduction in the aggregation rate of HEWL in the presence of super-paramagnetic iron oxide nanoparticles (SPIONs) with slowing of nucleation and amyloid fibril elongation. HEWL-amyloid had a predominantly fibrillar structure and was toxic to various cells. A significant attenuation of cytotoxicity was observed when cells were treated with SPION-interacted HEWL-amyloid. Ultra-structural differences were observed between the native and SPION-interacted HEWL-amyloids by SEM and TEM imaging. Our findings confirm that SPIONs perturb amyloid fibrillation, thereby reducing the cytotoxicity of amyloid. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Suppression of lysozyme aggregation at alkaline pH by tri-N-acetylchitotriose. (United States)

    Kumar, Satish; Ravi, Vijay Kumar; Swaminathan, Rajaram


    Inhibiting protein misfolding and aggregation is imperative for treatment of amyloid diseases. In this regard small molecules which bind to and stabilize the monomeric protein have invited attention owing to their ability to significantly slow down or inhibit aggregation and amyloid formation. We have earlier shown that hen egg-white lysozyme (HEWL) spontaneously forms soluble oligomers at pH 12.2, which are later stabilized by intermolecular disulphide bonds, eventually resulting in amyloid fibrils. In this work, we show that overnight ( approximately 12 h) pre-incubation of HEWL with its competitive inhibitor, N,N',N''-Triacetylchitotriose (chitotriose) at neutral pH, impairs its aggregation and fibrillogenesis at pH 12.2. Unlike in control or N-Acetyl-D-glucosamine (NAG) pre-incubated samples, HEWL-chitotriose complex displayed i) reduced thioflavin T and ANS fluorescence, ii) small oligomers but no amyloid fibrils in AFM, iii) absence of large aggregates in SDS-PAGE and gel-filtration elutions, iv) marginally more helical content in CD spectra and v) >70% enzymatic activity after 24 h and approximately 16% activity after week long incubation at alkaline pH. It is likely that strong binding in the HEWL-chitotriose complex, in contrast to weakly bound HEWL-NAG complex, raises the activation energy barrier for protein misfolding and subsequent aggregation, thereby retarding the aggregation kinetics substantially. These results hold promise for the therapy of human lysozyme amyloidosis.

  3. Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles. (United States)

    Fan, Jun-Bao; Chen, Jie; Liang, Yi


    The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.

  4. Socioeconomic status and the incidence of atrial fibrillation in whites and blacks: the Atherosclerosis Risk in Communities (ARIC) study. (United States)

    Misialek, Jeffrey R; Rose, Kathryn M; Everson-Rose, Susan A; Soliman, Elsayed Z; Clark, Cari J; Lopez, Faye L; Alonso, Alvaro


    No previous studies have examined the interplay among socioeconomic status, sex, and race with the risk of atrial fibrillation (AF). We prospectively followed 14 352 persons (25% black, 75% white, 55% women, mean age 54 years) who were free of AF and participating in the Atherosclerosis Risk in Communities (ARIC) study. Socioeconomic status was assessed at baseline (1987-1989) through educational level and total family income. Incident AF through 2009 was ascertained from electrocardiograms, hospitalizations, and death certificates. Cox regression was used to estimate hazard ratios and 95% CIs of AF for education and family income. Interactions were tested between socioeconomic status and age, race, or sex. Over a median follow-up of 20.6 years, 1794 AF cases occurred. Lower family income was associated with higher AF risk (hazard ratio 1.45, 95% CI 1.27 to 1.67 in those with income less than $25 000 per year compared with those with $50 000 or more per year). The association between education and AF risk varied by sex (P=0.01), with the lowest education group associated with higher AF risk in women (hazard ratio 1.88, 95% CI 1.55 to 2.28) but not in men (hazard ratio 1.15, 95% CI 0.97 to 1.36) compared with the highest education group. Adjustment for cardiovascular risk factors attenuated the associations. There were no interactions with race or age. Blacks had lower AF risk than whites in all income and education groups. Lower family income was associated with a higher AF risk overall, whereas the impact of education on AF risk was present only in women. Differences in socioeconomic status do not explain the lower risk of AF in blacks compared with whites. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  5. Linear correlation between thermal stability and folding kinetics of lysozyme. (United States)

    Cao, Aoneng; Wang, Gang; Tang, Youqi; Lai, Luhua


    We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).

  6. Solvent freeze out crystallization of lysozyme from a lysozyme-ovalbumin mixture

    Energy Technology Data Exchange (ETDEWEB)

    Diaz Borbon, V.; Ulrich, J. [Martin-Luther-Universitaet Halle-Wittenberg, Zentrum fuer Ingenieurwissenschaft, Verfahrenstechnik/TVT, 06099 Halle Saale (Germany)


    Hen egg white lysozyme (HEWL) crystallization conditions from an ovalbumin-lysozyme mixture were found by screening tests and further located in pseudo-phase diagrams. This information was used to set up the initial conditions for the solvent freeze out (SFO) process. The process uses the freezing of ice to create the supersaturation for the proteins to crystallize out of the solution. The crystallization of HEWL (15 mg/mL) out of a lysozyme-ovalbumin mixture (1.7 mg/mL) is carried out by SFO. Under the reported conditions, a crystallization yield of 69 % was obtained. A mean crystal size of 77.8 {mu}m was enhanced in a crystallization time of 15.1 h. The lysozyme nature of the crystals is proven by SDS PAGE and enzymatic activity tests. (copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  7. Aggregation of lysozyme and of poly(ethylene glycol)-modified lysozyme after adsorption to silica. (United States)

    Daly, Susan M; Przybycien, Todd M; Tilton, Robert D


    Surface-induced aggregation is a common instability during protein storage, delivery and purification. This aggregation can lead to the formation of fibrils rich in intermolecular beta-sheet structure. Techniques to probe surface-clustering are limited. Here we use protein intrinsic fluorescence and thioflavin T probe fluorescence in a total internal reflection fluorescence (TIRF) sampling geometry to simultaneously monitor the kinetics of adsorption and aggregation for chicken egg lysozyme on a silica surface. We observe a slow surface-induced aggregation process that continues well after the lysozyme adsorption kinetics have plateaued. The rate of surface-induced aggregation is independent of the lysozyme concentration in solution. Consistent with the clustering observed via thioflavin T fluorescence, infrared amide I band spectra also show a 1.5-fold increase in intermolecular beta-sheet content upon lysozyme adsorption. Tryptophan emission spectra show no evidence for any tertiary structural change upon adsorption. Furthermore, we observe that the covalent modification of lysozyme with a single poly(ethylene glycol) (PEG) grafted chain does not inhibit aggregation on the surface, but a second PEG graft significantly inhibits the intermolecular beta-sheet formation.

  8. Designing microcapsules based on protein fibrils and protein - polysaccharide complexes

    NARCIS (Netherlands)

    Hua, K.N.P.


    Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first

  9. Microcapsules with protein fibril reinforced shells: effect of fibril properties on mechanical strength of the shell. (United States)

    Humblet-Hua, Nam-Phuong K; van der Linden, Erik; Sagis, Leonard M C


    In this study, we produced microcapsules using layer-by-layer adsorption of food-grade polyelectrolytes on an emulsion droplet template. We compared the mechanical stability of microcapsules to shells consisting of alternating layers of ovalbumin-high methoxyl pectin (Ova-HMP) complexes and semi-flexible ovalbumin (Ova) fibrils (average contour length, L(c) ~ 200 nm), with microcapsules built of alternating layers of lysozyme-high methoxyl pectin (LYS-HMP) complexes and lysozyme (LYS) fibrils. Two types of LYS fibrils were used: short and rod-like (L(c) ~ 500 nm) and long and semi-flexible (L(c) = 1.2-1.5 μm). At a low number of layers (≤4), microcapsules from Ova complexes and fibrils were stronger than microcapsules prepared from LYS complexes and fibrils. With an increase of the number of layers, the mechanical stability of microcapsules from LYS-HMP/LYS fibrils increased significantly and capsules were stronger than those prepared from Ova-HMP/Ova fibrils with the same number of layers. The contour length of the LYS fibrils did not have a significant effect on mechanical stability of the LYS-HMP/LYS fibril capsules. The stiffer LYS fibrils produce capsules with a hard but more brittle shell, whereas the semi-flexible Ova fibrils produce capsules with a softer but more stretchable shell. These results show that mechanical properties of this type of capsule can be tuned by varying the flexibility of the protein fibrils.

  10. Rapid and simple purification of lysozyme from the egg shell membrane. (United States)

    Kozuka, Miyuki; Murao, Sato; Yamane, Takuya; Inoue, Tsutomu; Ohkubo, Iwao; Ariga, Hiroyoshi


    Lysozyme (EC is a hydrolytic enzyme that cleaves the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, a major bacterial cell wall polymer. In the food industry, lysozyme is used as an additive mainly in the production of wine and beer. Lysozyme was found to be localized in the egg shell membrane. In this study, we found that lysozyme was easily purified from the egg shell membrane and that the enzyme also had antibacterial activity. Furthermore, we found that the antibacterial activity of purified lysozyme from the egg shell membrane was lower than that of purified lysozyme from the egg white at alkaline pH. The method for rapid purification of lysozyme developed in this study should contribute to the food industry.

  11. The role of protonation in protein fibrillation

    DEFF Research Database (Denmark)

    Jeppesen, M.D.; Otzen, D.E.; Westh, P.


    buffer and the peptide hormone glucagon during fibrillation. Glucagon absorbs or releases protons to an extent which allows it to attain a net charge of zero in the fibrillar state, both at acidic and basic pH. Similar results are obtained for lysozyme. This suggests that side chain pKa values change...

  12. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)



    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To

  13. Large-Scale Candidate Gene Analysis in Whites and African Americans Identifies IL6R Polymorphism in Relation to Atrial Fibrillation The National Heart, Lung, and Blood Institute's Candidate Gene Association Resource (CARe) Project

    NARCIS (Netherlands)

    Schnabel, Renate B.; Kerr, Kathleen F.; Lubitz, Steven A.; Alkylbekova, Ermeg L.; Marcus, Gregory M.; Sinner, Moritz F.; Magnani, Jared W.; Wolf, Philip A.; Deo, Rajat; Lloyd-Jones, Donald M.; Lunetta, Kathryn L.; Mehra, Reena; Levy, Daniel; Fox, Ervin R.; Arking, Dan E.; Mosley, Thomas H.; Mueller-Nurasyid, Martina; Young, Taylor R.; Wichmann, H. -Erich; Seshadri, Sudha; Farlow, Deborah N.; Rotter, Jerome I.; Soliman, Elsayed Z.; Glazer, Nicole L.; Wilson, James G.; Breteler, Monique M. B.; Sotoodehnia, Nona; Newton-Cheh, Christopher; Kaeaeb, Stefan; Ellinor, Patrick T.; Alonso, Alvaro; Benjamin, Emelia J.; Heckbert, Susan R.


    Background-The genetic background of atrial fibrillation (AF) in whites and African Americans is largely unknown. Genes in cardiovascular pathways have not been systematically investigated. Methods and Results-We examined a panel of approximately 50 000 common single-nucleotide polymorphisms (SNPs)

  14. A setup for simultaneous measurement of infrared spectra and light scattering signals: Watching amyloid fibrils grow from intact proteins

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yang; Maurer, Jürgen; Roth, Andreas; Vogel, Vitali; Winter, Ernst; Mäntele, Werner, E-mail: [Institut für Biophysik, Goethe-Universität Frankfurt am Main, Max-von Laue-Straße 1, D-60438 Frankfurt am Main (Germany)


    A setup for the simultaneous measurement of mid-infrared spectra and static light scattering is described that can be used for the analysis of the formation of nanoscale and microscopic aggregates from smaller molecules to biopolymers. It can be easily integrated into sample chambers of infrared spectrometers or combined with laser beams from tunable infrared lasers. Here, its use for the analysis of the formation of amyloid fibrils from intact proteins is demonstrated. The formation of amyloid fibrils or plaques from proteins is a widespread and pathogenetic relevant process, and a number of diseases are caused and correlated with the deposition of amyloid fibrils in cells and tissues. The molecular mechanisms of these transformations, however, are still unclear. We report here the simultaneous measurement of infrared spectra and static light scattering for the analysis of fibril formation from egg-white lysozyme. The transformation of the native form into non-native forms rich in β-sheet structure is measured by analysis of the amide I spectral region in the infrared spectra, which is sensitive for local structures. At the same time, light scattering signals at forward direction as well as the forward/backward ratio, which are sensitive for the number of scattering centers and their approximate sizes, respectively, are collected for the analysis of fibril growth. Thermodynamic and kinetic parameters as well as mechanistic information are deduced from the combination of the two complementary techniques.

  15. A setup for simultaneous measurement of infrared spectra and light scattering signals: Watching amyloid fibrils grow from intact proteins (United States)

    Li, Yang; Maurer, Jürgen; Roth, Andreas; Vogel, Vitali; Winter, Ernst; Mäntele, Werner


    A setup for the simultaneous measurement of mid-infrared spectra and static light scattering is described that can be used for the analysis of the formation of nanoscale and microscopic aggregates from smaller molecules to biopolymers. It can be easily integrated into sample chambers of infrared spectrometers or combined with laser beams from tunable infrared lasers. Here, its use for the analysis of the formation of amyloid fibrils from intact proteins is demonstrated. The formation of amyloid fibrils or plaques from proteins is a widespread and pathogenetic relevant process, and a number of diseases are caused and correlated with the deposition of amyloid fibrils in cells and tissues. The molecular mechanisms of these transformations, however, are still unclear. We report here the simultaneous measurement of infrared spectra and static light scattering for the analysis of fibril formation from egg-white lysozyme. The transformation of the native form into non-native forms rich in β-sheet structure is measured by analysis of the amide I spectral region in the infrared spectra, which is sensitive for local structures. At the same time, light scattering signals at forward direction as well as the forward/backward ratio, which are sensitive for the number of scattering centers and their approximate sizes, respectively, are collected for the analysis of fibril growth. Thermodynamic and kinetic parameters as well as mechanistic information are deduced from the combination of the two complementary techniques.

  16. Studies on isolation and partial purification of lysozyme from egg ...

    African Journals Online (AJOL)



    Jan 5, 2009 ... and as an anti-cancer drug (Ghosh, 2003), as well as a germinative agent of bacterial spores (Ando, 1975). It is used for lysing Escherichia coli and .... Elli MSG, Camperi AS, Agus T, Navarrodel A, Caneizo OV (1999). Direct lysozyme separation from egg white by dye membrane affitinity chromatography.

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

    Directory of Open Access Journals (Sweden)

    Catarina Oliveira Silva

    Full Text Available 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

  18. Validation of PR interval length as a criterion for development of atrial fibrillation in non-Hispanic whites, African Americans and Hispanics. (United States)

    Shulman, Eric; Aagaard, Philip; Kargoli, Faraj; Hoch, Ethan; Zheng, Laura; Di Biase, Luigi; Fisher, John; Gross, Jay; Kim, Soo; Ferrick, Kevin; Krumerman, Andrew


    PR interval prolongation on electrocardiogram (ECG) increases the risk of atrial fibrillation (AF). Non-Hispanic Whites are at higher risk of AF compared to African Americans and Hispanics. However, it remains unknown if prolongation of the PR interval for the development of AF varies by race/ethnicity. Therefore, we determined whether race affects the PR interval length's ability to predict AF and if the commonly used criterion of 200 ms in AF prediction models can continue to be used for non-White cohorts. This is a retrospective epidemiological study of consecutive inpatient and outpatients. An ECG database was initially interrogated. Patients were included if their initial ECG demonstrated sinus rhythm and had two or more electrocardiograms and declared a race and/or ethnicity as non-Hispanic White, African American or Hispanic. Development of AF was stratified by race/ethnicity along varying PR intervals. Cox models controlled for age, gender, race/ethnicity, systolic blood pressure, BMI, QRS, QTc, heart rate, murmur, treatment for hypertension, heart failure and use of AV nodal blocking agents to assess PR interval's predictive ability for development of AF. 50,870 patients met inclusion criteria of which 5,199 developed AF over 3.72 mean years of follow-up. When the PR interval was separated by quantile, prolongation of the PR interval to predict AF first became significant in Hispanic and African Americans at the 92.5th quantile of 196-201 ms (HR: 1.42, 95% CI: 1.09-1.86, p=0.01; HR: 1.32, 95% CI: 1.07-1.64, p=0.01, respectively) then in non-Hispanic Whites at the 95th quantile at 203-212 ms (HR: 1.24, 95% CI: 1.24-1.53, p=0.04). For those with a PR interval above 200 ms, African Americans had a lower risk than non-Hispanic Whites to develop AF (HR: 0.80, 95% CI: 0.64-0.95, p=0.012), however, no significant difference was demonstrated in Hispanics. This is the first study to validate a PR interval value of 200 ms as a criterion in African Americans and

  19. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

    Directory of Open Access Journals (Sweden)

    Gui Hong Fu

    Full Text Available Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type and goose-type (g-type lysozymes from Asian seabass (Lates calcarifer. The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50 and Asp(67 and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL domain containing three conserved catalytic residues (Glu(71, Asp(84, Asp(95 essential for catalytic activity. Real time quantitative PCR (qRT-PCR revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  20. Metastable Amyloid Phases and their Conversion to Mature Fibrils (United States)

    Muschol, Martin; Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy

    Self-assembly of proteins into amyloid fibrils plays a key role in both functional biological responses and pathogenic disorders which include Alzheimer's disease and type II diabetes. Amyloid fibril assembly frequently generates compact oligomeric and curvilinear polymeric intermediates which are implicated to be toxic to cells. Yet, the relation between these early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. Our measurements indicate that lysozyme amyloid oligomers and their curvilinear fibrils only form after crossing a salt and protein concentration dependent threshold. These oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. Our experimental transition boundaries match well with colloidal model predictions accounting for salt-modulated charge repulsion. We also report our preliminary findings on the mechanism by which these metastable oligomeric phases are converted into stable amyloid fibrils.

  1. Release of lysozyme from the branched polyelectrolyte-lysozyme complexation. (United States)

    Ni, Ran; Cao, Dapeng; Wang, Wenchuan


    On the basis of the discretely charged sphere model of lysozyme, the release behavior of lysozyme from the branched polyelectrolyte-lysozyme complexation is investigated by adding salt and changing the pH values of the solution. It is found that, with the increase of the salt ionic strength of the solution, the lysozymes are gradually released from the oppositely charged polyelectrolyte as a result of the screening of electrostatic attraction between the two ionic species by adding the salt. Interestingly, there exists a critical salt ionic strength at which all proteins are released from the branched polyelectrolyte, and the polyelectrolyte-protein complexation is broken completely. Beyond the critical value, the increase of the salt ionic strength causes self-association of the proteins released from the branched polyelectrolyte-protein complexation. The self-association of the protein is detrimental in biological systems. By calculating the second virial coefficient, we found that the optimal salt content for the dispersion of proteins coincides with the critical ionic strength, because the second virial coefficient reaches its maximum at the critical ionic strength. Similarly, increasing the pH value of the solution can also release the lysozymes from the polyelectrolyte, because the increase of pH value of the solution changes the charge distribution and net charge of the lysozyme, weakens the attraction between lysozymes mediated by polyelectrolyte, and finally leads to the dissolution of the complexation of branched polyelectrolyte with lysozymes in strong alkaline solution. In addition, by exploring the effect of architecture of the polyelectrolyte on the release behavior of proteins, we found that it is more difficult to release proteins from the branched polyelectrolyte than from the linear polyelectrolyte.

  2. Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient (United States)

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


    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.

  3. Birth weight and the risk of atrial fibrillation in whites and African Americans: the Atherosclerosis Risk In Communities (ARIC) study. (United States)

    Lawani, Sherifat O; Demerath, Ellen W; Lopez, Faye L; Soliman, Elsayed Z; Huxley, Rachel R; Rose, Kathryn M; Alonso, Alvaro


    Low birth weight (LBW) has been associated with an increased risk of cardiovascular disease (CVD). A previous study, however, found higher risk of atrial fibrillation (AF) in individuals with higher birth weight (BW). To further understand this apparent paradox, we examined the relationship between AF and BW in the Atherosclerosis Risk in Communities (ARIC) cohort. The analysis included 10,132 individuals free of AF at baseline (1996-1998), who provided BW information, were not born premature, and were not a twin. Self-reported BW was categorized as low (4.0 kg). AF incidence was ascertained from hospital discharge codes and death certificates. We used multivariable Cox proportional hazard models to determine the hazard ratios (HR) and 95% confidence intervals (CI) of AF across BW groups. During an average follow-up of 10.3 years, we identified 882 incident AF cases. LBW was associated with higher risk of AF. Compared to individuals in the medium BW category, the HR (95% CI) of AF was 1.33 (0.99, 1.78) for LBW and 1.00 (0.81, 1.24) for high BW after adjusting for sociodemographic variables (p for trend = 0.29). Additional adjustment for CVD risk factors did not attenuate the associations (HR 1.42, 95% CI 1.06, 1.90 for LBW and HR 0.86, 95% CI 0.69-1.07 for high BW, compared to medium BW, p for trend = 0.01). LBW was associated with a higher risk of AF. This association was independent of known predictors of AF and is consistent with that observed for other cardiovascular diseases.

  4. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.


    Hughey, V L; Johnson, E A


    Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolate...

  5. Assessment of atrial fibrillation and vulnerability in patients with Wolff-Parkinson-White syndrome using two-dimensional speckle tracking echocardiography.

    Directory of Open Access Journals (Sweden)

    Jing-Jie Li

    Full Text Available PURPOSE: The aim was to assess atrial fibrillation (AF and vulnerability in Wolff-Parkinson-White (WPW syndrome patients using two-dimensional speckle tracking echocardiography (2D-STE. METHODS: All patients were examined via transthoracic echocardiography and 2D-STE in order to assess atrial function 7 days before and 10 days after RF catheter ablation. A postoperative 3-month follow-up was performed via outpatient visit or telephone calls. RESULTS: Results showed significant differences in both body mass index (BMI and supraventricular tachycardia (SVT duration between WPW patients and DAVNP patients (both P<0.05. Echocardiography revealed that the maximum left atrial volume (LAVmax and the left ventricular mass index (LVMI in diastole increased noticeably in patients with WPW compared to patients with DAVNP both before and after ablation (all P<0.05. Before ablation, there were obvious differences in the levels of SRs, SRe, and SRa from the 4-chamber view (LA in the WPW patients group compared with patients in the DAVNP group (all P<0.05. In the AF group, there were significant differences in the levels of systolic strain rate (SRs, early diastolic strain rate (SRe, and late diastolic strain rate (SRa from the 4-chamber view (LA both before and after ablation (all P<0.05. In the non-AF group, there were decreased SRe levels from the 4-chamber view (LA/RA pre-ablation compared to post-ablation (all P<0.05. CONCLUSION: Our findings provide convincing evidence that WPW syndrome may result in increased atrial vulnerability and contribute to the development of AF. Further, RF catheter ablation of AAV pathway can potentially improve atrial function in WPW syndrome patients. Two-dimensional speckle tracking echocardiography imaging in WPW patients would be necessary in the evaluation and improvement of the overall function of RF catheter ablation in a long-term follow-up period.

  6. The impact of B-type natriuretic peptide levels on the suppression of accompanying atrial fibrillation in Wolff-Parkinson-White syndrome patients after accessory pathway ablation. (United States)

    Kawabata, Mihoko; Goya, Masahiko; Takagi, Takamitsu; Yamashita, Shu; Iwai, Shinsuke; Suzuki, Masahito; Takamiya, Tomomasa; Nakamura, Tomofumi; Hayashi, Tatsuya; Yagishita, Atsuhiko; Sasaki, Takeshi; Takahashi, Yoshihide; Ono, Yuhichi; Hachiya, Hitoshi; Yamauchi, Yasuteru; Otomo, Kenichiro; Nitta, Junichi; Okishige, Kaoru; Nishizaki, Mitsuhiro; Iesaka, Yoshito; Isobe, Mitsuaki; Hirao, Kenzo


    Atrial fibrillation (AF) often coexists with Wolff-Parkinson-White (WPW) syndrome. We compared the efficacy of Kent bundle ablation alone and additional AF ablation on accompanying AF, and examined which patients would still have a risk of AF after successful Kent bundle ablation. This retrospective multicenter study included 96 patients (56±15 years, 72 male) with WPW syndrome and AF undergoing Kent bundle ablation. Some patients underwent simultaneous pulmonary vein isolation (PVI) for AF. The incidence of post-procedural AF was examined. Sixty-four patients underwent only Kent bundle ablation (Kent-only group) and 32 also underwent PVI (+PVI group). There was no significant difference in the basic patient characteristics between the groups. Additional PVI did not improve the freedom from residual AF compared to Kent bundle ablation alone (p=0.53). In the Kent-only group, AF episodes remained in 25.0% during the follow-up (709 days). A univariate analysis showed that age ≥60 years, left atrial dimension ≥38mm, B-type natriuretic peptide (BNP) ≥40pg/ml, and concomitant hypertension were predictive factors for residual AF. However, in the multivariate analysis, only BNP ≥40pg/ml remained as an independent predictive factor (HR=17.1 and CI: 2.3-128.2; p=0.006). Among patients with WPW syndrome and AF, Kent bundle ablation alone may have a sufficient clinical impact of preventing recurrence of AF in select patients. Screening the BNP level would help decide the strategy to manage those patients. Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

  7. Lysozyme in water-acetonitrile mixtures: Preferential solvation at the inner edge of excess hydration (United States)

    Sirotkin, Vladimir A.; Kuchierskaya, Alexandra A.


    Preferential solvation/hydration is an effective way for regulating the mechanism of the protein destabilization/stabilization. Organic solvent/water sorption and residual enzyme activity measurements were performed to monitor the preferential solvation/hydration of hen egg-white lysozyme at high and low water content in acetonitrile at 25 °C. The obtained results show that the protein destabilization/stabilization depends essentially on the initial hydration level of lysozyme and the water content in acetonitrile. There are three composition regimes for the dried lysozyme. At high water content, the lysozyme has a higher affinity for water than for acetonitrile. The residual enzyme activity values are close to 100%. At the intermediate water content, the dehydrated lysozyme has a higher affinity for acetonitrile than for water. A minimum on the residual enzyme activity curve was observed in this concentration range. At the lowest water content, the organic solvent molecules are preferentially excluded from the dried lysozyme, resulting in the preferential hydration. The residual catalytic activity is ˜80%, compared with that observed after incubation in pure water. Two distinct schemes are operative for the hydrated lysozyme. At high and intermediate water content, lysozyme is preferentially hydrated. However, in contrast to the dried protein, at the intermediate water content, the initially hydrated lysozyme has the increased preferential hydration parameters. At low water content, the preferential binding of the acetonitrile molecules to the initially hydrated lysozyme was detected. No residual enzyme activity was observed in the water-poor acetonitrile. Our data clearly show that the initial hydration level of the protein macromolecules is one of the key factors that govern the stability of the protein-water-organic solvent systems.

  8. Study on nucleation kinetics of lysozyme crystallization (United States)

    Lin, Chen; Zhang, Yang; Liu, Jing J.; Wang, Xue Z.


    The nucleation kinetics of hen egg-white lysozyme crystallization was investigated using a hot stage cooling crystallizer and a microscope to monitor the solution crystallization process in real time. Images of crystals were continuously recorded under varied precipitant and protein concentrations. The nucleation rate was found to be higher at higher precipitant concentration, and increase monotonically with protein concentration if the precipitant concentration was held constant. Attempt was made to interpret the experimental data using classical nucleation theory. It was found that the model predictions are lower than the experimental values at low supersaturations but agree well with experimental data at high supersaturations. The trends in the experimental data suggest that two nucleation mechanisms might co-exist: heterogeneous nucleation seems to be the dominant at low supersaturation while at higher supersaturation homogeneous nucleation seems to play the major role.

  9. Atrial Fibrillation (United States)

    ... A-Z Clinical Trials Publications and Resources Health Education and Awareness The Science Science Home Blood Disorders ... heart's two upper chambers—called the atria (AY-tree-uh)—to fibrillate. The ... a difficult decision concerning surgery for patients with atrial fibrillation, the ...

  10. Atrial fibrillation

    African Journals Online (AJOL)


    Objective: Atrial fibrillation is the commonest chronic arrhythmia and the etiology is widely varied. The aim of this study was to determine the etiology, clinical characteristics and treatment offered to adult patients with atrial fibrillation managed in a referral hospital in Port Harcourt, southern Nigeria. Methods:A retrospective ...

  11. Complex coacervates of hyaluronic acid and lysozyme

    DEFF Research Database (Denmark)

    Water, Jorrit J.; Schack, Malthe M.; Velazquez-Campoy, Adrian


    stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well...... with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined...

  12. Lysozymes in the animal kingdom

    Indian Academy of Sciences (India)

    In the animal kingdom, three major distinct lysozyme types have been identified – the c-type (chicken or conventional type), the g-type (goose-type) and the ... Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Center (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, ...

  13. Kinetic studies on the initial crystallization process of lysozyme in the presence of D2O and H2O. (United States)

    Liu, X Q; Sano, Y


    In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H2O was much slower than in D2O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H2O was lower than in D2O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H2O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCl. Therefore, the effect of lysozyme concentration on the aggregation process in H2O may be smaller than in D2O.

  14. Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane. (United States)

    Derde, Melanie; Nau, Françoise; Guérin-Dubiard, Catherine; Lechevalier, Valérie; Paboeuf, Gilles; Jan, Sophie; Baron, Florence; Gautier, Michel; Vié, Véronique


    Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Precrystallization structures in supersaturated lysozyme solutions studied by dynamic light scattering and scanning force microscopy (United States)

    Schaper, Achim; Georgalis, Yannis; Umbach, Patrick; Raptis, Jannis; Saenger, Wolfram


    A comparitive study of the nanostructure evolving during aggregation of hen-egg white lysozyme in supersaturated solution was carried out by dynamic light scattering (DLS) and scanning force microscopy (SFM). Lysozyme aggregate (cluster) formation was observed in solution in the presence of NaCl, (NH4)2SO4, and NaNO3 as precipitating agents. The growth kinetics were examined by DLS and revealed fractal growth of the clusters with a fractal dimension of 1.8 obtained independently of the type of inert salt. Such behavior is typical for diffusion-limited cluster-cluster (DLCA) aggregation. Initial lysozyme cluster sizes were in the range of 12-35 nm. SFM images of individual lysozyme clusters at the liquid-solid interface were obtained in the presence of NaCl and NaNO3 under crystallization conditions, and revealed cluster sizes in agreement with those determined by DLS. Extended domains of smaller sized clusters appeared on the mica surface after subjecting supersaturated lysozyme solutions to a dialysis step. The feasibility of DLS and SFM for monitoring the nano- and mesoscopic morphology of lysozyme aggregates in supersatured solutions and at the solid-liquid interface is discussed.

  16. Lysozyme-Mediated Formation of Protein-Silica Nano-Composites for Biosensing Applications (Postprint) (United States)


    reagents Lysozyme (from hen egg white), tetramethyl orthosilicate (TMOS) and tetraethyl orthosilicate (TEOS) were purchased from Sigma–Aldrich (St. Louis...Hydrochloric acid, acetone, bovine serum albumin (BSA, fract V, cold alcohol precipitated) and dimethyl sulfoxide (DMSO) were all purchased from

  17. Particle-loaded hollow-fiber membrane adsorbers for lysozyme separation

    NARCIS (Netherlands)

    Avramescu, M.E.; Borneman, Zandrie; Wessling, Matthias


    The separation of lysozyme (LZ), a valuable enzyme naturally present in chicken egg white, was carried out using a new type of ion exchange hollow-fiber membranes. Functionalities were incorporated into the polymeric membranes by dispersing ion-exchange resins (IERs) in a microporous structure

  18. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory (United States)

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


    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…

  19. Use of europium ions for SAD phasing of lysozyme at the Cu Kα wavelength


    Vijayakumar, Balakrishnan; Velmurugan, Devadasan


    Europium(III) ions bound to the surface of hen egg-white lysozyme were found to exhibit good anomalous signal facilitating SAD phasing using laboratory-source data and automated model building. The europium ion-binding sites were observed up to the 15σ level.

  20. The association of lysozyme with casein

    NARCIS (Netherlands)

    Roos, de A.L.; Walstra, P.; Geurts, T.J.


    The association of hen eggs’ lysozyme with caseins was studied by using three casein substrates: (I) solutions of the various caseins, (II) artificially made casein micelles of various compositions and (III) caseins adsorbed onto soya-oil emulsion droplets. In solution, lysozyme associated most

  1. Invertebrate lysozymes: Diversity and distribution, molecular ...

    Indian Academy of Sciences (India)

    Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type ... Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 ...

  2. Complex coacervation of lysozyme and heparin

    DEFF Research Database (Denmark)

    van de Weert, Marco; Andersen, Mia Bendix; Frokjaer, Sven


    To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability.......To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability....

  3. A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria.

    Directory of Open Access Journals (Sweden)

    Lien Callewaert


    Full Text Available Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme. A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria

  4. Novel squarylium dyes for detection of amyloid fibrils in vitro

    Directory of Open Access Journals (Sweden)

    K. O. Vus


    Full Text Available A series of novel symmetrical and asymmetrical squarylium dyes with the different substituents in the donor moieties have been tested for their ability to detect and characterize insulin and lysozyme amyloid fibrils prepared in acidic buffer at elevated temperature. The dye-protein binding parameters were estimated in terms of the one-site Langmuir adsorption model using the data of direct and reverse fluorimetric titrations. By comparing the dye quantum yields, binding affinities, and extents of the fluorescence enhancement in the protein-bound state, G6 and G7 were selected as the most prospective amyloid tracers. Furthermore, these probes provided evidence for the lower polarity of the lysozyme fibrillar grooves compared to insulin aggregates. The novel dyes G6 and G7 were recommended for amyloid fibril detection and characterization in the near-infrared region.

  5. Membrane crystallization of lysozyme: kinetic aspects (United States)

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


    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.

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

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


    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.

  7. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

    Directory of Open Access Journals (Sweden)

    Sinjan Choudhary

    Full Text Available We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  8. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. (United States)

    Hughey, V L; Johnson, E A


    Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.

  9. Comparison of two different lysozyme types under native and crystallization conditions using two-dimensional NMR and dynamic light scattering. (United States)

    Poznański, Jaroslaw; Georgalis, Yannis; Wehr, Lorin; Saenger, Wolfram; Zielenkiewicz, Piotr


    In order to elucidate differences observed in the aggregation kinetics of hen-egg white lysozyme under crystallization conditions we have undertaken a comparative study of the enzyme marketed by Seikagaku and Sigma companies. When the crystallization of the two lysozyme preparations is followed by time-resolved dynamic light scattering, the structural differences are also observed under native conditions in the early nucleation kinetics. The differences are manifested in the formation rates of macroscopic crystals, but do not influence the morphology of the typical tetragonal lysozyme crystal. Using two-dimensional NMR we have followed the differences in the native-like solution structure of the two preparations, while the primary sequence and molecular mass are identical. According to the published structure of tetragonal lysozyme crystal the largest deviations were found for the residues involved in the intermolecular interactions in crystal structure.

  10. Aptamer-Based SERS Detection of Lysozyme on a Food-Handling Surface. (United States)

    Boushell, Victoria; Pang, Shintaro; He, Lili


    Undeclared food allergens due to cross contamination of processing equipment is a leading cause for food product recalls. Therefore, there is a great need for developing rapid and sensitive methods to detect food allergens. In this paper, an aptamer highly specific to egg white lysozyme was coupled to dendritic silver nanoparticles in order to perform surface enhanced Raman spectroscopy (SERS). The procedure was successfully tested in water and on a stainless steel food-handling surface. The lowest detectable concentration for lysozyme was 0.5 μg/mL in water and 5 μg/mL on a stainless steel food-handling surface. Principal component analysis shows a significant change in SERS spectra when lysozyme was present, suggesting the successful capture of lysozyme by the aptamer. Quantification of lysozyme target was also shown from 0 to 6 μg/mL, that is, 0, 0.5, 2, 6 μg/mL. Overall method took less than 40 min. The developed method can be extended to detect other food allergens using specific aptamers. © 2016 Institute of Food Technologists®.

  11. Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix. (United States)

    Hincke, M T; Gautron, J; Panheleux, M; Garcia-Ruiz, J; McKee, M D; Nys, Y


    The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.

  12. Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Linda Helmfors

    Full Text Available Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT lysozyme and the disease-associated variant F57I in the central nervous system (CNS of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP. SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

  13. Deconstructing glucagon fibrillation

    DEFF Research Database (Denmark)

    Ghodke, Shirin

    Aggregation of misfolded proteins into ordered amyloid fibrils has deep implications in disease pathology as well as in pharmaceutical production of therapeutic proteins. This thesis elucidates the fibrillation pathway of the therapeutic peptide hormone, glucagon and explores the biophysical basis...... of its inherent fibril polymorphism by varying physicochemical factors like temperature, pressure and co-solvents. This work demonstrates the important role of hydration in the fibrillation process as well as in fibril polymorphism....

  14. Effect of macromolecular impurities on lysozyme solubility and crystallizability: dynamic light scattering, phase diagram, and crystal growth studies (United States)

    Skouri, M.; Lorber, B.; Giegé, R.; Munch, J.-P.; Candau, J. S.


    The effects of macromolecular impurities on protein solubility and crystallizability were investigated by dynamic light scattering and crystal growth experiments using hen egg-white lysozyme as the model protein. In the presence of traces of protein impurities, representing no more than 2% (w/w) of the total protein, the average diffusion coefficients of the macromolecular particles found in undersaturated lysozyme solutions are significantly lower than those measured with purest lysozyme preparations. This fact is explained by the simultaneous existence of individual molecules and of large size aggregates in contaminated solutions, as indicated by the bimodal light scattering autocorrelation function. Controlled contamination experiments in which ovalbumin or conalbumin were added to purest lysozyme indicate that aggregates result from heterogeneous association of lysozyme molecules with the structurally unrelated proteins. These aggregates might become starting points for heterogeneous nucleation leading to the growth of ill-shaped microcrystals. Aggregates in under- or supersaturated lysozyme solutions containing NaCl can be eliminated by filtration over microporous membranes. As a result the number of ill-shaped crystals diminishes drastically; that of well-shaped tetragonal crystals decreases also but their size increases.

  15. Who Is at Risk for Atrial Fibrillation (AF or AFib)? (United States)

    ... Anyone with heart disease, including valve problems , hypertrophic cardiomyopathy , acute coronary syndrome , Wolff-Parkinson-White (WPW) syndrome and history of heart attack . Additionally, atrial fibrillation is the most common complication after heart surgery. Drinking alcohol Binge drinking (having ...

  16. Study of lysozyme resistance in Rhodococcus equi. (United States)

    Hébert, Laurent; Bidaud, Pauline; Goux, Didier; Benachour, Abdellah; Laugier, Claire; Petry, Sandrine


    Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.


    Directory of Open Access Journals (Sweden)

    S. S. Dekina


    Full Text Available The lysozyme immobilization in cryogel of polyvinyl alcohol and physico-chemical properties of obtained preparation was investigated. Hydrolytic activity of lysozyme was determined by bacteriolytic method, using Micrococcus lysodeikticus cells acetone powder as substrate. Protein content was determined by the Lowry–Hartree method. Immobilization of lysozyme was conducted by entrapment in polyvinyl alcohol gel with subsequent cycles of freezing-thawing. Antimicrobial activity was studied by standard disk-diffusional method. The hydrogel filmic coatings with antimicrobial action, insoluble at physiological conditions, with quantitative retaining of protein and hydrolytic activity of lysozyme were obtained. The product is characterized by the widened pH-profile of activity at acidic pH values, stability in acidic medium (pH 5.5 and at storage. Its antimicrobial action against Staphylococcus aureus ATCC 25923 F-49, Pseudomonas aeruginosa 415, Escherichia coli 055 K 59912/4 and Candida albicans ATCC 885-653 was noted. The proposed method of lysozyme immobilization allows to obtain stable, highly effective product with antimicrobial activity, prospective for usage in biomedical investigations.

  18. Kinetics of Competitive Adsorption between Lysozyme and Lactoferrin on Silicone Hydrogel Contact Lenses and the Effect on Lysozyme Activity. (United States)

    Hall, Brad; Jones, Lyndon; Forrest, James A


    To determine the effect of competitive adsorption between lysozyme and lactoferrin on silicone hydrogel contact lenses and the effect on lysozyme activity. Three commercially available silicone hydrogel contact lens materials (senofilcon A, lotrafilcon B and balafilcon A) were examined, for time points ranging from 10 s to 2 h. Total protein deposition was determined by I(125) radiolabeling of lysozyme and lactoferrin, while the activity of lysozyme was determined by a micrococcal activity assay. Senofilcon A and balafilcon A did not show any relevant competitive adsorption between lysozyme and lactoferrin. Lotrafilcon B showed reduced protein deposition due to competitive adsorption for lactoferrin at all time points and lysozyme after 7.5 min. Co-adsorption of lactoferrin and lysozyme decreased the activity of lysozyme in solution for senofilcon A and lotrafilcon B, but co-adsorption had no effect on the surface activity of lysozyme for all lens types investigated. Competition between lysozyme and lactoferrin is material specific. Co-adsorption of lysozyme and lactoferrin does not affect the activity of surface-bound lysozyme but can reduce the activity of subsequently desorbed lysozyme.

  19. Crystallization of lysozyme from lysozyme - ovalbumin mixtures: Separation potential and crystal growth kinetics (United States)

    Maosoongnern, Somchai; Flood, Chalongsri; Flood, Adrian E.; Ulrich, Joachim


    Lysozyme was successfully separated from mixtures of lysozyme and ovalbumin by crystallization. The purity of the lysozyme product is more than 98%, the remaining activity is greater than 97%, and the yields of the crystal products were greater than 80%. The experimental conditions used were varied to study the effect of the operating parameters on the growth kinetics of lysozyme crystal and the separation ability of the process. The growth rates of lysozyme are second order with respect to the relative supersaturation. Therefore the growth kinetics of the crystallization process is controlled by the surface integration mechanism. The calculated growth rate constants were 5.4×10-6 cm/h and 2.5×10-6 cm/h for the crystallization process at 20 °C and 10 °C, respectively. There is no significant effect of the ovalbumin impurity up to the concentration of 67.5% ovalbumin (based on total protein) on the growth kinetics of lysozyme. Changing the NaCl concentration from 4% to 3% had no effect on the growth kinetics of lysozyme, although this does change the solubility and therefore the yield. The calculated activation energy was 53.08 kJ/mol which supports the hypothesis that the crystallization process is controlled by the surface integration mechanism.

  20. Michler’s Hydrol Blue: A Sensitive Probe for Amyloid Fibril Detection

    KAUST Repository

    Kitts, Catherine C.


    Michler\\'s hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14°-22° to the fibril axis and along the grooves of the β-sheet. © 2011 American Chemical Society.

  1. Processing of Lysozyme by Macrophages: Identification of the Determinant Recognized by Two T-Cell Hybridomas (United States)

    Allen, Paul M.; Strydom, Daniel J.; Unanue, Emil R.


    The purpose of this study was to identify the fragment of the hen egg-white lysozyme (HEL) molecule presented by macrophages to helper T cells. This was investigated by using T-cell hybridomas and macrophages prefixed in paraformaldehyde. We previously had shown that such prefixed macrophages could present a tryptic digest of HEL. The tryptic peptides were separated by HPLC and tested for their ability to stimulate the T-cell hybridomas. Only one tryptic peptide was found to be immunogenic. This immunogenic peptide was identified as the tryptic peptide T-8, containing amino acids 46-61. The precise determinant on the peptide T-8 being recognized was further defined by testing the response of the two T-cell hybridomas to human lysozyme. Neither clone responded to human lysozyme. From the amino acid sequence of human lysozyme, the determinant was localized to the four amino-terminal residues. Cleavage of the immunogenic peptide with either chymotrypsin or protease V-8 completely abolished the immunogenicity. This suggested that the T-cell determinant is located in the hydrophilic amino-terminal residues and that it must be associated with a hydrophobic stretch of amino acids, which allows the peptide to associate with the macrophage plasma membrane.

  2. Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water (United States)

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


    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.

  3. Spectroscopic studies on the interaction between novel polyvinylthiol-functionalized silver nanoparticles with lysozyme. (United States)

    Ali, Mohd Sajid; Al-Lohedan, Hamad A; Rafiquee, M Z A; Atta, Ayman M; Ezzat, Abdurrahman O


    Silver nanoparticles were functionalized with polyvinylthiol (Ag-PVT) and their effect on the conformation of hen-egg white lysozyme was seen by means of spectroscopic techniques, viz., UV visible, fluorescence (intrinsic and synchronous), resonance Rayleigh scattering and circular dichroism. UV absorption spectra of lysozyme show a hyperchromic shift on the addition of Ag-PVT nanoparticles indicating the complex formation between the two. The interaction between lysozyme and Ag-PVT nanoparticles was takes place via static quenching with 1:1 binding ratio as revealed by the analysis of fluorescence measurements. Circular dichroism spectroscopic data show a decrease in α-helical content of lysozyme on interaction with Ag-PVT nanoparticles which was due to the partial unfolding of the protein. Synchronous fluorescence spectroscopy disclosed that the microenvironments of both tryptophan and tyrosine residues were perturbed in the presence of Ag-PVT nanoparticles and perturbation in the tryptophan environment was more prominent. Rayleigh scattering (RRS) intensity increases on increasing the Ag-PVT nanoparticles concentration till it reaches to the saturation. The RRS intensity increases four times as compared to the native protein indicating the possibility of protein aggregation at higher concentrations of nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Amyloid Fibril Solubility. (United States)

    Rizzi, L G; Auer, S


    It is well established that amyloid fibril solubility is protein specific, but how solubility depends on the interactions between the fibril building blocks is not clear. Here we use a simple protein model and perform Monte Carlo simulations to directly measure the solubility of amyloid fibrils as a function of the interaction between the fibril building blocks. Our simulations confirms that the fibril solubility depends on the fibril thickness and that the relationship between the interactions and the solubility can be described by a simple analytical formula. The results presented in this study reveal general rules how side-chain-side-chain interactions, backbone hydrogen bonding, and temperature affect amyloid fibril solubility, which might prove to be a powerful tool to design protein fibrils with desired solubility and aggregation properties in general.

  5. Echinacoside inhibits amyloid fibrillization of HEWL and protects against Aβ-induced neurotoxicity. (United States)

    Zhang, Di; Li, Hua; Wang, Jian-Bo


    Amyloid protein depositions play crucial roles in a variety of degenerative disorders composing amyloidosis. There is a great interest in developing small molecule inhibitors of amyloidogenic processes. We examined the inhibitory effects of echinacoside (ECH) with different concentrations and at different fiber-forming stages in vitro utilizing the hen egg-white lysozyme (HEWL) model system. We also evaluated the antioxidant capacity of ECH by using elimination tests for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (HO) free radicals. We investigated the protection provided by ECH against neurotoxicity induced by β-amyloid protein (Aβ). Through spectroscopic analyses, electron microscopy, cell viability assay, and hemolysis assay, we found that ECH dose dependently inhibited HEWL aggregation, and this inhibition occurred in different fiber-forming stages. ECH could also scavenge the DPPH and OH free radicals in a concentration-dependent manner. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2',7'-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescent measurement results indicated that ECH could increase viability of rat pheochromocytoma PC12 cells injured by Aβ and suppress the increase in intracellular reactive oxygen species (ROS) triggered by Aβ. The present study findings facilitate a better understanding of the interaction between ECH and amyloid-forming proteins and also shed light on the protection of ECH against amyloid fibril-induced neuronal cell death. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Lysozyme resistance in Streptococcus suis is highly variable and multifactorial.

    Directory of Open Access Journals (Sweden)

    Paul J Wichgers Schreur

    Full Text Available BACKGROUND: Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen. RESULTS: The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed the involvement of autolysin activity and capsule structures in lysozyme resistance of S. suis. CONCLUSIONS: This study shows that lysozyme resistance levels are highly variable among S. suis isolates and serotypes. Furthermore, the results show that lysozyme resistance in S. suis can involve different mechanisms including OatA-mediated peptidolycan modification, autolysin

  7. Investigation of the interactions between aptamer and misfolded proteins: From monomer and oligomer to fibril by single-molecule force spectroscopy. (United States)

    Zheng, Yan; Wang, Qing; Yang, Xiaohai; Li, Zhiping; Gao, Lei; Zhang, Hua; Nie, Wenyan; Geng, Xiuhua; Wang, Kemin


    Increasing knowledge on the understanding interactions of aptamer with misfolded proteins (including monomer, oligomer, and amyloid fibril) is crucial for development of aggregation inhibitors and diagnosis of amyloid diseases. Herein, the interactions of lysozyme monomer-, oligomer-, and amyloid fibril-aptamer were investigated using single-molecule force spectroscopy. The results revealed that the aptamer screened against lysozyme monomer could also bind to oligomer and amyloid fibril, in spite of the recognition at a lower binding probability. It may be attributed to the inherent structural differences of misfolded proteins and the flexible conformation of aptamer. In addition, dynamic force spectra showed that there were similar dissociation paths in the dissociation process of lysozyme monomer-, oligomer-, and amyloid fibril-aptamer complexes. It showed that the dissociation only passed 1 energy barrier from the binding state to the detachment. However, the dynamic parameters suggested that the oligomer- and amyloid fibril-aptamer were more stable than lysozyme monomer-aptamer. The phenomena may result from the exposure of aptamer-recognized sequences on the surface and the electrostatic interactions. This work demonstrated that single-molecule force spectroscopy could be a powerful tool to study the binding behavior of the aptamer with misfolded proteins at single-molecule level, providing abundant information for researches and comprehensive applications of aptamer probes in diagnosis of amyloid diseases. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Influence of the ionic liquid 1-butyl-3-methylimidazolium bromide on amyloid fibrillogenesis in lysozyme: Evidence from photophysical and imaging studies. (United States)

    Basu, Anirban; Bhattacharya, Subhash Chandra; Kumar, Gopinatha Suresh


    Many proteins can abnormally fold to form pathological amyloid deposits/aggregates that are responsible for various degenerative disorders called amyloidosis. Here we have examined the anti-amyloidogenic potency of an ionic liquid, 1-butyl-3-methylimidazolium bromide, using lysozyme as a model system. Thioflavin T fluorescence assay demonstrated that the ionic liquid suppressed the formation of lysozyme fibrils significantly. This observation was further confirmed by the Congo red assay. Fluorescence microscopy, intrinsic fluorescence studies, nile red fluorescence assay, ANS binding assay and circular dichroism studies also testified diminishing of the fibrillogenesis in the presence of ionic liquid. Formation of amyloid fibrils was also characterized by α to β conformational transition. From far-UV circular dichroism studies it was observed that the β-sheet content of the lysozyme samples decreased in the presence of the ionic liquid which in turn implied that fibrillogenesis was supressed by the ionic liquid. Atomic force microscopy imaging unequivocally established that the ionic liquid attenuated fibrillogenesis in lysozyme. These results may be useful for the development of more effective therapeutics for amyloidosis. Copyright © 2017. Published by Elsevier B.V.

  9. Aptamer-Based Electrochemical Sensing of Lysozyme

    Directory of Open Access Journals (Sweden)

    Alina Vasilescu


    Full Text Available Protein analysis and quantification are required daily by thousands of laboratories worldwide for activities ranging from protein characterization to clinical diagnostics. Multiple factors have to be considered when selecting the best detection and quantification assay, including the amount of protein available, its concentration, the presence of interfering molecules, as well as costs and rapidity. This is also the case for lysozyme, a 14.3-kDa protein ubiquitously present in many organisms, that has been identified with a variety of functions: antibacterial activity, a biomarker of several serious medical conditions, a potential allergen in foods or a model of amyloid-type protein aggregation. Since the design of the first lysozyme aptamer in 2001, lysozyme became one of the most intensively-investigated biological target analytes for the design of novel biosensing concepts, particularly with regards to electrochemical aptasensors. In this review, we discuss the state of the art of aptamer-based electrochemical sensing of lysozyme, with emphasis on sensing in serum and real samples.

  10. Sodium dodecyl sulphate (SDS) induced changes in propensity and kinetics of α-lactalbumin fibrillation. (United States)

    Kumar, E Kiran; Qumar, Shamsul; Prabhu, N Prakash


    Understanding surfactants induced changes on protein folding, aggregation, and fibrillation has a lot of implications in their laboratory and industrial applications. The effect of an anionic surfactant, sodium dodecyl sulphate (SDS), on fibrillation of an acidic protein α-lactalbumin (α-LA) at neutral pH condition was investigated. SDS at lower concentrations increased the lag time by nearly two-fold whereas the fibril elongation rate was not significantly altered. At the concentrations above 0.2mM, SDS lengthened the lag time by many-fold (∼60), but fibril elongation was accelerated by 3-6 fold. At the concentrations above 2mM, SDS inhibited α-LA fibrillation and led it to the formation of amorphous aggregates. These results were compared with the effect of SDS on the fibrillation of lysozyme, a basic protein. Though fibril inhibition was observed on both the proteins at the micellar concentrations of SDS, there were differences in the effect on lag time and elongation rate at the lower concentrations of SDS. This suggests that the inhibition of protein fibrillation by SDS-micelles might be a common mechanism irrespective of the surface charges on protein. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Atrial Ectopics Precipitating Atrial Fibrillation

    Directory of Open Access Journals (Sweden)

    Johnson Francis


    Full Text Available Holter monitor tracing showing blocked atrial ectopics and atrial ectopic precipitating atrial fibrillation is being demonstrated. Initially it was coarse atrial fibrillation, which rapidly degenerated into fine atrial fibrillation.

  12. Lysozyme pattern formation in evaporating droplets (United States)

    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 Ph

  13. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells. (United States)

    Sowa-Jasiłek, Aneta; Zdybicka-Barabas, Agnieszka; Stączek, Sylwia; Wydrych, Jerzy; Skrzypiec, Krzysztof; Mak, Paweł; Deryło, Kamil; Tchórzewski, Marek; Cytryńska, Małgorzata


    The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Effects of molecular weight of hyaluronic acid on its viscosity and enzymatic activities of lysozyme and peroxidase. (United States)

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


    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.

  15. Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod. (United States)

    Seppola, Marit; Bakkemo, Kathrine Ryvold; Mikkelsen, Helene; Myrnes, Bjørnar; Helland, Ronny; Irwin, David M; Nilsen, Inge W


    Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

  16. Amyloid Fibrils from Hemoglobin. (United States)

    Jayawardena, Nadishka; Kaur, Manmeet; Nair, Smitha; Malmstrom, Jenny; Goldstone, David; Negron, Leonardo; Gerrard, Juliet A; Domigan, Laura J


    Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of β-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-β quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (-20 to 80 °C) and pHs (2-10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.

  17. Amyloid Fibrils from Hemoglobin

    Directory of Open Access Journals (Sweden)

    Nadishka Jayawardena


    Full Text Available Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM and atomic force microscopy (AFM, with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of β-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-β quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (−20 to 80 °C and pHs (2–10, and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.

  18. The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates (United States)

    Forsythe, Elizabeth; Pusey, Marc Lee


    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.

  19. The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates (United States)

    Forsythe, Elizabeth; Lee Pusey, Marc


    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 ˜4 to ˜63. Both decreasing temperature and increasing salt concentrations shifted plots of the growth rate versus C/ Csat 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.

  20. [Atrial fibrillation and stroke]. (United States)

    Aamodt, Anne Hege; Sandset, Per Morten; Atar, Dan; Tveit, Arnljot; Russell, David


    More than 70,000 Norwegians have atrial fibrillation, which is a major risk factor for ischemic stroke. A large proportion of ischemic strokes caused by atrial fibrillation could be prevented if patients receive optimal prophylactic treatment. This article describes the risk for ischemic stroke in patients with atrial fibrillation, and discusses who should receive prophylactic treatment and which therapy provides the best prevention. The article is based on recently published European, American and Canadian guidelines, a search in PubMed and the authors' own clinical experience. The new risk score CHA2DS2-VASc is better than the CHADS2 score for identifying patients with atrial fibrillation who have a truly low risk of ischemic stroke and are not in need of antithrombotic treatment. Oral anticoagulation therapy is recommended for patients with two or more risk factors for thromboembolism in addition to atrial fibrillation (CHA2DS2-VASc ≥ 2). Patients with atrial fibrillation and a single additional risk factor (CHA2DS2-VASc =1) an individual assessment should be made as to who should receive oral anticoagulants, and for patients with CHA2DS2-VASc = 0 antithrombotic treatment is not recommended. New oral anticoagulants are at least as effective as warfarin for preventing ischemic stroke in patients with nonvalvular atrial fibrillation, they carry a lower risk of cerebral haemorrhage, especially intracranial haemorrhage and are more practical in use. Platelet inhibitors have a minimal role in stroke prevention in patients with atrial fibrillation. Risks stratifying patients using the CHA2DS2-VASc score is a better method for assessing which patients with atrial fibrillation who should receive oral anticoagulation. The introduction of new oral anticoagulants will simplify preventive treatment and hopefully lead to a more efficient anticoagulation treatment in a larger number of patients with atrial fibrillation.

  1. Influence of lysozyme treatments on champagne base wine foaming properties. (United States)

    Marchal, R; Chaboche, D; Douillard, R; Jeandet, P


    The objective of this study was to estimate the effect of lysozyme on the foaming characteristics of Champagne base wine. Lysozyme additions were made to the musts and also to the wines before and after bentonite or charcoal treatments, which remove endogenous proteins. Treatments with bentonite diminished foamability and foam stability of wines, whatever the dose (30 or 80 g/hL) and variety [Chardonnay, -28%; Pinot noir, -20% (at 30 g/hL)]. An addition of lysozyme in must raised Pinot noir wine foamability by 21%, whereas the difference is hardly perceptible for Chardonnay wine (+3%). Pinot noir and Chardonnay wines, originating from lysozyme-treated musts, in addition to bentonite treatment on the wine, presented higher foamability than wines treated only with bentonite. Lysozyme was removed (91-100%) by the bentonite treatment. Then, it was not responsible for the increase in foamability but seemed to have a protective effect on the wine proteins. When wines were initially treated with bentonite (150 g/hL) and then enriched with 80 g/hL lysozyme, this enzyme was not able to restore foaming properties. Treatments with charcoal always diminished foamability. The average increase in foamability due to an addition of lysozyme after charcoal treatment (80 g/hL) was 23%. Results showed a real positive effect of lysozyme on foam stability when wines have to be treated with charcoal (+25% and +56% for the Pinot noir wine and the Chardonnay wine, respectively, at 30 g/hL).

  2. Expression of T4 Lysozyme Gene (gene e) in Streptococcus ...

    African Journals Online (AJOL)

    The lysozyme enzymes expressing by these bacteria were found to be active on Micrococcus luteus cells and thereby preventing their growth on assay plates. Thermostability of these enzymes from the recombinant bacteria was also found different from each other. The lysozyme expressed by S. salivarius subsp.

  3. Diversification and adaptive sequence evolution of Caenorhabditis lysozymes (Nematoda: Rhabditidae). (United States)

    Schulenburg, Hinrich; Boehnisch, Claudia


    Lysozymes are important model enzymes in biomedical research with a ubiquitous taxonomic distribution ranging from phages up to plants and animals. Their main function appears to be defence against pathogens, although some of them have also been implicated in digestion. Whereas most organisms have only few lysozyme genes, nematodes of the genus Caenorhabditis possess a surprisingly large repertoire of up to 15 genes. We used phylogenetic inference and sequence analysis tools to assess the evolution of lysozymes from three congeneric nematode species, Caenorhabditis elegans, C. briggsae, and C. remanei. Their lysozymes fall into three distinct clades, one belonging to the invertebrate-type and the other two to the protist-type lysozymes. Their diversification is characterised by (i) ancestral gene duplications preceding species separation followed by maintenance of genes, (ii) ancestral duplications followed by gene loss in some of the species, and (iii) recent duplications after divergence of species. Both ancestral and recent gene duplications are associated in several cases with signatures of adaptive sequence evolution, indicating that diversifying selection contributed to lysozyme differentiation. Current data strongly suggests that genetic diversity translates into functional diversity. Gene duplications are a major source of evolutionary innovation. Our analysis provides an evolutionary framework for understanding the diversification of lysozymes through gene duplication and subsequent differentiation. This information is expected to be of major value in future analysis of lysozyme function and in studies of the dynamics of evolution by gene duplication.

  4. Diversification and adaptive sequence evolution of Caenorhabditis lysozymes (Nematoda: Rhabditidae

    Directory of Open Access Journals (Sweden)

    Boehnisch Claudia


    Full Text Available Abstract Background Lysozymes are important model enzymes in biomedical research with a ubiquitous taxonomic distribution ranging from phages up to plants and animals. Their main function appears to be defence against pathogens, although some of them have also been implicated in digestion. Whereas most organisms have only few lysozyme genes, nematodes of the genus Caenorhabditis possess a surprisingly large repertoire of up to 15 genes. Results We used phylogenetic inference and sequence analysis tools to assess the evolution of lysozymes from three congeneric nematode species, Caenorhabditis elegans, C. briggsae, and C. remanei. Their lysozymes fall into three distinct clades, one belonging to the invertebrate-type and the other two to the protist-type lysozymes. Their diversification is characterised by (i ancestral gene duplications preceding species separation followed by maintenance of genes, (ii ancestral duplications followed by gene loss in some of the species, and (iii recent duplications after divergence of species. Both ancestral and recent gene duplications are associated in several cases with signatures of adaptive sequence evolution, indicating that diversifying selection contributed to lysozyme differentiation. Current data strongly suggests that genetic diversity translates into functional diversity. Conclusion Gene duplications are a major source of evolutionary innovation. Our analysis provides an evolutionary framework for understanding the diversification of lysozymes through gene duplication and subsequent differentiation. This information is expected to be of major value in future analysis of lysozyme function and in studies of the dynamics of evolution by gene duplication.

  5. Adaptive functional diversification of lysozyme in insectivorous bats. (United States)

    Liu, Yang; He, Guimei; Xu, Huihui; Han, Xiuqun; Jones, Gareth; Rossiter, Stephen J; Zhang, Shuyi


    The role of gene duplication in generating new genes and novel functions is well recognized and is exemplified by the digestion-related protein lysozyme. In ruminants, duplicated chicken-type lysozymes facilitate the degradation of symbiotic bacteria in the foregut. Chicken-type lysozyme has also been reported to show chitinase-like activity, yet no study has examined the molecular evolution of lysozymes in species that specialize on eating insects. Insectivorous bats number over 900 species, and lysozyme expression in the mouths of some of these species is associated with the ingestion of insect cuticle, suggesting a chitinase role. Here, we show that chicken-type lysozyme has undergone multiple duplication events in a major family of insect-eating bats (Vespertilionidae) and that new duplicates have undergone molecular adaptation. Examination of duplicates from two insectivorous bats-Pipistrellus abramus and Scotophilus kuhlii-indicated that the new copy was highly expressed in the tongue, whereas the other one was less tissue-specific. Functional assays applied to pipistrelle lysozymes confirmed that, of the two copies, the tongue duplicate was more efficient at breaking down glycol chitin, a chitin derivative. These results suggest that the evolution of lysozymes in vespertilionid bats has likely been driven in part by natural selection for insectivory. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail:

  6. Small angle neutron scattering study of lysozyme solutions (United States)

    Boué, F.; Lefaucheux, F.; Robert, M. C.; Rosenman, I.


    In order to investigate how macromolecular aggregation proceeds for obtaining nucleation and crystal growth, a series of hen egg white (HEW) lysozyme solutions representative of a large supersaturation range have been investigated by small angle neutron scattering (SANS). In all these solutions, the signal well corresponds to species of radii of gyration Rg below 50 Å. Moreover, the effective values Rg are compatible with a rather monodisperse distribution of species the size of which increases when the supersaturation increases; for example, saturated solutions correspond to dimer populations. The largest size which has been identified corresponds to octomers which seems a limit beyond which nucleation and growth occur. The growth units are larger than dimers and probably correspond to tetramers or octomers. SANS allows one to study kinetic aspects. We observe that when a given supersaturation is quickly established, the radius of gyration increases with time. In the light of these results, it appears that SANS affords a powerful tool to study aggregation phenomena occurring in the metastable zone.

  7. Atrial Fibrillation and Stroke (United States)

    ... faulty heart valves, lung disease, and stimulant or alcohol abuse. Some people will have no identifiable cause for their AF. × Definition Atrial fibrillation (AF) describes the rapid, irregular beating ...

  8. Atrial fibrillation - discharge (United States)

    Auricular fibrillation - discharge; A-fib - discharge; AF - discharge; Afib - discharge ... Avoid salty and fatty foods. Stay away from fast-food restaurants. ... how to check your pulse, and check it every day. It is better ...

  9. Molecular dynamics simulations of lysozyme in water/sugar solutions

    Energy Technology Data Exchange (ETDEWEB)

    Lerbret, A. [Department of Food Science, Cornell University, 101 Stocking Hall, Ithaca, NY 14853 (United States); Affouard, F. [Laboratoire de Dynamique et Structure des Materiaux Moleculaires, UMR CNRS 8024, Universite Lille I, 59655 Villeneuve d' Ascq Cedex (France)], E-mail:; Bordat, P. [Laboratoire de Chimie Theorique et de Physico-Chimie Moleculaire, UMR 5624, Universite de Pau et des Pays de l' Adour, 64000 Pau (France); Hedoux, A.; Guinet, Y.; Descamps, M. [Laboratoire de Dynamique et Structure des Materiaux Moleculaires, UMR CNRS 8024, Universite Lille I, 59655 Villeneuve d' Ascq Cedex (France)


    Structural and dynamical properties of the solvent at the protein/solvent interface have been investigated by molecular dynamics simulations of lysozyme in trehalose, maltose and sucrose solutions. Results are discussed in the framework of the bioprotection phenomena. The analysis of the relative concentration of water oxygen atoms around lysozyme suggests that lysozyme is preferentially hydrated. When comparing the three sugars, trehalose is seen more excluded than maltose and sucrose. The preferential exclusion of sugars from the protein surface induces some differences in the behavior of trehalose and maltose, particularly at 50 and 60 wt% concentrations, that are not observed experimentally in binary sugar/mixtures. The dynamical slowing down of the solvent is suggested to mainly arise from the homogeneity of the water/sugar matrices controlled by the percolation of the sugar hydrogen bonds networks. Furthermore, lysozyme strongly increases relaxation times of solvent molecules at the protein/solvent interface.

  10. The direct piezoelectric effect in the globular protein lysozyme (United States)

    Stapleton, A.; Noor, M. R.; Sweeney, J.; Casey, V.; Kholkin, A. L.; Silien, C.; Gandhi, A. A.; Soulimane, T.; Tofail, S. A. M.


    Here, we present experimental evidence of the direct piezoelectric effect in the globular protein, lysozyme. Piezoelectric materials are employed in many actuating and sensing applications because they can convert mechanical energy into electrical energy and vice versa. Although originally studied in inorganic materials, several biological materials including amino acids and bone, also exhibit piezoelectricity. The exact mechanisms supporting biological piezoelectricity are not known, nor is it known whether biological piezoelectricity conforms strictly to the criteria of classical piezoelectricity. The observation of piezoelectricity in protein crystals presented here links biological piezoelectricity with the classical theory of piezoelectricity. We quantify the direct piezoelectric effect in monoclinic and tetragonal aggregate films of lysozyme using conventional techniques based on the Berlincourt Method. The largest piezoelectric effect measured in a crystalline aggregate film of lysozyme was approximately 6.5 pC N-1. These findings raise fundamental questions as to the possible physiological significance of piezoelectricity in lysozyme and the potential for technical applications.

  11. Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance. (United States)

    Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah


    Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The DeltaEF_0783 mutant and DeltaEF_0783 DeltaEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and DeltaEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of DeltaEF_0783 and DeltaEF_0783 DeltaEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis.

  12. Modeling the Mechanical Response of Tetragonal Lysozyme Crystals


    Zamiri, Amir; De, Suvranu


    In this paper we investigate the mechanical response of tetragonal lysozyme crystals based on existing experimental data and a continuum-based crystal plasticity model. Compression analyses along different crystallographic directions reveal that the mechanical response of lysozyme crystals is highly anisotropic and orientation dependent. While the response is purely elastic along the [110] direction, it is elasto-plastic along the [100] and [2̄ 12] directions. The yield stress and critical re...

  13. Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces. (United States)

    Subramanian, Palaniappan; Lesniewski, Adam; Kaminska, Izabela; Vlandas, Alexis; Vasilescu, Alina; Niedziolka-Jonsson, Joanna; Pichonat, Emmanuelle; Happy, Henri; Boukherroub, Rabah; Szunerits, Sabine


    The paper reports on a surface plasmon resonance (SPR)-based approach for the sensitive and selective detection of lysozyme. The SPR sensor consists of a 50 nm gold film coated with a thin film of reduced graphene oxide (rGO) functionalized with anti-lysozyme DNA aptamer. The SPR chip coating with rGO matrix was achieved through electrophoretic deposition of graphene oxide (GO) at 150 V. Electrophoretic deposition resulted in partial reduction of GO to rGO with a thickness depending on the deposition time. For very short time pulses of 20 s, the resulting rGO film had a thickness of several nanometers and was appropriate for SPR sensing. The utility of the graphene-based SPR sensor for the selective and sensitive detection of proteins was demonstrated using lysozyme as model protein. Functionalization of rGO matrix with anti-lysozyme DNA aptamer through π-stacking interactions allowed selective SPR detection of lysozyme. The graphene-based SPR biosensor provides a means for the label-free, concentration-dependent and selective detection of lysozymes with a detection limit of 0.5 nM. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Atrial Fibrillation and Hyperthyroidism

    Directory of Open Access Journals (Sweden)

    Jayaprasad N


    Full Text Available Atrial fibrillation occurs in 10 – 15% of patients with hyperthyroidism. Low serum thyrotropin concentration is an independent risk factor for atrial fibrillation. Thyroid hormone contributes to arrythmogenic activity by altering the electrophysiological characteristics of atrial myocytes by shortening the action potential duration, enhancing automaticity and triggered activity in the pulmonary vein cardio myocytes. Hyperthyroidism results in excess mortality from increased incidence of circulatory diseases and dysrhythmias. Incidence of cerebral embolism is more in hyperthyroid patients with atrial fibrillation, especially in the elderly and anti-coagulation is indicated in them. Treatment of hyperthyroidism results in conversion to sinus rhythm in up to two-third of patients. Beta-blockers reduce left ventricular hypertrophy and atrial and ventricular arrhythmias in patients with hyperthyroidism. Treatment of sub clinical hyperthyroidism is controversial. Optimizing dose of thyroxine treatment in those with replacement therapy and beta-blockers is useful in exogenous subclinical hyperthyroidism.

  15. Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media

    Directory of Open Access Journals (Sweden)

    Woo Jeong-Im


    Full Text Available Abstract Background Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity. Methods Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M-/- mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of S. pneumoniae 6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation. Results Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M-/- mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M-/- mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with S. pneumoniae 6B and resulted in severe middle ear inflammation, compared to wild type mice. Conclusion The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.

  16. Salt-specific effects in lysozyme solutions

    Directory of Open Access Journals (Sweden)

    T. Janc


    Full Text Available The effects of additions of low-molecular-mass salts on the properties of aqueous lysozyme solutions are examined by using the cloud-point temperature, T_{cloud}, measurements. Mixtures of protein, buffer, and simple salt in water are studied at pH=6.8 (phosphate buffer and pH=4.6 (acetate buffer. We show that an addition of buffer in the amount above I_{buffer} = 0.6 mol dm^{-3} does not affect the T_{cloud} values. However, by replacing a certain amount of the buffer electrolyte by another salt, keeping the total ionic strength constant, we can significantly change the cloud-point temperature. All the salts de-stabilize the solution and the magnitude of the effect depends on the nature of the salt. Experimental results are analyzed within the framework of the one-component model, which treats the protein-protein interaction as highly directional and of short-range. We use this approach to predict the second virial coefficients, and liquid-liquid phase diagrams under conditions, where T_{cloud} is determined experimentally.

  17. Structural characteristics of hydration sites in lysozyme. (United States)

    Soda, Kunitsugu; Shimbo, Yudai; Seki, Yasutaka; Taiji, Makoto


    A new method is presented for determining the hydration site of proteins, where the effect of structural fluctuations in both protein and hydration water is explicitly considered by using molecular dynamics simulation (MDS). The whole hydration sites (HS) of lysozyme are composed of 195 single HSs and 38 clustered ones (CHS), and divided into 231 external HSs (EHS) and 2 internal ones (IHS). The largest CHSs, 'Hg' and 'Lβ', are the IHSs having 2.54 and 1.35 mean internal hydration waters respectively. The largest EHS, 'Clft', is located in the cleft region. The real hydration structure of a CHS is an ensemble of multiple structures. The transition between two structures occurs through recombinations of some H-bonds. The number of the experimental X-ray crystal waters is nearly the same as that of the estimated MDS hydration waters for 70% of the HSs, but significantly different for the rest of HSs. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress. (United States)

    Abey, Sarah K; Yuana, Yuana; Joseph, Paule V; Kenea, Natnael D; Fourie, Nicolaas H; Sherwin, LeeAnne B; Gonye, Gregory E; Smyser, Paul A; Stempinski, Erin S; Boulineaux, Christina M; Weaver, Kristen R; Bleck, Christopher K E; Henderson, Wendy A


    Stress has demonstrated effects on inflammation though underlying cell-cell communication mechanisms remain unclear. We hypothesize that circulating RNAs and extracellular vesicles (EVs) in patients with chronic stress contain signals with functional roles in cell repair. Blood transcriptome from patients with Irritable Bowel Syndrome versus controls were compared to identify signaling pathways and effectors. Plasma EVs were isolated (size-exclusion chromatography) and characterized for effectors' presence (immunogold labelling-electron microscopy). Based on transcriptome pathways and EV-labelling, lysozyme's effects on cell migration were tested in human colon epithelial CRL-1790 cells and compared to the effects of CXCL12, a migration inducer (wound assay). The effect of lysozyme on immune-linked mRNA and protein levels in cells which survived following serum starvation and scratch wound were investigated (NanoString). Blood transcriptomes revealed pyridoxal 5'phosphate salvage, pyrimidine ribonucleotides salvage pathways, atherosclerosis, and cell movement signaling with membrane CD9 and extracellular lysozyme as effectors. Plasma EVs showed labelling with CD9, mucins, and lysozyme. This is the first identification of lysozyme on plasma EVs. In CRL-1790 cells, lysozyme induced migration and repaired scratch wound as well as CXCL12. Immune mRNA and protein expressions were altered in cells which survived following serum starvation and scratch wound, with or without lysozyme in serum-free media post-wounding: CD9, IL8, IL6 mRNAs and CD9, NT5E, PD-L1 proteins. Repair and inflammatory signals are identified in plasma EVs and circulating RNAs in chronic stress. Registered #NCT00824941. This study highlights the role of circulating RNAs and EVs in stress.

  19. Encapsulation and controlled release of lysozyme from electrospun poly(epsilon-caprolactone)/poly(ethylene glycol) non-woven membranes by formation of lysozyme-oleate complexes. (United States)

    Li, Yan; Jiang, Hongliang; Zhu, Kangjie


    In this study, the concept of hydrophobic ion pairing was adopted for incorporating lysozyme into electrospun poly(epsilon-caprolactone) (PCL)/poly(ethylene glycol) (PEG) non-woven membranes. The solubility of lysozyme in organic solvent was enhanced through the formation of lysozyme-oleate complexes, which could be directly loaded into PCL/PEG membranes using electrospinning technique. The resultant PCL/PEG nanofibers have a compact structure with an average diameter ranged from about 0.4 microm to 0.9 microm. The addition of PEG into the PCL nanofibers not only improved the hydrophilicity of the membrane, but also played an important role on in vitro lysozyme release rate. It was found that the release rate of lysozyme was enhanced with the increase of PEG content. In addition, the increase of salt concentration in the release medium accelerated lysozyme release. It has also been shown that the released lysozyme retained most of its enzymatic activity.

  20. Crystallization of lysozyme in pores of etched heavy-ion tracks (United States)

    Dobrev, D.; Baur, D.; Neumann, R.


    Intensive studies of protein crystallization are provoked by the need to accumulate structural information and to open up novel potential applications in science and technology. This work focuses on the crystallization of hen egg white lysozyme (HEWL) in micrometer-size templates of etched swift-ion tracks produced in foils of polycarbonate, polyimide, and mica. HEWL deposition was accomplished by the salting with sodium chloride method at 20 and 6 °C. The crystals overgrowing the pore orifices showed no orientation when crystallized at room temperature. At 6 °C, they presented a regular habit and were uniaxially oriented on a polycarbonate substrate. Biaxial orientation was observed on polyimide membranes, and epitaxial orientation dominated on mica. However, no crystallization was observed on the mica membrane covered with a thin gold layer. The epitaxial growth of HEWL was particularly pronounced in the case of ordered substrates and low crystallization temperature, i.e. at higher supersaturation. By computer simulation it was possible to determine the surface lattice planes and thus provide the preferential crystal-growth orientation. Crystallization of lysozyme and other proteins in ion-track templates of submicrometer channels should be of high practical interest.

  1. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism. (United States)

    Jin, Yi; Petricevic, Marija; John, Alan; Raich, Lluís; Jenkins, Huw; Portela De Souza, Leticia; Cuskin, Fiona; Gilbert, Harry J; Rovira, Carme; Goddard-Borger, Ethan D; Williams, Spencer J; Davies, Gideon J


    The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.

  2. Assessment of antimicrobial activity of c-type lysozyme from Indian shrimp Fenneropenaeus indicus

    Directory of Open Access Journals (Sweden)

    Viswanathan Karthik


    Full Text Available Objective: To assess the multitudinal antimicrobial effects of recombinant lysozyme from Fenneropenaeus indicus (rFi-Lyz in comparison with commercially available recombinant hen egg white lysozyme (rHEWL. Methods: Antimicrobial activity of the recombinant rFi-Lyz using several Gram positive, Gram negative bacteria and fungi in comparison with rHEWL has been evaluated. rFi-Lyz was expressed and purified using Ni2+ affinity chromatography. The effect of rFi-Lyz in the growth of yeast Candida krusei, plant molds Rhizoctonia solani and Fusarium solani was assessed by well diffusion assay in petri plates with potato dextrose agar. Results: rFi-Lyz exhibited high inhibitory activity on Gram positive bacteria such as Staphylococcus aureus and Bacillus subtilis. Among various Gram negative bacteria tested Klebsiella pneumoniae exhibited the highest inhibition followed by Pseudomonas aeruginosa and Shigella dysenteriae. rFi-Lyz also exhibited significant inhibition on two marine pathogens Aeromonas veronii and Vibrio alginolyticus. Among the various fungal strains tested, rFi-Lyz inhibited the growth of budding yeast Candida krusei significantly. Further the growth of two other plants fungus Rhizoctonia solani and Fusarium oxysporum were retarded by rFi-Lyz in the plate inhibition assay. Conclusions: rFi-Lyz exhibits a broad spectrum of antimicrobial activity like a natural antibiotic on various pathogenic bacteria and fungal strains.

  3. Metal-assisted and microwave accelerated-evaporative crystallization: Application to lysozyme protein (United States)

    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.

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

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


    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.

  5. Use of europium ions for SAD phasing of lysozyme at the Cu Kα wavelength. (United States)

    Vijayakumar, Balakrishnan; Velmurugan, Devadasan


    Europium is shown to be a good anomalous scatterer in SAD phasing for solving the structure of biological macromolecules. The large value of the anomalous contribution of europium, f'' = 11.17 e(-), at the Cu Kα wavelength is an advantage in de novo phasing and automated model building. Tetragonal crystals of hen egg-white lysozyme (HEWL) incorporating europium(III) chloride (50 mM) were obtained which diffracted to a resolution of 2.3 Å at a wavelength of 1.54 Å (Cu Kα). The master data set (360° frames) was split and analyzed for anomalous signal-to-noise ratio, multiplicity, completeness, SAD phasing and automated building. The structure solution and model building of the split data sets were carried out using phenix.autosol and phenix.autobuild. The contributions of the Eu ions to SAD phasing using in-house data collection are discussed. This study revealed successful lysozyme phasing by SAD using laboratory-source data involving Eu ions, which are mainly coordinated by the side chains of Asn46, Asp52 and Asp101 together with some water molecules.

  6. Unfolding and aggregation of lysozyme: a thermodynamic and kinetic study by FTIR spectroscopy. (United States)

    Sassi, Paola; Giugliarelli, Alessandra; Paolantoni, Marco; Morresi, Assunta; Onori, Giuseppe


    The unfolding of hen egg-white lysozyme dissolved both in D(2)O and CH(3)CH(2)OD/D(2)O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Kinetics of surfactant-induced aggregation of lysozyme studied by fluorescence spectroscopy. (United States)

    Jain, Neha; Bhattacharya, Mily; Mukhopadhyay, Samrat


    The study of protein conformational changes in the presence of surfactants and lipids is important in the context of protein folding and misfolding. In the present study, we have investigated the mechanism of the protein conformational change coupled with aggregation leading to size growth of Hen Egg White Lysozyme (HEWL) in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS) in alkaline pH. We have utilized intrinsic protein fluorescence (tryptophan) and extrinsic fluorescent reporters such as 8-anilinonaphthalene-1-sulfonic acid (ANS), dansyl and fluorescein to follow the protein conformational change in real-time. By analyzing the kinetics of fluorescence intensity and anisotropy of multiple fluorescent reporters, we have been able to delineate the mechanism of surfactant-induced aggregation of lysozyme. The kinetic parameters reveal that aggregation proceeds with an initial fast-phase (conformational change) followed by a slow-phase (self-assembly). Our results indicate that SDS, below critical micelle concentration, induces conformational expansion that triggers the aggregation process at a micromolar protein concentration range. © Springer Science+Business Media, LLC 2010


    Directory of Open Access Journals (Sweden)

    Dekina S. S.


    Full Text Available The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells acetone powder was a substrate. Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, protein-polymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years, and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

  9. Modeling Tetragonal Lysozyme Crystal Growth Rates (United States)

    Gorti, Sridhar; Forsythe, Elizabeth L.; Pusey, Marc L.


    Tetragonal lysozyme 110 face crystal growth rates, measured over 5 orders of magnitude in range, can be described using a model where growth occurs by 2D nucleation on the crystal surface for solution supersaturations of c/c(sub eq) less than or equal to 7 +/- 2. Based upon the model, the step energy per unit length, beta was estimated to be approx. 5.3 +/- 0.4 x 10(exp -7) erg/mol-cm, which for a step height of 56 A corresponds to barrier of approx. 7 +/- 1 k(sub B)T at 300 K. For supersaturations of c/c(sub eq) > 8, the model emphasizing crystal growth by 2D nucleation not only could not predict, but also consistently overestimated, the highest observable crystal growth rates. Kinetic roughening is hypothesized to occur at a cross-over supersaturation of c/c(sub eq) > 8, where crystal growth is postulated to occur by a different process such as adsorption. Under this assumption, all growth rate data indicated that a kinetic roughening transition and subsequent crystal growth by adsorption for all solution conditions, varying in buffer pH, temperature and precipitant concentration, occurs for c/c(sub eq)(T, pH, NaCl) in the range between 5 and 10, with an energy barrier for adsorption estimated to be approx. 20 k(sub B)T at 300 K. Based upon these and other estimates, we determined the size of the critical surface nucleate, at the crossover supersaturation and higher concentrations, to range from 4 to 10 molecules.

  10. Ecofriendly gold nanoparticles - Lysozyme interaction: Thermodynamical perspectives. (United States)

    Roy, Swarup; Saxena, Shailendra K; Mishra, Suryakant; Yogi, Priyanka; Sagdeo, P R; Kumar, Rajesh


    In the featured work interaction between biosynthesized gold nanoparticles (GNP) and lysozyme (Lys) has been studied using multi-spectroscopic approach. A moderate association constant (Kapp) of 2.66×104L/mol has been observed indicative of interactive nature. The binding constant (Kb) was 1.99, 6.30 and 31.6×104L/mol at 291, 298 and 305K respectively and the number of binding sites (n) was found to be approximately one. Estimated values of thermodynamic parameters (Enthalpy change, ΔH=141.99kJ/mol, entropy change, ΔS=570J/mol/K, Gibbs free energy change, ΔG=-27.86kJ/mol at 298K) suggest hydrophobic force as the main responsible factor for the Lys-GNP interaction and also the process of interaction is spontaneous. The average binding distance (r=3.06nm) and the critical energy transfer distance (Ro=1.84nm) between GNP and Lys was also evaluated using Förster's non-radiative energy transfer (FRET) theory and results clearly indicate that non-radiative type energy transfer is possible. Moreover, the addition of GNP does not show any significant change in the secondary structure of Lys as confirmed from circular dichroism (CD) spectra. Furthermore, NMR spectroscopy also indicates interaction between Lys and GNP. The resulting insight is important for the better understanding of structural nature and thermodynamic aspects of binding between the Lys and GNP. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth (United States)

    Foley, Joseph; Hill, Shannon E.; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin


    Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the


    Directory of Open Access Journals (Sweden)

    I. M. Marusenko


    Full Text Available Review on a problem of the development of atrial fibrillation in patients with thyrotoxicosis is presented. Thyrotoxicosis is one of the most frequent endocrine diseases, conceding only to a diabetes mellitus. The most frequent reasons of hyperthyroidism are Graves’ disease and functional thyroid autonomy. The authors give an analysis of data on the cardiac effects of thyrotoxicosis, features of heart remodeling under the influence of thyroid hyperfunction, prevalence of atrial fibrillation in thyrotoxicosis, depending on age, as well as the possibility of restoring sinus rhythm in the combination of these diseases. Particular attention is paid to the effect on the heart of subclinical thyrotoxicosis, which is defined as a dysfunction of the thyroid gland, characterized by low serum concentration of thyrotropin, normal values of free thyroxine and free triiodothyronine. Subclinical hyperthyroidism is also capable of causing heart remodeling and diastolic dysfunction.Prevalence of thyrotoxicosis in elderly people is higher in areas of iodine deficiency; it is relevant for our country due to the large territory of iodine deficiency. In elderly patients, the cardiac effects of thyrotoxicosis prevail in the clinical picture, that makes it difficult to diagnose endocrine disorders, and correction of thyrotoxicosis is critically important for the successful control of the heart rhythm. The article also discusses the problem of thyrotoxic cardiomyopathy, caused by the toxic effect of excess thyroid hormones: features of this heart disorder, factors affecting its formation, clinical significance and contribution to the development of rhythm disturbances. The greatest significance is the development of atrial fibrillation as a result of thyrotox-icosis in older patients who already have various cardiovascular diseases.Atrial fibrillation is the most frequent heart rhythm disorder in thyrotoxicosis. The main cause of arrhythmia in hyperthyroidism is the

  13. Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme (United States)

    Nadarajah, Arunan


    This project was originally conceived to analyze the extensive data of tetragonal lysozyme crystal growth rates collected at NASA/MSFC by Dr. Marc L. Pusey's research group. At that time the lack of analysis of the growth rates was hindering progress in understanding the growth mechanism of tetragonal lysozyme and other protein crystals. After the project was initiated our initial analysis revealed unexpected complexities in the growth rate behavior. This resulted in an expansion in the scope of the project to include a comprehensive investigation of the growth mechanisms of tetragonal lysozyme crystals. A discussion of this research is included as well a list of presentations and publications resulting from the research. This project contributed significantly toward the education of several students and fostered extensive collaborations between investigators.

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

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


    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.

  15. Analysis of the nucleation and crystal growth kinetics of lysozyme by a theory of self-assembly


    Ataka, M.; Asai, M.


    Concentration changes in supersaturated solutions during the nucleation and growth of the orthorhombic form of hen egg-white lysozyme crystals have been observed for 121 d at 35 degrees C and pH 4.6, and with 3% NaCl. The effect of a variation in the initial protein concentration on the rate of approach to solubility in equilibrium is analyzed, by applying a model, originally developed for the understanding of protein self-assembly. It is shown that the observed kinetics can be explained fair...

  16. Spectrophotometric studies on the interaction between (-)-epigallocatechin gallate and lysozyme (United States)

    Ghosh, Kalyan Sundar; Sahoo, Bijaya Ketan; Dasgupta, Swagata


    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.


    NARCIS (Netherlands)



    Low molecular weight proteins (LMWPs), such as lysozyme, may be suitable carriers to target drugs to the kidney. In this study the antiinflammatory drug naproxen was covalently bound to lysozyme (1:1). Pharmacokinetics of the conjugate, naproxen-lysozyme (nap-LYSO), were compared to that of an

  18. A Vibrational Circular Dichroism Microsampling Accessory: Mapping Enhanced Vibrational Circular Dichroism in Amyloid Fibril Films. (United States)

    Lu, Xuefang; Li, Honggang; Nafie, Jordan W; Pazderka, Tomáš; Pazderková, Markéta; Dukor, Rina K; Nafie, Laurence A


    We report the first vibrational circular dichroism (VCD) measurement of spatial heterogeneity in a sample using infrared (IR) microsampling. Vibrational circular dichroism spectra are typically measured using a standard IR cell with an IR beam diameter of 10 mm or greater making it impossible to investigate the spatial heterogeneity of a solid film sample. We have constructed a VCD sampling assembly with either 3 mm or 1 mm spatial resolution. An XY-translation stage was used to measure spectra at different spatial locations producing IR and VCD maps of the sample. In addition, a rotating sample stage was employed using a dual photoelastic modulator (PEM) setup to suppress artifacts due to linear birefringence in solid-phase or film samples. Infrared and VCD mapping of an insulin fibril film has been carried out at both 3 and 1 mm spatial resolution, and lysozyme films were mapped at 1 mm resolution. The IR spectra of different spots vary in intensity due primarily to sample thickness. The changes in the VCD intensity across the map largely correlate to corresponding changes in the IR map. Closer inspection of the insulin map revealed changes in the relative intensities of the VCD spectra not present in the parent IR spectra, which indicated differences in the degree of supramolecular chirality of the fibrils in the various spatial regions. For lysozyme films, in addition to different degrees of supramolecular chirality, reversal of the net fibril chirality was observed. The large signal-to-noise ratio observed at 1 mm resolution implies the feasibility of further increasing the spatial resolution by one or two orders of magnitude for protein fibril film samples.

  19. Antimicrobial activity of lysozyme with special relevance to milk ...

    African Journals Online (AJOL)

    Lysozyme is a hydrolytic enzyme which has been purified from cells, secretions and tissues of virtually all living organisms and viruses. While this protein has been recognized to possess many physiological and functional properties, its high microbicidal activity remains, by far, the main virtue that explains the high attention ...

  20. Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial

    NARCIS (Netherlands)

    Wichgers, P.J.; Weeghel, van C.; Rebel, J.M.J.; Smits, M.A.; Putten, van J.P.M.; Smith, H.E.


    Background Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a

  1. Binding of Lysozyme to Spherical Poly(styrenesulfonate Gels

    Directory of Open Access Journals (Sweden)

    Martin Andersson


    Full Text Available Polyelectrolyte gels are useful as carriers of proteins and other biomacromolecules in, e.g., drug delivery. The rational design of such systems requires knowledge about how the binding and release are affected by electrostatic and hydrophobic interactions between the components. To this end we have investigated the uptake of lysozyme by weakly crosslinked spherical poly(styrenesulfonate (PSS microgels and macrogels by means of micromanipulator assisted light microscopy and small angle X-ray scattering (SAXS in an aqueous environment. The results show that the binding process is an order of magnitude slower than for cytochrome c and for lysozyme binding to sodium polyacrylate gels under the same conditions. This is attributed to the formation of very dense protein-rich shells in the outer layers of the microgels with low permeability to the protein. The shells in macrogels contain 60 wt % water and nearly charge stoichiometric amounts of lysozyme and PSS in the form of dense complexes of radius 8 nm comprising 30–60 lysozyme molecules. With support from kinetic modelling results we propose that the rate of protein binding and the relaxation rate of the microgel are controlled by the protein mass transport through the shell, which is strongly affected by hydrophobic and electrostatic interactions. The mechanism explains, in turn, an observed dependence of the diffusion rate on the apparent degree of crosslinking of the networks.

  2. Laser ablation dynamics and production of thin films of lysozyme

    DEFF Research Database (Denmark)

    Canulescu, Stela; Schou, Jørgen; Amoruso, S.

    Lysozyme is a well-known protein, which is used in food processing because of its bactericidal properties. The mass (14307 amu) is in the range in which it easily can be monitored by mass spectrometric methods, for example by MALDI (Matrix assisted laser desorption ionization). We have recently p...

  3. Laser ablation dynamics and production of thin films of lysozyme

    DEFF Research Database (Denmark)

    Schou, Jørgen; Canulescu, Stela; Matei, Andreea

    Lysozyme is a well-known protein, which is used in food processing because of its bacteriocidal properties. The mass (14307 u) is in the range, in which it easily can be controlled by mass spectrometric methods, for example by MALDI (Matrix assisted laser desorption ionisation). We have recently ...

  4. Complex coacervate core micelles with a lysozyme-modified corona

    NARCIS (Netherlands)

    Danial, M.; Klok, H.A.; Norde, W.; Cohen Stuart, M.A.


    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.

  5. Role of PAMAM-OH dendrimers against the fibrillation pathway of biomolecules. (United States)

    Sekar, Gajalakshmi; Florance, Ida; Sivakumar, A; Mukherjee, Amitava; Chandrasekaran, Natarajan


    The binding behavior of nanoparticle with proteins determines its biocompatibility. This study reports the interaction of ten different biomolecules (proteins-BSA, HSA, haemoglobin, gamma globulin, transferrin and enzymes-hog and bacillus amylase, lysozyme from chicken and human and laccases from Tramates versicolor) with a surface group hydroxylated Poly AMido AMide dendrimer (PAMAM) of generation 5. The study has utilized various spectroscopic methods like UV-vis spectroscopy, Fluorescence emission, Synchronous, 3-D spectroscopy and Circular Dichroism to detect the binding induced structural changes in biomolecules that occur upon interaction with mounting concentration of the dendrimers. Aggregation of proteins results in the formation of amyloid fibrils causing several human diseases. In this study, fibrillar samples of all ten biomolecules formed in the absence and the presence of dendrimers were investigated with Congo Red absorbance and ThT Assay to detect fibril formation, Trp Emission and 3-D scan to evaluate the effect of fibrillation on aromatic environment of biomolecules, and CD spectroscopy to measure the conformational changes in a quantitative manner. These assays have generated useful information on the role of dendrimers in amyloid fibril formation of biomolecules. The outcomes of the study remain valuable in evaluating the biological safety of PAMAM-OH dendrimers for their biomedical application in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Lactoferrin, lysozyme and immunoglobulin G content in milk of four breeds of cows managed under intensive production system. (United States)

    Król, J; Litwińczuk, Z; Brodziak, A; Barłowska, J


    The aim of the study was to analyze chosen antibacterial protein content, i.e. immunoglobulin G, lactoferrin and lysozyme in bovine milk obtained from cows of four breeds raised and maintained under intensive production technology, that is in the free stall system, and fed total mixed ration diet (TMR system). The studies were conducted on milk from four breeds of dairy cows maintained in Poland, i.e. Polish Holstein-Friesian Black-White and Red-White variety, Jersey and Simental. Milk samples were collected solely from healthy cows, having performed the TOK test with Mastirapid. Further analyses were made only on the milk samples with SCC determined under 2.0 x 10(5) cells/ml, i.e. 423 samples. Each sample was examined for lactoferrin and lysozyme determination using reversed-phase high-performance liquid chromatography (RP-HPLC) with UV-VIS detector. The immunoglobulin G (IgG) levels were established by the aid of radial immunodiffusion technique with Bovine IgG LL tests (The Binding Site, Birmingham, UK). It should be stated that a breed of cow had a significant effect on a content of the antimicrobial proteins analyzed in bovine milk evaluated. Milk obtained from Simental and Jersey cows prove to be their excellent source, i.e. lactoferrin (116.74 and 103.48 mg/l), lysozyme (9.84 and 13.02 microg/l) and immunoglobulin G (579.9 and 508.6 mg/l). The highest content of these protein was stated in milk of multiparous cows at the late stage of lactation.

  7. Use of Lysozyme as a Feed Additive on Rumen Fermentation and Methane Emission

    Directory of Open Access Journals (Sweden)

    Ashraf A. Biswas


    Full Text Available This study was conducted to determine the effect of lysozyme addition on in vitro rumen fermentation and to identify the lysozyme inclusion rate for abating methane (CH4 production. An in vitro ruminal fermentation technique was done using a commercial concentrate to rice straw ratio of 8:2 as substrate. The following treatments were applied wherein lysozyme was added into 1 mg dry matter substrate at different levels of inclusion: Without lysozyme, 2,000, 4,000, and 8,000 U lysozyme. Results revealed that, lysozyme addition had a significant effect on pH after 24 h of incubation, with the highest pH (p<0.01 observed in 8,000 U lysozyme, followed by the 4,000 U, 2,000 U, and without lysozyme. The highest amounts of acetic acid, propionic acid (p<0.01 and total volatile fatty acid (TVFA (p<0.05 were found in 8,000 U after 24 h of incubation. The CH4 concentration was the lowest in the 8,000 U and the highest in the without lysozyme addition after 24 h of incubation. There was no significant differences in general bacteria, methanogen, or protozoan DNA copy number. So far, addition of lysozyme increased the acetate, propionate, TVFA, and decreased CH4 concentration. These results suggest that lysozyme supplementation may improve in vitro rumen fermentation and reduce CH4 emission.

  8. Atrial fibrillation: inflammation in disguise?

    NARCIS (Netherlands)

    Lappegard, K.T.; Hovland, A.; Pop, G.A.M.; Mollnes, T.E.


    Atrial fibrillation is highly prevalent, and affected patients are at an increased risk of a number of complications, including heart failure and thrombo-embolism. Over the past years, there has been increasing interest in the role of inflammatory processes in atrial fibrillation, from the first

  9. Atrial fibrillation and female sex. (United States)

    Anselmino, Matteo; Battaglia, Alberto; Gallo, Cristina; Gili, Sebastiano; Matta, Mario; Castagno, Davide; Ferraris, Federico; Giustetto, Carla; Gaita, Fiorenzo


    Atrial fibrillation is the most common supraventricular arrhythmia. Its prevalence increases with age and preferentially affects male patients. Over 75 years of age, however, female patients being more prevalent, the absolute number of patients affected is similar between sexes. Despite this, few data are available in the literature concerning sex-related differences in atrial fibrillation patients. The present systematic review therefore considers comorbidities, referring symptoms, quality of life, pharmacological approaches and trans-catheter ablation in female rather than in male atrial fibrillation patients in search of parameters that may have an impact on the treatment outcome. In brief, female atrial fibrillation patients more commonly present comorbidities, leading to a higher prevalence of persistent atrial fibrillation; moreover, they refer to hospital care later and with a longer disease history. Atrial fibrillation symptoms relate to low quality of life in female patients; in fact, atrial fibrillation paroxysm usually presents higher heart rate, leading to preferentially adopt a rate rather than a rhythm-control strategy. Female atrial fibrillation patients present an increased risk of stroke, worsened by the lower oral anticoagulant prescription rate related to the concomitant higher haemorrhagic risk profile. Trans-catheter ablation is under-used in female patients and, on the contrary, they are more commonly affected by anti-arrhythmic drug side effects.

  10. Impact of lysozyme on stability mechanism of nanozirconia aqueous suspension

    Energy Technology Data Exchange (ETDEWEB)

    Szewczuk-Karpisz, Katarzyna, E-mail:; Wiśniewska, Małgorzata


    Highlights: • Adsorption and stabilization-destabilization properties of lysozyme (LSZ) in the nanozirconia-biopolymer solution system were determined. • The stability measurements were performed using turbidimetric method. • Lysozyme macromolecules undergo adsorption on the ZrO{sub 2} surface under electrostatic adsorbent-adsorbate attraction, i.e. at pH 6 and 9. • The biopolymer adsorption impact on the zirconia stability varies at different pH values. - Abstract: The effect of lysozyme (LSZ) presence on the zirconium(IV) oxide (ZrO{sub 2}) aqueous suspension stability was examined. The applied zirconia contains mesopores (with a diameter about 30 nm) and its mean particle size is about 100 nm. To determine the stability mechanism of ZrO{sub 2} suspension in the biopolymer presence, the adsorption and electrokinetic (surface charge density and zeta potential) measurements were performed in the pH range 3–10. The lysozyme adsorption on the nanozirconia surface proceeds mainly through electrostatic forces. Under solid-polymer repulsion conditions, there is no adsorption of lysozyme (pH < 6, C{sub NaCl} 0.01 mol/dm{sup 3}). The increase of solution ionic strength to 0.2 mol/dm{sup 3} causes screening of unfavourable forces and biopolymer adsorption becomes possible. The LSZ addition to the ZrO{sub 2} suspension influences its stability. At pH 3, 4.6 and 7.6, slight improvement of the system stability was obtained. In turn, at pH 9 considerable destabilization of nanozirconia particles covered by polymeric layers occurs.

  11. On the characterization of intermediates in the isodesmic aggregation pathway of hen lysozyme at alkaline pH.

    Directory of Open Access Journals (Sweden)

    Vijay Kumar Ravi

    Full Text Available Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer's, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM-0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6 ± 0.6 × 10(-5 s(-1. Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.

  12. Dementia and Atrial Fibrillation

    DEFF Research Database (Denmark)

    Pastori, Daniele; Miyazawa, Kazuo; Lip, Gregory Y H


    The risk of developing dementia is increased in patients with atrial fibrillation (AF), with the incidence of both conditions increasing with aging. Patients with dementia frequently do not receiving adequate thrombo-prophylaxis, because of the inability to monitor INR and/or to achieve...... in therapeutic range during VKAs therapy, the assessment of cognitive impairment may help identify those patients who may benefit from switching to NOACs. In conclusion, patients with AF and dementia benefit from anticoagulation and should not be denied receiving adequate stroke prevention. Cognitive function...

  13. Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Lise Vanderkelen

    Full Text Available Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme, and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme. Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors.

  14. Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion


    Hill, Shannon E.; Robinson, Joshua; Matthews, Garrett; Muschol, Martin


    The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relev...

  15. Modeling the Mechanical Response of Tetragonal Lysozyme Crystals (United States)

    Zamiri, Amir; De, Suvranu


    In this paper we investigate the mechanical response of tetragonal lysozyme crystals based on existing experimental data and a continuum-based crystal plasticity model. Compression analyses along different crystallographic directions reveal that the mechanical response of lysozyme crystals is highly anisotropic and orientation dependent. While the response is purely elastic along the [110] direction, it is elasto-plastic along the [100] and [2̄ 12] directions. The yield stress and critical resolved shear stress are observed to be sensitive to temperature and the amount of intracrystalline water. An increase in temperature and the amount of intracrystalline water molecules leads to a decrease in the critical resolved shear stress of the slip systems and makes the crystal softer. The analysis presented in this paper may be applied to the study of other protein crystal systems as well as their optimal design for biotechnological applications. PMID:20039601

  16. Screening for Atrial Fibrillation

    DEFF Research Database (Denmark)

    Freedman, Ben; Camm, A. John; Calkins, Hugh


    in September 2015 to promote discussion and research about AF screening as a strategy to reduce stroke and death and to provide advocacy for implementation of country-specific AF screening programs. During 2016, 60 expert members of AF-SCREEN, including physicians, nurses, allied health professionals, health......Approximately 10% of ischemic strokes are associated with atrial fibrillation (AF) first diagnosed at the time of stroke. Detecting asymptomatic AF would provide an opportunity to prevent these strokes by instituting appropriate anticoagulation. The AF-SCREEN international collaboration was formed...... or by intermittent ECG recordings over 2 weeks is not a benign condition and, with additional stroke factors, carries sufficient risk of stroke to justify consideration of anticoagulation. With regard to the methods of mass screening, handheld ECG devices have the advantage of providing a verifiable ECG trace...

  17. Tear lipocalin, lysozyme and lactoferrin concentrations in postmenopausal women. (United States)

    Careba, I; Chiva, A; Totir, M; Ungureanu, E; Gradinaru, Sinziana


    Among the most frequently encountered pathologies examined by the ophthalmologist is dry eye syndrome (DE), which can be discovered particularly in the elderly. The initial diagnosis of DE is of high importance, but also challenging. This is because the biochemical changes in the tear film often develop before any detectable signs. In this study, the possible relationship between ocular symptomatology, tear volume and tear break-up time (TBUT) and lipocalin, lactoferrin and lysozyme concentrations in the tear film were explored in a group of symptomatic dry-eyed postmenopausal (PM) women compared to age-matched controls. Sixty-six healthy PM females with ages of at least 50 years were grouped in two homogeneous lots (by age, post-menopause, co-morbidities) of 33 females each, one lot presenting mild or moderate dry eye syndrome (DE) and one asymptomatic non-dry eye (NDE), based on their feedback to the Ocular Surface Disease Index (OSDI) questionnaire and noninvasive TBUT and Schirmer test results. Tears were collected via capillary tubes and an eye wash method. Tear lysozyme, lactoferrin and lipocalin concentrations were determined via electrophoresis. OSDI responses revealed 3 mild DE, 30 moderate DE and 33 NDE. The OSDI total score and sub scores for the DE group were significantly greater than for the NDE group (p lactoferrin concentration (plactoferrin concentrations. In a PM population, lipocalin and lysozyme are invariable, irrespective of the presence and severity of DE symptoms. However, lactoferrin shows a significant decrease. This is a comprehensive study of lipocalin, lactoferrin and lysozyme in dry-eyed PM women and our results suggested that lactoferrin could be used as a biomarker of DE in postmenopausal women. PM = postmenopausal; DE = dry eye disease; NDE = non-dry eye; ELISA = Enzyme-linked immunosorbent assay.

  18. Pharmacological Treatment for Atrial Fibrillation

    Directory of Open Access Journals (Sweden)

    Kaoru Sugi, MD PhD


    Full Text Available Pharmacological treatment for atrial fibrillation has a variety of purposes, such as pharmacological defibrillation, maintenance of sinus rhythm, heart rate control to prevent congestive heart failure and prevention of both cerebral infarction and atrial remodeling. Sodium channel blockers are superior to potassium channel blockers for atrial defibrillation, while both sodium and potassium channel blockers are effective in the maintenance of sinus rhythm. In general, digitalis or Ca antagonists are used to control heart rate during atrial fibrillation to prevent congestive heart failure, while amiodarone or bepridil also reduce heart rates during atrial fibrillation. Anticoagulant therapy with warfarin is recommended to prevent cerebral infarction and angiotensin converting enzyme antagonists or angiotensin II receptor blockers are also used to prevent atrial remodeling. One should select appropriate drugs for treatment of atrial fibrillation according to the patient's condition.

  19. Nonlinear dynamics in ventricular fibrillation. (United States)

    Hastings, H M; Evans, S J; Quan, W; Chong, M L; Nwasokwa, O


    Electrogram recordings of ventricular fibrillation appear complex and possibly chaotic. However, sequences of beat-to-beat intervals obtained from these recordings are generally short, making it difficult to explicitly demonstrate nonlinear dynamics. Motivated by the work of Sugihara on atmospheric dynamics and the Durbin-Watson test for nonlinearity, we introduce a new statistical test that recovers significant dynamical patterns from smoothed lag plots. This test is used to show highly significant nonlinear dynamics in a stable canine model of ventricular fibrillation.

  20. Study on the expression of human lysozyme in oviduct bioreactor mediated by recombinant avian adeno-associated virus. (United States)

    Wang, A P; Wang, Y J; Wu, S; Zuo, W Y; Guo, C M; Hong, W M; Zhu, S Y


    Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins. © 2017 Poultry Science Association Inc.

  1. Fibril fragmentation enhances amyloid cytotoxicity. (United States)

    Xue, Wei-Feng; Hellewell, Andrew L; Gosal, Walraj S; Homans, Steve W; Hewitt, Eric W; Radford, Sheena E


    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.

  2. Rapid and Simple Purification of Lysozyme from the Egg Shell Membrane


    Kozuka, Miyuki; Murao, Sato; Yamane, Takuya; Inoue, Tsutomu; Ohkubo, Iwao; Ariga, Hiroyoshi


    Lysozyme (EC is a hydrolytic enzyme that cleaves the β-(1, 4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, a major bacterial cell wall polymer. In the food industry, lysozyme is used as an additive mainly in the production of wine and beer. Lysozyme was found to be localized in the egg shell membrane. In this study, we found that lysozyme was easily purified from the egg shell membrane and that the enzyme also had an antibacterial activity. ...

  3. Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease. (United States)

    Helmfors, Linda; Boman, Andrea; Civitelli, Livia; Nath, Sangeeta; Sandin, Linnea; Janefjord, Camilla; McCann, Heather; Zetterberg, Henrik; Blennow, Kaj; Halliday, Glenda; Brorsson, Ann-Christin; Kågedal, Katarina


    The hallmarks of Alzheimer disease are amyloid-β plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-β1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease. Copyright © 2015. Published by Elsevier Inc.

  4. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme.

    Directory of Open Access Journals (Sweden)

    Stephanie A Ragland


    Full Text Available Lysozyme is a cornerstone of innate immunity. The canonical mechanism for bacterial killing by lysozyme occurs through the hydrolysis of cell wall peptidoglycan (PG. Conventional type (c-type lysozymes are also highly cationic and can kill certain bacteria independently of PG hydrolytic activity. Reflecting the ongoing arms race between host and invading microorganisms, both gram-positive and gram-negative bacteria have evolved mechanisms to thwart killing by lysozyme. In addition to its direct antimicrobial role, more recent evidence has shown that lysozyme modulates the host immune response to infection. The degradation and lysis of bacteria by lysozyme enhance the release of bacterial products, including PG, that activate pattern recognition receptors in host cells. Yet paradoxically, lysozyme is important for the resolution of inflammation at mucosal sites. This review will highlight recent advances in our understanding of the diverse mechanisms that bacteria use to protect themselves against lysozyme, the intriguing immunomodulatory function of lysozyme, and the relationship between these features in the context of infection.

  5. Estimation of the initial equilibrium constants in the formation of tetragonal lysozyme nuclei (United States)

    Pusey, Marc L.


    Results are presented from a study of the equilibria, kinetic rates, and the aggregation pathway which leads from a lysozyme monomer crystal to a tetragonal crystal, using dialyzed and recrystallized commercial hen eggwhite lysozyme. Relative light scattering intensity measurements were used to estimate the initial equilibrium constants for undersaturated lysozyme solutions in the tetragonal regime. The K1 value was estimated to be (1-3) x 10 exp 4 L/mol. Estimates of subsequent equilibrium constants depend on the crystal aggregation model chosen or determined. Experimental data suggest that tetragonal lysozyme crystal grows by addition of aggregates preformed in the bulk solution, rather than by monomer addition.

  6. The interaction of lysozyme with caffeine, theophylline and theobromine in solution. (United States)

    Zhang, Hong-Mei; Tang, Bo-Ping; Wang, Yan-Qing


    The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV-Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)-lysozyme complex. The binding constants (K(A)) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.

  7. Bacteriolytic Activity Of Human Interleukin-2, Chicken Egg Lysozyme In The Presence Of Potential Effectors (United States)

    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.


    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

  8. Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme (United States)

    Iniesta, Jesús; Esclapez-Vicente, María Deseada; Heptinstall, John; Walton, David J.; Peterson, Ian R.; Mikhailov, Victor A.; Cooper, Helen J.


    In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20. PMID:21760652

  9. Atrial fibrillation in elite athletes. (United States)

    Furlanello, F; Bertoldi, A; Dallago, M; Galassi, A; Fernando, F; Biffi, A; Mazzone, P; Pappone, C; Chierchia, S


    Atrial fibrillation (AF) is a rare event in people younger than 25 years of age, but is probably more frequent in competitive athletes. We analyzed the presence of AF, paroxysmal or chronic, in a population of young elite athletes, including previous Olympic and World champions, who were studied for arrhythmias that endangered their athletic careers. From 1974 to June 1977, 1,772 athletes identified with arrhythmias (1,464 males and 308 females; mean age 21 years) underwent individualized work-ups. Among these, 146 (122 males and 24 females; mean age 24 years) were young elite athletes. They were studied from 1985 to 1997, with a mean follow-up of 62 months. Of the 146 young elite athletes, 13 (9%) had AF (paroxysmal in 11 and chronic in 2); all were male. The paroxysmal AF occurred during effort (n = 7), after effort (n = 1), or at rest (n = 3) and was reinduced by transesophageal pacing or endocavitary electrophysiologic testing under the same clinical circumstances. AF was the cause of symptoms in 13 (40%) of 22 young elite athletes with long-lasting palpitations. Five young elite athletes had a substrate for AF: Wolff-Parkinson-White syndrome (WPW) in 3, arrhythmogenic right ventricular dysplasia (ARVD) in 1, healed myocarditis in 1, and was considered idiopathic in 8. All elite athletes are alive with a mean follow-up of 62 months and 7 continue in their sports: 3 after radiofrequency catheter ablation (of WPW in 2 and AF with maze-type nonfluoroscopic approach in 1) and 4 after a period of de-training. AF, occurring in young elite athletes and affecting only males, is one of the most frequent causes of prolonged palpitations and is reproduced easily by transesophageal atrial pacing or electrophysiologic testing. AF may be a cause of disqualification from sports eligibility, but may disappear if the athletic activity is stopped for an adequate period of time, if trigger mechanisms are corrected (i.e., WPW), or if the substrate is modified.

  10. Manipulation of lysozyme phase behavior by additives as function of conformational stability. (United States)

    Galm, Lara; Morgenstern, Josefine; Hubbuch, Jürgen


    Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity. In this work the potency of different additives, namely glycerol, PEG 1000, and glycine, to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated. The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior, lysozyme solubility, crystal size and morphology. This work aims to link this pH dependent impact to a protein-specific parameter, the conformational stability of lysozyme. At pH 3 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine stabilized or destabilized lysozymes' native conformation resulting in a modified size of the crystallization area without influencing lysozyme solubility, crystal size and morphology. Addition of 1 M glycine even promoted non-native aggregation at pH 3 whereas addition of PEG 1000 completely inhibited non-native aggregation. At pH 5 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine did not influence lysozymes' native conformation, but strongly influenced the position of the crystallization area, lysozyme solubility, crystal size and morphology. The observed pH dependent impact of the additives could be linked to a differing lysozyme conformational stability in the binary systems without additives at pH 3 and pH 5. However, in any case lysozyme phase behavior could selectively be manipulated by addition of glycerol, PEG 1000 and glycine. Furthermore, at pH 5 crystal size and morphology could selectively be manipulated. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Mapping the solid-state properties of crystalline lysozyme during pharmaceutical unit-operations. (United States)

    Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T


    Bulk crystallisation of protein therapeutic molecules towards their controlled drug delivery is of interest to the biopharmaceutical industry. The complexity of biotherapeutic molecules is likely to lead to complex material properties of crystals in the solid state and to complex transitions. This complexity is explored using batch crystallised lysozyme as a model. The effects of drying and milling on the solid-state transformations of lysozyme crystals were monitored using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), FT-Raman, and enzymatic assay. XRPD was used to characterise crystallinity and these data supported those of crystalline lysozyme which gave a distinctive DSC thermogram. The apparent denaturation temperature (Tm) of the amorphous lysozyme was ∼201 °C, while the Tm of the crystalline form was ∼187 °C. Raman spectra supported a more α-helix rich structure of crystalline lysozyme. This structure is consistent with reduced cooperative unit sizes compared to the amorphous lysozyme and is consistent with a reduction in the Tm of the crystalline form. Evidence was obtained that milling also induced denaturation in the solid-state, with the denatured lysozyme showing no thermal transition. The denaturation of the crystalline lysozyme occurred mainly through its amorphous form. Interestingly, the mechanical denaturation of lysozyme did not affect its biological activity on dissolution. Lysozyme crystals on drying did not become amorphous, while milling-time played a crucial role in the crystalline-amorphous-denatured transformations of lysozyme crystals. DSC is shown to be a key tool to monitor quantitatively these transformations. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. White Toenails (United States)

    ... this page. Please enable Javascript in your browser. White Toenails White toenails can develop for several reasons. Trauma, such ... trauma does not cause broken blood vessels, a white spot may appear under the nail. The spot ...

  13. Effects of hesperidin, a flavanone glycoside interaction on the conformation, stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies? (United States)

    Ratnaparkhi, Aditi; Muthu, Shivani A; Shiriskar, Sonali M; Pissurlenkar, Raghuvir R S; Choudhary, Sinjan; Ahmad, Basir


    Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and possess ability to go through the blood-brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer's, Parkinson's, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP-HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (Ka, ~ 5 × 10(4) M(-1)) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.

  14. Atrial fibrillation in KCNE1-null mice

    NARCIS (Netherlands)

    Temple, Joel; Frias, Patricio; Rottman, Jeffrey; Yang, Tao; Wu, Yuejin; Verheijck, E. Etienne; Zhang, Wei; Siprachanh, Chanthaphaychith; Kanki, Hideaki; Atkinson, James B.; King, Paul; Anderson, Mark E.; Kupershmidt, Sabina; Roden, Dan M.


    Although atrial fibrillation is the most common serious cardiac arrhythmia, the fundamental molecular pathways remain undefined. Mutations in KCNQ1, one component of a sympathetically activated cardiac potassium channel complex, cause familial atrial fibrillation, although the mechanisms in vivo are

  15. Effects of prolonged running in the heat and cool environments on selected physiological parameters and salivary lysozyme responses

    Directory of Open Access Journals (Sweden)

    Nur S. Ibrahim


    Conclusion: This study found similar lysozyme responses between both hot and cool trials. Thus, room/ambient temperature did not affect lysozyme responses among recreational athletes. Nevertheless, the selected physiological parameters were significantly affected by room temperature.

  16. Salivary Lysozyme in Relation to Dental Caries among Thai Preschoolers. (United States)

    Lertsirivorakul, J; Petsongkram, B; Chaiyarit, P; Klaynongsruang, S; Pitiphat, W


    The objective of this study was to analyze salivary lysozyme levels and activities in Thai preschoolers with different dental caries status. Unstimulated saliva samples were collected from 64 preschoolers, divided into a caries free group (n = 32) and a severe early childhood caries (S-ECC) group (n = 32). Both groups were similar regarding gender, age, dental caries status, salivary flow rate, and salivary protein concentrations. No differences were also in the caregivers' characteristics, oral health behaviors, and feeding habits. Only professional fluoride application was less frequently found in the S-ECC group (p caries free group (pcaries.

  17. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates. (United States)

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E


    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  18. Production of active lysozyme films by matrix assisted pulsed laser evaporation at 355 nm

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Kingshott, P.


    Thin lysozyme films have been produced in a dry environment by MAPLE (matrix assisted pulsed laser evaporation) from a water ice matrix irradiated by laser light at 355 nm above the absorption threshold of the protein. A significant part of the lysozyme molecules are transferred to the film without...

  19. Midgut lysozymes of Lucilia sericata - new antimicrobials involved in maggot debridement therapy. (United States)

    Valachova, I; Takac, P; Majtan, J


    Larvae of Lucilia sericata are used for maggot debridement therapy (MDT) because of their ability to remove necrotic tissue and eradicate bacterial pathogens of infected wounds. So far, very few antibacterial factors have been fully characterized (eg lucifensin). Using a molecular approach, some other putative antimicrobial compounds, including three novel lysozymes, have been previously identified and predicted to be involved in MDT. Nevertheless, data on lysozymes tissue origin and their functions have never been elucidated. Therefore, the aim of this study was to investigate the expression of three lysozymes in L. sericata and confirm their antibacterial effects within MDT. Moreover, we characterized the eradication process of bacteria within the digestive system of maggots and determined the role of lysozymes in this process. We found that three lysozymes are expressed in specific sections of the L. sericata midgut. Recombinant lysozymes displayed comparable antibacterial activity against Micrococcus luteus. Furthermore, the majority of Gram-positive bacteria were destroyed in vivo within the particular section of the L. sericata midgut where lysozymes are produced. Larval ingestion and subsequent eradication of wound pathogens during their passage through the intestine of maggots are due to, at least in part, antibacterial action of three midgut lysozymes. © 2014 The Royal Entomological Society.

  20. Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Jiwoo [College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu 41566 (Korea, Republic of); Ku, Sae-Kwang [Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 38610 (Korea, Republic of); Lee, Suyeon [College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu 41566 (Korea, Republic of); Bae, Jong-Sup, E-mail: [College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu 41566 (Korea, Republic of)


    Lysozyme, found in relatively high concentration in blood, saliva, tears, and milk, protects us from the ever-present danger of bacterial infection. Previous studies have reported proinflammatory responses of endothelial cells to the release of polyphosphate(PolyP). In this study, we examined the anti-inflammatory responses and mechanisms of lysozyme and its effects on PolyP-induced septic activities in human umbilical vein endothelial cells (HUVECs) and mice. The survival rates, septic biomarker levels, behavior of human neutrophils, and vascular permeability were determined in PolyP-activated HUVECs and mice. Lysozyme suppressed the PolyP-mediated vascular barrier permeability, upregulation of inflammatory biomarkers, adhesion/migration of leukocytes, and activation and/or production of nuclear factor-κB, tumor necrosis factor-α, and interleukin-6. Furthermore, lysozyme demonstrated protective effects on PolyP-mediated lethal death and the levels of the related septic biomarkers. Therefore, these results indicated the therapeutic potential of lysozyme on various systemic inflammatory diseases, such as sepsis or septic shock. -- Highlights: •PolyP is shown to be an important mediator of vascular inflammation. •Lysozyme inhibited PolyP-mediated hyperpermeability. •Lysozyme inhibited PolyP-mediated septic response. •Lysozyme reduced PolyP-induced septic mortality.

  1. Identification of lysozyme activity from two edible bivalves - Perna viridis (Linnaeus) and Meretrix casta (Chemnitz)

    Digital Repository Service at National Institute of Oceanography (India)

    Sharma, S.; Tanu; Chatterji, A.

    the mantle fluid and meat, had more lysozyme activity as compared to the acetic acid extract. The extract of P. viridis showed a better activity than M. casta. Partial purification of lysozyme was carried out using gel filtration chromatography and initial...

  2. Surface morphology of thin lysozyme films produced by matrix-assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Pryds, Nini


    Thin films of the protein, lysozyme, have been deposited by the matrix-assisted pulsed laser evaporation (MAPLE) technique. Frozen targets of 0.3-1.0 wt.% lysozyme dissolved in ultrapure water were irradiated by laser light at 355 mn with a fluence of 2 J/cm(2). The surface quality of the thin...

  3. Isothermal Titration Calorimetry and Macromolecular Visualization for the Interaction of Lysozyme and Its Inhibitors (United States)

    Wei, Chin-Chuan; Jensen, Drake; Boyle, Tiffany; O'Brien, Leah C.; De Meo, Cristina; Shabestary, Nahid; Eder, Douglas J.


    To provide a research-like experience to upper-division undergraduate students in a biochemistry teaching laboratory, isothermal titration calorimetry (ITC) is employed to determine the binding constants of lysozyme and its inhibitors, N-acetyl glucosamine trimer (NAG[subscript 3]) and monomer (NAG). The extremely weak binding of lysozyme/NAG is…

  4. Analysis of the nucleation and crystal growth kinetics of lysozyme by a theory of self-assembly. (United States)

    Ataka, M; Asai, M


    Concentration changes in supersaturated solutions during the nucleation and growth of the orthorhombic form of hen egg-white lysozyme crystals have been observed for 121 d at 35 degrees C and pH 4.6, and with 3% NaCl. The effect of a variation in the initial protein concentration on the rate of approach to solubility in equilibrium is analyzed, by applying a model, originally developed for the understanding of protein self-assembly. It is shown that the observed kinetics can be explained fairly well by this model, whose basic assumptions are that (a) the nucleation is induced by aggregation of i0 molecules into particular geometry, and (b) the growth proceeds via attachment of a monomer. The i0 value for this process is four, which agrees with the number of molecules in a unit cell. Similarity and dissimilarity of the observed crystal growth to that of low molecular weight substances are discussed.

  5. Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae). (United States)

    Kwon, Hyojung; Bang, Kyeongrin; Lee, Minsup; Cho, Saeyoull


    A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 µM, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms.

  6. Molecular mechanism of lysozyme adsorption onto chemically modified alginate guar gum matrix. (United States)

    Brassesco, Ma Emilia; Woitovich Valetti, Nadia; Picó, Guillermo


    The equilibrium isotherms and adsorption kinetics of lysozyme (LZ) on epichlorohydrin (Epi) cross-linked alginate-guar gum (Alg-GG) matrix were studied. Adsorption kinetics followed a pseudo-first-order model while the equilibrium isotherm could be represented by the Freundlich equation. The maximal amount of LZ adsorbed onto this matrix was around 2.4mg per g of hydrated matrix at pH 7.00. The adsorption mechanism was associated to a simple diffusion process with a weak columbic interaction between LZ and the matrix. The presence of NaCl 0.3M induced a total displacement of the LZ from the matrix. Under this condition, the percentage of desorbed protein was 95%. Successive cycles of adsorption-washing-elution were performed and the results showed the reversibility of the process and the usefulness of the method for enzyme purification and separation. A last successful step was carried out for the purification of LZ from egg white as natural source. The model proved to be useful applied as a platform design in the isolation and purification of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster. (United States)

    Sandin, Linnea; Bergkvist, Liza; Nath, Sangeeta; Kielkopf, Claudia; Janefjord, Camilla; Helmfors, Linda; Zetterberg, Henrik; Blennow, Kaj; Li, Hongyun; Nilsberth, Camilla; Garner, Brett; Brorsson, Ann-Christin; Kågedal, Katarina


    Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1-42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  8. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Directory of Open Access Journals (Sweden)

    Jessica L Hastie


    Full Text Available σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP. In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme.

  9. Lysozyme's lectin-like characteristics facilitates its immune defense function

    KAUST Repository

    Zhang, Ruiyan


    Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

  10. Terahertz mechanical vibrations in lysozyme: Raman spectroscopy vs modal analysis (United States)

    Carpinteri, Alberto; Lacidogna, Giuseppe; Piana, Gianfranco; Bassani, Andrea


    The mechanical behaviour of proteins is receiving an increasing attention from the scientific community. Recently it has been suggested that mechanical vibrations play a crucial role in controlling structural configuration changes (folding) which govern proteins biological function. The mechanism behind protein folding is still not completely understood, and many efforts are being made to investigate this phenomenon. Complex molecular dynamics simulations and sophisticated experimental measurements are conducted to investigate protein dynamics and to perform protein structure predictions; however, these are two related, although quite distinct, approaches. Here we investigate mechanical vibrations of lysozyme by Raman spectroscopy and linear normal mode calculations (modal analysis). The input mechanical parameters to the numerical computations are taken from the literature. We first give an estimate of the order of magnitude of protein vibration frequencies by considering both classical wave mechanics and structural dynamics formulas. Afterwards, we perform modal analyses of some relevant chemical groups and of the full lysozyme protein. The numerical results are compared to experimental data, obtained from both in-house and literature Raman measurements. In particular, the attention is focused on a large peak at 0.84 THz (29.3 cm-1) in the Raman spectrum obtained analyzing a lyophilized powder sample.

  11. White Ring; White ring

    Energy Technology Data Exchange (ETDEWEB)

    Aoki, H.; Yuzawa, H. [Nikken Sekkei Ltd., Osaka (Japan)


    White Ring is a citizen`s gymnasium used for figure skating and short track speed skating games of 18th Winter Olympic Games in 1998. White Ring is composed of a main-arena and a sub-arena. For the main-arena with an area 41mtimes66m, an ice link can be made by disengaging the potable floor and by flowing brine in the bridged polystyrene pipes embedded in the concrete floor. Due to the fortunate groundwater in this site, well water is used for the outside air treatment energy in 63% during heating and in 35% during cooling. Ammonia is used as a cooling medium for refrigerating facility. For the heating of audience area in the large space, heat load from the outside is reduced by enhancing the heat insulation performance of the roof of arena. The audience seats are locally heated using heaters. For the White Ring, high quality environment is realized for games through various functions of the large-scale roof of the large space. Success of the big event was expected. 15 figs., 4 tabs.

  12. Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC. (United States)

    Holen, E; Elsayed, S


    Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.

  13. Atrial fibrillation and delayed gastric emptying.

    Directory of Open Access Journals (Sweden)

    Isadora C Botwinick

    Full Text Available BACKGROUND: Atrial fibrillation and delayed gastric emptying (DGE are common after pancreaticoduodenectomy. Our aim was to investigate a potential relationship between atrial fibrillation and DGE, which we defined as failure to tolerate a regular diet by the 7(th postoperative day. METHODS: We performed a retrospective chart review of 249 patients who underwent pancreaticoduodenectomy at our institution between 2000 and 2009. Data was analyzed with Fisher exact test for categorical variables and Mann-Whitney U or unpaired T-test for continuous variables. RESULTS: Approximately 5% of the 249 patients included in the analysis experienced at least one episode of postoperative atrial fibrillation. Median age of patients with atrial fibrillation was 74 years, compared with 66 years in patients without atrial fibrillation (p = 0.0005. Patients with atrial fibrillation were more likely to have a history of atrial fibrillation (p = 0.03. 92% of the patients with atrial fibrillation suffered from DGE, compared to 46% of patients without atrial fibrillation (p = 0.0007. This association held true when controlling for age. CONCLUSION: Patients with postoperative atrial fibrillation are more likely to experience delayed gastric emptying. Interventions to manage delayed gastric function might be prudent in patients at high risk for postoperative atrial fibrillation.

  14. Managing atrial fibrillation in active patients and athletes. (United States)

    Reiss, R A


    Atrial fibrillation in young or middle-aged active patients can often be managed with medication. Evaluation should address associated conditions and predisposing factors such as idiopathic hypertrophic subaortic stenosis, Wolff-Parkinson-White syndrome, congenital heart disease, hyperthyroidism, excess alcohol or other drug use, and exercise-induced catecholamine release. Diagnostic studies may include an ECG, 24-hour Holter or event monitoring, exercise treadmill testing, stress echocardiography, electrophysiologic studies, and laboratory testing. Electrocardioversion provides rapid, predictable treatment, but ablation therapy is sometimes needed.

  15. Personalized management of atrial fibrillation

    DEFF Research Database (Denmark)

    Kirchhof, Paulus; Breithardt, Günter; Aliot, Etienne


    The management of atrial fibrillation (AF) has seen marked changes in past years, with the introduction of new oral anticoagulants, new antiarrhythmic drugs, and the emergence of catheter ablation as a common intervention for rhythm control. Furthermore, new technologies enhance our ability to de...

  16. Fracture mechanics of collagen fibrils

    DEFF Research Database (Denmark)

    Svensson, Rene B; Mulder, Hindrik; Kovanen, Vuokko


    Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fib...

  17. Genetic aspects of atrial fibrillation

    NARCIS (Netherlands)

    Wiesfeld, ACP; Hemels, MEW; Van Tintelen, JP; Van den Berg, MP; Van Veldhuisen, DJ; Van Gelder, IC


    Atrial fibrillation (AF) occurs predominantly in the elderly and is commonly associated with underlying cardiac diseases. A significant number of patients, however, have early onset AF that is not associated with any underlying disease. At present, it is unknown how often this form of AF is familial

  18. Deglutition-Induced Atrial Fibrillation (United States)

    Malik, Amyn; Ali, Syed Sohail; Rahmatullah, Amin


    We present the case of 38-year-old woman who experienced palpitations on swallowing, which were later found to be atrial fibrillation. Her symptoms improved on treatment with disopyramide and verapamil. Within 9 months, she was weaned from both medications without recurrence of symptoms. PMID:16429915

  19. Multistate Mechanism of Lysozyme Denaturation through Synchronous Analysis of Raman Spectra. (United States)

    Xing, Lei; Lin, Ke; Zhou, Xiaoguo; Liu, Shilin; Luo, Yi


    The denaturation mechanism of hen egg lysozyme is still controversial. In this study, Raman spectroscopy was employed to study the thermal and chemical denaturation mechanisms of lysozyme. All of the Raman bands were synchronously recorded and analyzed during the denaturation process. It was found that the Raman bands of the side groups changed before the bands of skeleton groups. This directly reveals the three-state mechanism of thermal denaturation of lysozyme. The preferential change of the side groups was also observed in the chemical denaturation of lysozyme by guanidine hydrochloride. Moreover, it was found that the Raman bands of the groups on the surface of lysozyme changed before those of the other groups. This indicates that the chemical denaturants interact with the protein surface before the protein core in each step and the chemical denaturation of lysozyme conforms to the multistate and outside-in mechanisms. The synchronous Raman study not only reveals the multistate mechanism of lysozyme denaturation but also demonstrates that this synchronous Raman analysis is a powerful method to study the denaturation mechanisms of other proteins.

  20. Microbial community related to lysozyme digestion process for boosting waste activated sludge biodegradability. (United States)

    Xin, Xiao-Dong; He, Jun-Guo; Qiu, Wei; Tang, Jian; Liu, Tian-Tian


    Waste activated sludge from a lab-scale sequencing batch reactor was used to investigate the potential relation of microbial community with lysozyme digestion process for sludge solubilization. The results showed the microbial community shifted conspicuously as sludge suffered lysozyme digestion. Soluble protein and polysaccharide kept an increasing trend in solution followed with succession of microbial community. The rise of lysozyme dosage augmented the dissimilarity among communities in various digested sludge. A negative relationship presented between community diversity and lysozyme digestion process under various lysozyme/TS from 0 to 240min (correlation coefficient R(2) exceeded 0.9). Pareto-Lorenz curves demonstrated that microbial community tended to be even with sludge disintegration process by lysozyme. Finally, with diversity (H) decrease and community distribution getting even, the SCOD/TCOD increased steadily in solution which suggested the sludge with high community diversity and uneven population distribution might have tremendous potential for improving their biodegradability by lysozyme digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation. (United States)

    De Bernardez Clark, E; Hevehan, D; Szela, S; Maachupalli-Reddy, J


    Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species. On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields. Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation. Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation. Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.

  2. Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine. (United States)

    Dias, Rita; Vilas-Boas, Eduardo; Campos, Francisco M; Hogg, Tim; Couto, José António


    This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme. (United States)

    Sowa-Jasiłek, Aneta; Zdybicka-Barabas, Agnieszka; Stączek, Sylwia; Wydrych, Jerzy; Mak, Paweł; Jakubowicz, Teresa; Cytryńska, Małgorzata


    The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Selectivity and localization of lysozyme uptake in contemporary hydrogel contact lens materials. (United States)

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


    The purpose of this study was to investigate the early and selective uptake of lysozyme and the location of deposited lysozyme on contemporary hydrogel contact lens (CL) materials after exposure to an artificial tear solution (ATS) for 16 h. Seven different hydrogel CL materials [polymacon, omafilcon A, nelfilcon A, nesofilcon A, ocufilcon B, etafilcon A (Acuvue Moist), and etafilcon A (Acuvue Define)] were incubated in an ATS for various times. Total protein deposition was determined using a modified Bradford technique. Lysozyme, lactoferrin, and albumin deposition on CLs were determined using (125)I-radiolabeling method. A confocal laser scanning microscopy (CLSM) technique was utilized to map the location of lysozyme uptake in an asymmetric environment. All lens materials had significant amounts of lysozyme after 1 min of exposure to ATS. After 16 h of incubation, higher levels of total protein deposited on the two etafilcon A-based lenses (Moist and Define), followed by ocufilcon B and both were significantly higher than all other CLs tested (p = 0.0001). The two etafilcon A materials (Moist and Define) also deposited the highest amounts of lysozyme (514.8 ± 28.4 and 527.1 ± 14.7 μg/lens respectively) when compared to other test CLs (p = 0.0001). The CLSM technique revealed that the non-ionic CLs tended to have symmetric distribution of lysozyme throughout the lens materials, while the ionic CLs had an asymmetric distribution, with the highest concentration of lysozyme on and near the exposed surface. The quantity and nature of proteins deposited on CLs varies, depending upon the chemical composition of the material. Among the various lenses tested, etafilcon A deposited the highest amount of total protein, most of it represented by lysozyme, which was largely located near the surface of the lens.

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

    Directory of Open Access Journals (Sweden)

    Kenji Matsuura

    Full Text Available 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

  6. Pulsed laser deposition of lysozyme: the dependence on shot numbers and the angular distribution

    DEFF Research Database (Denmark)

    Constantinescu, C.; Matei, A.; Schou, Jørgen


    The ejection of molecules from a pressed solid target of lysozyme induced by laser ablation in the UV-regime at a wavelength of 355 nm was investigated. The ablation studies were carried out in vacuum at a laser fluence of 2 J/cm2 for which a significant fraction of proteins remains intact....... This was verified by matrix-assisted laser desorption ionization (MALDI) spectrometry of thin films deposited on silicon substrates. The deposition rate of lysozyme was found to decrease with the number of shots and was correlated with increasing thermal damage of the lysozyme. This was monitored by measurements...

  7. Modeling the Growth Rates of Tetragonal Lysozyme Crystal Faces (United States)

    Li, Meirong; Nadarajah, Arunan; Pusey, Marc L.


    The measured macroscopic growth rates of the (110) and (101) faces of tetragonal lysozyme show an unexpectedly complex dependence on the supersaturation. The growth rates decay asymptotically to zero when the supersaturation is lowered to zero and increase rapidly when the supersaturation is increased. When supersaturations are increased still further the growth rates attain a maximum before starting to decrease. However, growth of these crystals is known to proceed by the classical dislocation and 2D nucleation growth mechanisms. This anomaly can be explained if growth is assumed to occur not by monomer units but by lysozyme aggregates. Analysis of the molecular packing of these crystals revealed that they were constructed of strongly bonded 4(sub 3) helices, while weaker bonds were responsible for binding the helices to each other. It follows that during crystal growth the stronger bonds are formed before the weaker ones. Thus, the growth of these crystals could be viewed as a two step process: aggregate growth units corresponding to the 4(sub 3) helix are first formed in the bulk solution by stronger intermolecular bonds and then attached to the crystal face by weaker bonds on dislocation hillocks or 2D islands. This will lead to a distribution of aggregates in the solution with monomers and lower order aggregates being predominant at low supersaturations and higher order aggregates being predominant at high supersaturations. If the crystal grows mostly by higher order aggregates, such as tetramers and octamers, it would explain the anomalous dependence of the growth rates on the supersaturation. Besides the analysis of molecular packing, a comprehensive analysis of the measured (110) and (101) growth rates was also undertaken in this study. The distribution of aggregates in lysozyme nutrient solutions at various solution conditions were determined from reversible aggregation reactions at equilibrium. The supersaturation was defined for each aggregate species

  8. Measuring the nucleation rate of Lysozyme using microfluidics (United States)

    Selimović, Šeila; Jia, Yanwei; Fraden, Seth


    We employ the PhaseChip, a (poly)dimethylsiloxane (PDMS) microfluidic device, for statistical studies of protein crystal nucleation. The PhaseChip is designed to decouple nucleation and growth of protein crystals and so improve their yield and quality. Two layers of fluidic channels containing salt reservoirs and nanoliter-sized wells for protein drops in oil are separated by a thin PDMS membrane, which is permeable to water, but not to salt or macromolecules such as protein. We reversibly vary the supersaturation of protein inside the stored droplets by controlling the chemical potential of the reservoir. Lysozyme in the presence of sodium chloride is used as a model system. We determine the crystal nucleation rate as a function of protein supersaturation by counting the number of crystal nuclei per droplet, as demonstrated by Galkin and Vekilov.1 PMID:20161207

  9. 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. (United States)

    Panja, Sudipta; Halder, Mintu


    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

  10. White lies

    NARCIS (Netherlands)

    Erat, S.; Gneezy, U.


    In this paper we distinguish between two types of white lies: those that help others at the expense of the person telling the lie, which we term altruistic white lies, and those that help both others and the liar, which we term Pareto white lies. We find that a large fraction of participants are

  11. Chondroitin Sulfate Perlecan Enhances Collagen Fibril Formation

    DEFF Research Database (Denmark)

    Kvist, A. J.; Johnson, A. E.; Mörgelin, M.


    in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters...... produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated...... disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional...

  12. Alginate-Collagen Fibril Composite Hydrogel

    Directory of Open Access Journals (Sweden)

    Mahmoud Baniasadi


    Full Text Available We report on the synthesis and the mechanical characterization of an alginate-collagen fibril composite hydrogel. Native type I collagen fibrils were used to synthesize the fibrous composite hydrogel. We characterized the mechanical properties of the fabricated fibrous hydrogel using tensile testing; rheometry and atomic force microscope (AFM-based nanoindentation experiments. The results show that addition of type I collagen fibrils improves the rheological and indentation properties of the hydrogel.

  13. Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods (United States)

    Wang, Yan-Qing; Chen, Ting-Ting; Zhang, Hong-Mei


    The interaction between bisphenol A (BPA) and lysozyme (or trypsin) was investigated by UV-vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. BPA effectively quenched the intrinsic fluorescence of lysozyme and trypsin via static quenching. H-bonds and van der Waals interactions played a major role in stabilizing the BPA-proteinase complex. The distance r between donor and acceptor was obtained to be 1.65 and 2.26 nm for BPA-lysozyme and BPA-trypsin complexes, respectively. The effect of BPA on the conformation of lysozyme and trypsin was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra.

  14. Collagen fibril diameter and leather strength. (United States)

    Wells, Hannah C; Edmonds, Richard L; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T; Haverkamp, Richard G


    The main structural component of leather and skin is type I collagen in the form of strong fibrils. Strength is an important property of leather, and the way in which collagen contributes to the strength is not fully understood. Synchrotron-based small angle X-ray scattering (SAXS) is used to measure the collagen fibril diameter of leather from a range of animals, including sheep and cattle, that had a range of tear strengths. SAXS data were fit to a cylinder model. The collagen fibril diameter and tear strength were found to be correlated in bovine leather (r(2) = 0.59; P = 0.009), with stronger leather having thicker fibrils. There was no correlation between orientation index, i.e., fibril alignment, and fibril diameter for this data set. Ovine leather showed no correlation between tear strength and fibril diameter, nor was there a correlation across a selection of other animal leathers. The findings presented here suggest that there may be a different structural motif in skin compared with tendon, particularly ovine skin or leather, in which the diameter of the individual fibrils contributes less to strength than fibril alignment does.

  15. Viscoelastic behavior of discrete human collagen fibrils

    DEFF Research Database (Denmark)


    Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon...... on the strain. The slope of the viscous response showed a strain rate dependence corresponding to a power function of powers 0.242 and 0.168 for the two patellar tendon fibrils, respectively. In conclusion, the present work provides direct evidence of viscoelastic behavior at the single fibril level, which has...

  16. Dynamics of Fibril Growth and Feedback Motifs

    DEFF Research Database (Denmark)

    Cordsen, Pia

    in the literature were found, such as length distribution and apparant persistence lengths. It is found that at all concentrations, fibril growth is characterized by Poissonian stop-go dynamics where the fibril either grows (``go'') or does not grow (``stop''). A monomer-trimer model is proposed in which monomers...... chemical reaction rates of the model, and the theoretical and experimental growth probabilities are found to be in good agreement. Speed distributions of fibrils are also analysed and found to be in good agreement with the predictions of the model. Fibrils of the protein alpha-synuclein which are involved...

  17. Affinity purification of hen egg lysozyme using sephadex G75 | Islam ...

    African Journals Online (AJOL)

    We found lysozyme binds Sephadex G75, a dextran-based matrix routinely used for Gel-filtration chromatography, in a pH dependent manner. The binding is rapid and specific in a buffer containing 25 mM NaCl at pH 8.0, and requires only 0.1 ml of swollen Sephadex G75 suspension per mg of lysozyme. The bound ...

  18. Exogenous lysozyme influences Clostridium perfringens colonization and intestinal barrier function in broiler chickens. (United States)

    Liu, Dan; Guo, Yuming; Wang, Zhong; Yuan, Jianmin


    Necrotic enteritis is a worldwide poultry disease caused by the overgrowth of Clostridium perfringens in the small intestine. An experiment with a 2x2 factorial design (supplementation with or without 40 mg lysozyme/kg diet for chickens challenged with or without C. perfringens) was conducted to investigate the inhibitory efficacy of exogenous lysozyme against intestinal colonization by C. perfringens in chickens subject to oral inoculation of C. perfringens type A on days 17 to 20. The C. perfringens challenge resulted in significant increase of C. perfringens, Escherichia coli and Lactobacillus populations in the ileum, bacteria translocation to the spleen, the intestinal lesion scores , There was significantly lower intestinal lysozyme activity in the duodenum and jejunum and weight gain during days 14 to 28 of the experiment. The addition of exogenous lysozyme significantly reduced the concentration of C. perfringens in the ileum and the intestinal lesion scores, inhibited the overgrowth of E. coli and Lactobacillus in the ileum and intestinal bacteria translocation to the spleen, and improved intestinal lysozyme activity in the duodenum and the feed conversion ratio of chickens. These findings suggest that exogenous lysozyme could decrease C. perfringens colonization and improve intestinal barrier function and growth performance of chickens.

  19. Engineered regulation of lysozyme by the SH3-CB1 binding interaction. (United States)

    Pham, Elizabeth; Truong, Kevin


    The ability to design proteins with desired properties by using protein structural information will allow us to create high-value therapeutic and diagnostic products. Using the protein structures of lambda lysozyme and the SH3 domain of human Crk, we designed a synthetic protein switch that controls the activity of lysozyme by sterically hindering its active cleft through the binding of SH3 to its CB1 peptide-binding partner. First, several fusion protein designs with lysozyme and CB1 were modeled to determine the one with greatest steric effect in the presence of SH3. Next, the selected fusion protein was created and tested in vitro. In the absence of SH3, the lysozyme-CB1 fusion protein functioned normally. In the presence of SH3, the lysozyme activity was inhibited and with the addition of excess CB1 peptides to compete for SH3 binding, the lysozyme activity was restored. Lastly, this structure-based strategy can be used to engineer synthetic regulation by peptide-domain-binding interfaces into a variety of proteins.

  20. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle (United States)

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning


    Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

  1. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    Directory of Open Access Journals (Sweden)

    E.V. Seliverstova


    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  2. Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies (United States)

    Jing, Mingyang; Song, Wei; Liu, Rutao


    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.

  3. Biological and Clinical Implications of Lysozyme Deposition on Soft Contact Lenses. (United States)

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


    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.

  4. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Directory of Open Access Journals (Sweden)

    Bin Yang

    Full Text Available BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  5. Raman mapping of mannitol/lysozyme particles produced via spray drying and single droplet drying. (United States)

    Pajander, Jari Pekka; Matero, Sanni; Sloth, Jakob; Wan, Feng; Rantanen, Jukka; Yang, Mingshi


    This study aimed to investigate the effect of a model protein on the solid state of a commonly used bulk agent in spray-dried formulations. A series of lysozyme/mannitol formulations were spray-dried using a lab-scale spray dryer. Further, the surface temperature of drying droplet/particles was monitored using the DRYING KINETICS ANALYZER™ (DKA) with controllable drying conditions mimicking the spray-drying process to estimate the drying kinetics of the lysozyme/mannitol formulations. The mannitol polymorphism and the spatial distribution of lysozyme in the particles were examined using X-ray powder diffractometry (XRPD) and Raman microscopy. Partial Least Squares Discriminant Analysis was used for analyzing the Raman microscopy data. XRPD results indicated that a mixture of β-mannitol and α-mannitol was produced in the spray-drying process which was supported by the Raman analysis, whereas Raman analysis indicated that a mixture of α-mannitol and δ-mannitol was detected in the single particles from DKA. In addition Raman mapping indicated that the presence of lysozyme seemed to favor the appearance of α-mannitol in the particles from DKA evidenced by close proximity of lysozyme and mannitol in the particles. It suggested that the presence of lysozyme tend to induce metastable solid state forms upon the drying process.

  6. Investigating the Interaction of Fe Nanoparticles with Lysozyme by Biophysical and Molecular Docking Studies.

    Directory of Open Access Journals (Sweden)

    Zahra Aghili

    Full Text Available Herein, the interaction of hen egg white lysozyme (HEWL with iron nanoparticle (Fe NP was investigated by spectroscopic and docking studies. The zeta potential analysis revealed that addition of Fe NP (6.45±1.03 mV to HEWL (8.57±0.54 mV can cause to greater charge distribution of nanoparticle-protein system (17.33±1.84 mV. In addition, dynamic light scattering (DLS study revealed that addition of Fe NP (92.95±6.11 nm to HEWL (2.68±0.37 nm increases suspension potential of protein/nanoparticle system (51.17±3.19 nm. Fluorescence quenching studies reveled that both static and dynamic quenching mechanism occur and hydrogen bond and van der Waals interaction give rise to protein-NP system. Synchronous fluorescence spectroscopy of HEWL in the presence of Fe NP showed that the emission maximum wavelength of tryptophan (Trp residues undergoes a red-shift. ANS fluorescence data indicated a dramatic exposure of hydrophobic residues to the solvent. The considerable reduction in melting temperature (T(m of HEWL after addition of Fe NP determines an unfavorable interaction system. Furthermore circular dichoroism (CD experiments demonstrated that, the secondary structure of HEWL has not changed with increasing Fe NP concentrations; however, some conformational changes occur in tertiary structure of HEWL. Moreover, protein-ligand docking study confirmed that the Fe NP forms hydrogen bond contacts with HEWL.

  7. Partially folded intermediates in insulin fibrillation. (United States)

    Ahmad, Atta; Millett, Ian S; Doniach, Sebastian; Uversky, Vladimir N; Fink, Anthony L


    Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.

  8. Protein fibrillation due to elongation and fragmentation of initially appeared fibrils: A simple kinetic model (United States)

    Kashchiev, Dimo


    The assembly of various proteins into fibrillar aggregates is an important phenomenon with wide implications ranging from human disease to nanoscience. Employing a new model, we analyze the kinetics of protein fibrillation in the case when the process occurs by elongation of initially appeared fibrils which multiply solely by fragmentation, because fibril nucleation is negligible. Owing to its simplicity, our model leads to mathematically friendly and physically clear formulas for the time dependence of the fibrillation degree and for a number of experimental observables such as the maximum fibrillation rate, the fibrillation lag time, and the half-fibrillation time. These formulas provide a mechanistic insight into the kinetics of fragmentation-affected fibrillation of proteins. We confront theory with experiment and find that our model allows a good global description of a large dataset [W.-F. Xue, S. W. Homans, and S. E. Radford, Proc. Natl. Acad. Sci. U.S.A. 105, 8926 (2008)], 10.1073/pnas.0711664105 for the fibrillation kinetics of beta-2 microglobulin. Our analysis leads to new methods for experimental determination of the fibril solubility, elongation rate constant, and nucleation rate from data for the time course of protein fibrillation.

  9. Viscoelastic behavior of discrete human collagen fibrils

    DEFF Research Database (Denmark)

    Svensson, René; Hassenkam, Tue; Hansen, Philip


    Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon fibr...

  10. Graphene oxide strongly inhibits amyloid beta fibrillation

    NARCIS (Netherlands)

    Mahmoudi, Morteza; Akhavan, Omid; Ghavami, Mahdi; Rezaee, Farhad; Ghiasi, Seyyed Mohammad Amin


    Since amyloid beta fibrillation (AbF) plays an important role in the development of neurodegenerative diseases, we investigated the effect of graphene oxide (GO) and their protein-coated surfaces on the kinetics of Ab fibrillation in the aqueous solution. We showed that GO and their protein-covered

  11. Fibril assembly in whey protein mixtures

    NARCIS (Netherlands)

    Bolder, S.G.


    The objective of this thesis was to study fibril assembly in mixtures of whey proteins. The effect of the composition of the protein mixture on the structures and the resulting phase behaviour was investigated. The current work has shown that beta-lactoglobulin is responsible for the fibril assembly

  12. Kinetic theory of amyloid fibril templating. (United States)

    Schmit, Jeremy D


    The growth of amyloid fibrils requires a disordered or partially unfolded protein to bind to the fibril and adapt the same conformation and alignment established by the fibril template. Since the H-bonds stabilizing the fibril are interchangeable, it is inevitable that H-bonds form between incorrect pairs of amino acids which are either incorporated into the fibril as defects or must be broken before the correct alignment can be found. This process is modeled by mapping the formation and breakage of H-bonds to a one-dimensional random walk. The resulting microscopic model of fibril growth is governed by two timescales: the diffusion time of the monomeric proteins, and the time required for incorrectly bound proteins to unbind from the fibril. The theory predicts that the Arrhenius behavior observed in experiments is due to off-pathway states rather than an on-pathway transition state. The predicted growth rates are in qualitative agreement with experiments on insulin fibril growth rates as a function of protein concentration, denaturant concentration, and temperature. These results suggest a templating mechanism where steric clashes due to a single mis-aligned molecule prevent the binding of additional molecules.

  13. Atrial fibrillation in patients with ischemic stroke

    DEFF Research Database (Denmark)

    Thygesen, Sandra Kruchov; Frost, Lars; Eagle, Kim A


    BACKGROUND: Atrial fibrillation is a major risk factor for ischemic stroke. However, the prognostic impact of atrial fibrillation among patients with stroke is not fully clarified. We compared patient characteristics, including severity of stroke and comorbidity, quality of in-hospital care and o...

  14. Formation and properties of whey protein fibrils

    NARCIS (Netherlands)

    Kroes-Nijboer, A.


    Protein fibrils are threadlike aggregates that are about one molecule thick and more than thousand molecules long. Due to their threadlike structure they could potentially be used to form meat-like structures. Protein fibrils can be produced from milk protein and plant protein, opening opportunities

  15. Genetics Home Reference: familial atrial fibrillation (United States)

    ... to be associated with familial atrial fibrillation was KCNQ1 , which provides instructions for making a channel that ... atrial fibrillation ABCC9 GJA5 KCNA5 KCNE2 KCNH2 KCNJ2 KCNQ1 LMNA MYL4 NKX2-5 NPPA NUP155 PRKAG2 RYR2 ...

  16. Lysozyme-like activity in eggs and in some tissues of land snails Helix aspersa maxima and Achatina achatina. (United States)

    Fiołka, Marta J; Witkowski, Andrzej


    Antibacterial lysozyme-like activity against Micrococcus luteus in eggs and some tissues of snails Helix aspers maxima and Achatina achatina was detected in a turbidimetric standard assay. The bacteriolytic activity in Helix aspersa maxima was higher than in Achatina achatina. After the application of the bioautography technique, several lytic zones of Micrococcus luteus were observed in both studied species. Electrophoresis in denaturing conditions followed by immunodetection of lysozyme using EWL antibodies indicated the presence of several lysozyme forms in the tested snails.

  17. Biophysical and computational characterization of vandetanib-lysozyme interaction (United States)

    Kabir, Md. Zahirul; Hamzah, Nur Aziean Binti; Ghani, Hamidah; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad


    Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka = 4.96-3.14 × 103 M-1). Thermodynamic data (ΔS = + 12.82 J mol-1 K-1; ΔH = - 16.73 kJ mol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.

  18. Cetirizine-Induced atrial fibrillation

    Directory of Open Access Journals (Sweden)

    Altuğ Osken


    Full Text Available Atrial fibrillation (AF is the most common observed arrhythmia in clinical practice. In the literature, AF events associated with drug induction are available. Cetirizine is a second-generation histamine antagonist used in the treatment of allergies, angioedema, and urticaria. We wish to present an atypical case who took cetirizine medication for relieving symptoms of upper tract respiratory system infection, experienced rapid ventricular response AF and treated successfully. To best of our knowledge, this is the first case of cetirizine-induced AF.

  19. A magnetic nanoparticle-based aptasensor for selective and sensitive determination of lysozyme with strongly scattering silver nanoparticles. (United States)

    Li, Chun Mei; Zhan, Lei; Zheng, Lin Ling; Li, Yuan Fang; Huang, Cheng Zhi


    Qualitative and quantitative determination of lysozyme concentrations in urine and serum with high selectivity and sensitivity is important for diagnosing the progression of several diseases. In this report, we devised an improved method for specifically detecting lysozyme by combining magnetic nanoparticles (for separation and enrichment), an aptamer (for selective binding of lysozyme) and strongly scattering silver nanoparticles (AgNPs, for detection by light scattering, but also providing another level of selectivity due to their electrostatic binding with lysozyme). In this system, 0.4-30 nM lysozyme could be simply detected owing to the decreased light scattering of AgNPs in solution after magnetic separation, with a detection limit of 100 pM. In addition, lysozyme was also able to be semi-quantified by using the dark-field light scattering images of AgNPs after enrichment by the MNP-apt-lysozyme complex. Moreover, this design shows great promise for the robust and reliable detection of lysozyme in real samples, with a recovery rate ranging from 98.6% to 101.3% in human serum samples. Therefore, this assay provided robust measurements with good specificity, sensitivity, and tolerance of changes in the sample matrix. We expect that this MNP-based aptasensor may find utility in the accurate diagnosis of lysozyme-related diseases.

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

    National Research Council Canada - National Science Library

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


    .... 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...

  1. IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions. (United States)

    Shimakura, Hidekatsu; Uchiyama, Jumpei; Saito, Taku; Miyaji, Kazuki; Fujimura, Masato; Masuda, Kenichi; Okamoto, Noriaki; DeBoer, Douglas J; Sakaguchi, Masahiro


    Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

    Directory of Open Access Journals (Sweden)

    Ming Feng Jiang


    Full Text Available Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type milk lysozyme gene (YML, was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75 which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

  3. The interaction of flavonoid-lysozyme and the relationship between molecular structure of flavonoids and their binding activity to lysozyme. (United States)

    Yang, Ran; Yu, Lanlan; Zeng, Huajin; Liang, Ruiling; Chen, Xiaolan; Qu, Lingbo


    In this work, the interactions of twelve structurally different flavonoids with Lysozyme (Lys) were studied by fluorescence quenching method. The interaction mechanism and binding properties were investigated. It was found that the binding capacities of flavonoids to Lys were highly depend on the number and position of hydrogen, the kind and position of glycosyl. To explore the selectivity of the bindings of flavonoids with Lys, the structure descriptors of the flavonoids were calculated under QSAR software package of Cerius2, the quantitative relationship between the structures of flavonoids and their binding activities to Lys (QSAR) was performed through genetic function approximation (GFA) regression analysis. The QSAR regression equation was K(A) = 37850.460 + 1630.01Dipole +3038.330HD-171.795MR. (r = 0.858, r(CV)(2) = 0.444, F((11,3)) = 7.48), where K(A) is binding constants, Dipole, HD and MR was dipole moment, number of hydrogen-bond donor and molecular refractivity, respectively. The obtained results make us understand better how the molecular structures influencing their binding to protein which may open up new avenues for the design of the most suitable flavonoids derivatives with structure variants.

  4. Prevention of COPD exacerbation by lysozyme: a double-blind, randomized, placebo-controlled study

    Directory of Open Access Journals (Sweden)

    Fukuchi Y


    Full Text Available Yoshinosuke Fukuchi,1 Koichiro Tatsumi,2 Hiromasa Inoue,3 Yukinori Sakata,4 Kai Shibata,4 Hideaki Miyagishi,4 Yasuhiro Marukawa,4 Masakazu Ichinose5 1Department of Respiratory Medicine, Graduate School of Medicine, Juntendo University, Tokyo, 2Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, 3Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, 4Eisai Co., Ltd., Tokyo, 5Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Background/aim: Lysozyme (mucopeptide N-acetyl-muramyl hydrolase is widely used as a mucolytic and anti-inflammatory agent in Japan. We evaluated the effects of long-term lysozyme administration on COPD exacerbation. Methods: In a 1-year, randomized, double-blind, placebo-controlled, parallel trial, patients with moderate-to-severe COPD and one or more episodes of COPD exacerbation in the previous year before enrollment were selected. Lysozyme (270 mg or placebo was administered orally for 52 weeks as an add-on to the standard therapies such as bronchodilators. COPD exacerbation, pulmonary function, and COPD assessment test scores were analyzed. An exacerbation was defined as worsening of more than one symptom of COPD (cough, sputum volume, purulent sputum, or breathlessness leading to a change in medication. The primary endpoint was exacerbation rate. Results: A total of 408 patients were randomly assigned to the lysozyme and placebo groups. The baseline characteristics were similar between the two groups. The exacerbation rate was not significantly different between the two groups (1.4 vs 1.2; P=0.292, Poisson regression. However, a subgroup analysis showed that lysozyme might reduce exacerbation rate in patients with airway-dominant phenotype (1.2 vs 1.6. Moreover, the median time to first exacerbation was longer in patients with airway-dominant phenotype in the lysozyme group than that

  5. [Lysozyme activities of Oncomelania hupensis in different seasons and ages of snails]. (United States)

    Huang, Chun-lan; Zhou, Shu-lin; Li, Chao-pin


    To explore the relationship between the lysozyme activities of Oncomelania hupensis snails among different seasons, and the difference of the lysozyme activities among the different ages of the snails. The homogenate soft tissues of O. hupensis were dissolved with Tris-HCl-TritonX-114 buffer and concentrated, and the enzyme was extracted by centrifugation. There were significant differences of lysozyme activities among different seasons (Spring, Summer, Autumn, Winter) with the results of 71.33 +/- 4.16 U/mg, 93.67 +/- 7.10 U/mg, 150.33 +/- 10.01 U/mg, 358 +/- 35.38 U/mg,respectively (F = 144.455, P snails) with the results of 93.67 +/- 7.10 U/mg and 69.33 +/- 5.03 U/mg,respectively (t = 4.845, P snails, and the highest lysozyme activity is in winter. The lysozyme activity of adult snails is higher than that of the youth.

  6. Expression and Activity of Lysozyme in Apis Mellifera Carnica Brood Infested with Varroa Destructor

    Directory of Open Access Journals (Sweden)

    Zaobidna Ewa A.


    Full Text Available Varroa destructor is a parasitic mite that attacks the honey bee, and previous studies have suggested that parasitosis caused by this mite is accompanied by immunosuppresion in the host. In this study, the effect of mite infestation on the expression of the lysozyme-1 (lys-1 gene and lysozyme activity in Apis mellifera carnica was determined. The experiment was carried out on the five developmental stages of honey bee workers and drones. Developmental and gender-related differences in gene expression and lysozyme activity were observed in a Varroa destructor-infested brood. The relative expression of the lys-1 gene increased in a infested worker brood and decreased in a drone brood except for P3 pupae. In the final stage of development, the lys-1 gene expression was significantly lower in infested newly emerged workers and drones. Changes in the relative expression of the lys-1 gene in infested individuals was poorly manifested at the level of enzyme activity, whereas at the two final stages of development (P5 and I there was a positive correlation between relative lys-1 expression and lysozyme activity in infested bees of both genders (r=0.988, r=0.999, respectively. The results of this study indicate that V. destructor influences the lysozyme-linked immune response in bees.

  7. Single-Molecule Measurements of T4 Lysozyme using Carbon Nanotube Electronic Circuits (United States)

    Sims, Patrick Craig

    Because of their unique electronic and chemical properties, single-walled carbon nanotubes (SWNTs) are attractive candidates for label-free, single-molecule sensing and detection applications. In this work, a field-effect transistor (FET) architecture comprised of an individual SWNT is used to transduce the conformational motion of a single T4 lysozyme protein, conjugated to the SWNT side wall, into a corresponding electrical current signal. The SWNTs are grown using chemical vapor deposition, and metal electrical contacts are formed using electron beam evaporation. Using N-(1-Pyrene)maleimide, the protein is conjugated to the SWNT side wall. After conjugation, the sensing area of the device is submerged in an electrolyte solution, and the source-drain current is measured while applying an electrolyte-gate. Analysis of the signal provided single-molecule resolution of the dynamical activity of lysozyme as it hydrolyzes macromolecular peptidoglycan, a component of bacterial cell walls. This analysis revealed seven different independent time scales that govern the activity of lysozyme, the pH dependence of these time scales, and a lower limit on the number rate-limiting steps in lysozyme's hinge opening and closing motions. Furthermore, the signals elucidated differences in how lysozyme traverses and catalyzes structurally varying peptidoglycan constructs.

  8. Antimicrobial lysozyme-chitosan coatings affect functional properties and shelf life of chicken eggs during storage. (United States)

    Yuceer, Muhammed; Caner, Cengiz


    The interior quality, shell impact strength and functional characteristics of eggs coated with chitosan and lysozyme-chitosan combinations were evaluated for enhancing egg freshness during storage. A 10% (w/w) lysozyme solution was incorporated into 1% (w/w) chitosan film-forming solution at ratios of 0, 10, 20 and 60% (w/w). Storage time and coating had significant effects on Haugh unit, yolk index, weight loss, albumen pH, dry matter, relative whipping capacity (RWC) and albumen viscosity. Uncoated eggs had higher albumen pH and weight loss and lower albumen viscosity. All coated eggshells showed greater puncture strength than uncoated eggshells, resulting in extended shelf life. The 20 and 60% lysozyme-chitosan coatings were more effective in maintaining the internal quality of eggs (e.g. pH, dry matter and RWC). Attributes such as pH, dry matter and RWC were better after the 20% lysozyme-chitosan treatment than after the other treatments. The 10, 20 and 60% lysozyme-chitosan coatings, considered active packaging, showed promising attributes. They could be a viable alternative to existing techniques for maintaining the internal quality of fresh eggs during long-term storage. Chitosan coatings also improved shell strength. This study also confirms that measurements of albumen quality (pH, dry matter, viscosity and RWC) are excellent indicators of egg freshness. © 2013 Society of Chemical Industry.

  9. Controlled Release of Lysozyme from Double-Walled Poly(Lactide-Co-Glycolide (PLGA Microspheres

    Directory of Open Access Journals (Sweden)

    Rezaul H. Ansary


    Full Text Available Double-walled microspheres based on poly(lactide-co-glycolide (PLGA are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide (Glu-PLGA, and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w1/o/o/w2 emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52% ± 3.28% was achieved when 1% (w/v polyvinyl alcohol (PVA and 2.5% (w/v trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose provide a controlled and sustained release for lysozyme.

  10. Impact of a Reducing Agent on the Dynamic Surface Properties of Lysozyme Solutions. (United States)

    Tihonov, Michael M; Kim, Viktoria V; Noskov, Boris A


    Disulfide bond shuffling in the presence of the reducing agents dithiothreitol (DTT) or β-mercaptoethanol (BME) strongly affects the surface properties of lysozyme solutions. The addition of 0.32 mM DTT substantially alters the kinetic dependencies of the dynamic surface elasticity and surface tension relative to those of pure protein solutions. The significant increase in the dynamic surface elasticity likely relates to the cross-linking between lysozyme molecules and the formation of a dense layer of protein globules stabilized by intermolecular disulfide bonds at the liquid/gas interface. This effect differs from the previously described influence of chaotropic denaturants, such as guanidine hydrochloride (GuHCl) and urea, on the surface properties of lysozyme solutions. If both chaotropic and reducing agents are added to protein solutions simultaneously, their effects become superimposed. In the case of mixed lysozyme/GuHCl/DTT solutions, the dynamic surface elasticity near equilibrium decreases as the GuHCl concentration increases because of the gradual loosening of the cross-linked layer of protein globules but remains much higher than that of lysozyme/GuHCl solutions.

  11. Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses. (United States)

    Chung, Jiwoo; Ku, Sae-Kwang; Lee, Suyeon; Bae, Jong-Sup


    Lysozyme, found in relatively high concentration in blood, saliva, tears, and milk, protects us from the ever-present danger of bacterial infection. Previous studies have reported proinflammatory responses of endothelial cells to the release of polyphosphate(PolyP). In this study, we examined the anti-inflammatory responses and mechanisms of lysozyme and its effects on PolyP-induced septic activities in human umbilical vein endothelial cells (HUVECs) and mice. The survival rates, septic biomarker levels, behavior of human neutrophils, and vascular permeability were determined in PolyP-activated HUVECs and mice. Lysozyme suppressed the PolyP-mediated vascular barrier permeability, upregulation of inflammatory biomarkers, adhesion/migration of leukocytes, and activation and/or production of nuclear factor-κB, tumor necrosis factor-α, and interleukin-6. Furthermore, lysozyme demonstrated protective effects on PolyP-mediated lethal death and the levels of the related septic biomarkers. Therefore, these results indicated the therapeutic potential of lysozyme on various systemic inflammatory diseases, such as sepsis or septic shock. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. In vitro collagen fibril assembly: thermodynamic studies. (United States)

    Na, G C; Phillips, L J; Freire, E I


    The in vitro fibril assembly of calf skin collagen was examined as a function of ionic strength and temperature. In a 0.03 M NaPi, pH 7.0, buffer, fibril assembly required a minimum critical concentration of collagen. At nearly physiological ionic strengths and temperatures, the critical concentration was less than 1 microgram/mL and required a very sensitive method for measurement. Raising the ionic strength of the buffer resulted first in higher and then lower critical concentrations. Raising the temperature led to lower critical concentrations. A van't Hoff plot of the fibril growth constant calculated from the critical concentration gave positive enthalpy changes and positive heat capacity changes which indicate that the fibril growth is driven by both hydrophobic and ionic inter-collagen interactions. Sedimentation equilibrium studies showed the collagen to be monomeric at subcritical concentrations. Differential scanning microcalorimetric studies showed only one very sharp heat absorption peak for the fibril assembly which coincided with the appearance of solution turbidity. Within experimental error, the enthalpy changes of the fibril assembly measured with the microcalorimeter were of the same magnitude as the van't Hoff enthalpy changes. These results are discussed in light of a cooperative nucleation-growth mechanism of collagen fibril assembly proposed earlier.

  13. Exercise increases lactoferrin, but decreases lysozyme in salivary granulocytes. (United States)

    Gillum, Trevor; Kuennen, Matthew; McKenna, Zachary; Castillo, Micaela; Jordan-Patterson, Alex; Bohnert, Caitlin


    Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise. To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45+CD15+ cells. 11 males (20.3 ± 0.8 years, 57.2 ± 7.6 mL/kg/min V̇O2pk, 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO2pk. 12 mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45+) and granulocytes (CD45+CD15+) using flow cytometry. Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p <0.05). Lys MFI decreased with exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p <0.05). Acute running resulted in an increased Lac concentration which could lead to a decrease in inflammation, adding further evidence of the anti-inflammatory effects of exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

  14. Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study. (United States)

    Wizert, Alicja; Iskander, D Robert; Cwiklik, Lukasz


    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.

  15. Characterization of lysozyme films produced by matrix assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Purice, Andreea; Schou, Jørgen; Kingshott, Peter


    Thin lysozyme films of thickness up to more than 100 nm have been produced in a dry environment by MAPLE (matrix assisted pulsed laser evaporation) from a water ice matrix. Analysis of the films demonstrates that a significant part of the lysozyme molecules is transferred to the substrate without...... decomposition and that the protein activity is preserved. The film deposition rate for 1 wt% lysozyme has a maximum at 2 J/cm(2) of about 1 ng/cm(2) per laser shot. During the film production the deposition rate is constant without any sign of depletion or accumulation effects in the water ice target...... that from a water ice matrix. (C) 2007 Elsevier B.V. All rights reserved....

  16. Viscometric study of lysozyme solution with sugar and urea at various temperatures

    Directory of Open Access Journals (Sweden)

    Jamal Akhter Siddique


    at temperatures (293.15, 303.15, 313.13 and 323.15 K at various concentrations of glucose, maltose and urea. Change in entropy (ΔH, enthalpy (ΔS and free energy of activation (ΔG have also been evaluated for these systems. Value of B-coefficient of d (− glucose, maltose and urea has also been calculated from viscosity data in aqueous lysozyme solution. Viscosity B-coefficients of glucose and maltose in aqueous lysozyme solution are positive while that of the urea–lysozyme water system it is negative due to the structure breaking effect of urea. The values of entropy of activation are negative due to attainment of transition state for viscous flow, which is accompanied by bond formation and increase in order.

  17. Raman mapping of mannitol/lysozyme particles produced via spray drying and single droplet drying

    DEFF Research Database (Denmark)

    Pekka Pajander, Jari; Matero, Sanni Elina; Sloth, Jakob


    PURPOSE: This study aimed to investigate the effect of a model protein on the solid state of a commonly used bulk agent in spray-dried formulations. METHODS: A series of lysozyme/mannitol formulations were spray-dried using a lab-scale spray dryer. Further, the surface temperature of drying droplet....../particles was monitored using the DRYING KINETICS ANALYZER™ (DKA) with controllable drying conditions mimicking the spray-drying process to estimate the drying kinetics of the lysozyme/mannitol formulations. The mannitol polymorphism and the spatial distribution of lysozyme in the particles were examined using X......-ray powder diffractometry (XRPD) and Raman microscopy. Partial Least Squares Discriminant Analysis was used for analyzing the Raman microscopy data. RESULTS: XRPD results indicated that a mixture of β-mannitol and α-mannitol was produced in the spray-drying process which was supported by the Raman analysis...

  18. Psychosomatic correlations in atrial fibrillations

    Directory of Open Access Journals (Sweden)

    Vladimir Ernstovich Medvedev


    Full Text Available Patients with atrial fibrillations (AF and comorbid mental disorders were examined. Two patient groups differing in the structure of psychosomatic ratios were identified. Group 1 comprised patients with AF and signs of reactivity lability that manifested itself as psychopathological reactions to the primary manifestations of AF; Group 2 included those who had developed mental disorders mainly in end-stage cardiovascular disease (predominantly a permanent form of AF in the presence of such events as chronic heart failure (CHF. The results of the study suggest that the patients with AF have frequently anxiety and hypochondriacal disorders, which agrees with the data available in the literature. In addition, end-stage AF is marked by depressive syndromes caused by the severe course of cardiovascular diseases resulting in CHF.

  19. Dronedarone for atrial fibrillation therapy. (United States)

    Marzocchi, Michele; Lombardi, Federico


    Dronedarone is a new benzofuran derivative that has been developed as an antiarrhythmic agent on the basis of the amiodarone molecular structure with the intent of maintaining the same pharmacological effects while reducing thyroid and pulmonary toxicity. The drug is a multichannel blocker with antiadrenergic properties: it reduces heart rate and prolongs the action potential duration. Dronedarone is primarily metabolized by cytochrome P450; its half-life is much shorter than that of amiodarone because of a lower lipophilicity. As a consequence, only 7 days are needed to reach steady-state plasma levels. It has been tested in clinical trials both for rate and rhythm control and, even if its antiarrhythmic efficacy seems to be somehow lower than that of amiodarone, dronedarone is less often discontinued due to adverse reactions or organic toxicity. For these reasons, dronedarone can be very useful in long-term treatment of atrial fibrillation, by reducing hospitalizations and mortality.

  20. May Fever Trigger Ventricular Fibrillation?

    Directory of Open Access Journals (Sweden)

    Jean Luc Pasquié


    Full Text Available The clinical precipitants of ventricular fibrillation (VF remain poorly understood. Clinical factors such as hypoxemia, acidosis or electrolyte imbalance, drug-related toxicity, autonomic nervous system disorders as well as viral myocarditis have been proposed to be associated with sudden cardiac death particularly in patients with structural heart disease. However, In the Brugada syndrome, concurrent febrile illness has been reported to unmask the electrocardiographic features of the Brugada syndrome and be associated with an increased propensity for VF. More recently, a febrile illnesses of infectious etiology was associated to polymorphic ventricular tachycardia or VF in patients with normal hearts and without known repolarization abnormality. In this review we detail this phenomenon and its putative mechanisms.

  1. In Vitro Effect of Lysozyme on Albumin Deposition to Hydrogel Contact Lens Materials. (United States)

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


    Albumin deposition on contact lenses could be detrimental to contact lens (CL) wear because this may increase the risk of bacterial binding and reduce comfort. Lysozyme deposition on selected lens materials would reduce albumin deposition on lenses. This study aims to determine if lysozyme deposition on CLs could act as a barrier against subsequent albumin adsorption, using an in vitro model. Six hydrogel CL materials (etafilcon A, polymacon, nelfilcon A, omafilcon A, ocufilcon B, and nesofilcon A) were evaluated. Four CLs of each type were soaked in lysozyme solution for 16 hours at 37°C. Lysozyme-coated lenses were then placed in vials with 1.5 mL of artificial tear solution containing I-labeled albumin for 16 hours at 37°C with shaking. Four uncoated lenses of each type were used as controls. Lenses soaked in radiolabeled albumin were rinsed in a phosphate-buffered saline solution, and radioactive counts were measured directly on lenses using a gamma counter. Albumin uptake on lenses was measured using a calibration curve by plotting radioactive counts versus protein concentration. Results are reported as mean ± SD. Lysozyme-coated etafilcon A lenses exhibited lower levels of deposited albumin than uncoated etafilcon A lenses (58 ± 12 vs. 84 ± 5 ng/lens; P .05). Uncoated nesofilcon A lenses deposited the highest amount of albumin when compared with other uncoated lenses (P < .05). This study demonstrates that lysozyme deposited onto etafilcon A resists the deposition of albumin, which may potentially be beneficial to CL wearers.

  2. Lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs. (United States)

    Oliver, W T; Wells, J E


    Lysozyme is a 1,4-β-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine if lysozyme in nursery diets improved growth performance and gastrointestinal health of pigs weaned from the sow at 24 d of age. Two replicates of 96 pigs (192 total; 96 males, 96 females) were weaned from the sow at 24 d of age, blocked by BW and gender, and then assigned to 1 of 24 pens (4 pigs/pen). Each block was randomly assigned 1 of 3 dietary treatments for 28 d: control (two 14-d phases), control + antibiotics (carbadox/copper sulfate), or control + lysozyme (100 mg/kg diet). Pigs were weighed and blood sampled on d 0, 14, and 28 of treatment. Blood was analyzed for plasma urea nitrogen (PUN) and IgA. At 28 d, pigs were killed, and samples of jejunum and ileum were collected and fixed for intestinal morphology measurements. An additional jejunum sample was taken from the 12 pigs with the median BW per treatment to determine transepithelial electrical resistance (TER). Pigs consuming antibiotics or lysozyme grew at a faster rate than control pigs (0.433 ± 0.009 and 0.421 ± 0.008 vs. 0.398 ± 0.008 kg/d, respectively; P 0.48), but G:F was improved in pigs consuming antibiotics or lysozyme (0.756 ± 0.014, 0.750 ± 0.021, and 0.695 ± 0.019 kg/kg; P 0.48). Dietary treatment did not affect TER (P > 0.37), but gilts had lower TER compared with barrows (P 0.53). However, jejunum villi height was increased and crypt depth was decreased in pigs consuming antibiotics or lysozyme (P pigs weaned from the sow at 24 d of age.

  3. Alcohol consumption and risk of atrial fibrillation

    DEFF Research Database (Denmark)

    Tolstrup, Janne Schurmann; Wium-Andersen, Marie Kim; Ørsted, David Dynnes


    BACKGROUND: The aim of this study was to test the hypothesis that alcohol consumption, both observational (self-reported) and estimated by genetic instruments, is associated with a risk of atrial fibrillation and to determine whether people with high cardiovascular risk are more sensitive towards...... register. As a measure of alcohol exposure, both self-reported consumption and genetic variations in alcohol metabolizing genes (ADH1B/ADH1C) were used as instrumental variables. The endpoint was admission to hospital for atrial fibrillation as recorded in a validated hospital register. RESULTS: A total...... of 3493 cases of atrial fibrillation occurred during follow-up. High alcohol consumption was associated with a risk of atrial fibrillation among men, but not among women. Among the men who drank 28-35 and 35+ drinks/week, the hazards ratios were 1.40 (95% confidence interval 1.09-1.80) and 1.62 (95...

  4. Functionalization of α-synuclein fibrils

    Directory of Open Access Journals (Sweden)

    Simona Povilonienė


    Full Text Available The propensity of peptides and proteins to form self-assembled structures has very promising applications in the development of novel nanomaterials. Under certain conditions, amyloid protein α-synuclein forms well-ordered structures – fibrils, which have proven to be valuable building blocks for bionanotechnological approaches. Herein we demonstrate the functionalization of fibrils formed by a mutant α-synuclein that contains an additional cysteine residue. The fibrils have been biotinylated via thiol groups and subsequently joined with neutravidin-conjugated gold nanoparticles. Atomic force microscopy and transmission electron microscopy confirmed the expected structure – nanoladders. The ability of fibrils (and of the additional components to assemble into such complex structures offers new opportunities for fabricating novel hybrid materials or devices.

  5. Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme

    DEFF Research Database (Denmark)

    Ji, Shuying; Thulstrup, Peter Waaben; Mu, Huiling


    In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co...... the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme......-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed...

  6. Binary Vector Construction and Agrobacterium Tumefaciens-mediated Transformation of Lysozyme Gene in Seaweed Kappaphycus Alvarezii


    Handayani, Tri; Alimuddin, Alimuddin; Widyastuti, Utut; Suryati, Emma; Parenrengi, Andi


    Ice-ice disease is the biggest problem in the cultivation of seaweed Kappaphycus alvarezii. The disease is caused by bacterial infection and induced by drastic changes of water quality. Lysozyme has the ability to break down bacterial cell wall. The purpose of this research was to construct of a binary vector pMSH1-Lys carrying chicken Lysozyme (Lis) gene and introduce pMSH1-Lis on K. alvarezii. The binary vector expression was transformed into Agrobacterium tumefaciens LBA4404 by triparental...

  7. Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge. (United States)

    Oliver, W T; Wells, J E; Maxwell, C V


    Lysozyme is a 1,4-β-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect immune challenge. Two replicates of 600 pigs each were weaned from the sow at 26 d of age, blocked by litter and sex, and then randomly assigned to 1 of 24 pens in either a nursery room that had been fully disinfected or a nursery room left unclean since the previous group of pigs. Within a room, pigs were randomly assigned to either control diets (2 phase nursery regime), control diets + antibiotics (chlortetracycline/tiamulin hydrogen fumarate), or control diets + lysozyme (100 mg/kg diet). Pig weights and feed disappearance were measured and blood was collected on d 0, 14, and 28 of treatment. A group of 20 pigs were killed at 24 d of age for initial body composition analysis and 10 pigs of median weight were killed per diet room combination for body composition analysis after 28 d of treatment. Control + antibiotics and control + lysozyme-fed pigs grew at a faster rate for the 28-d study compared to control pigs (318 ± 14,320 ± 15 vs. 288 ± 15 g/d, respectively; P 0.05). The indirect immune challenge did not alter growth performance from d 0 to 14 of treatment but decreased ADG from d 14 to 28 of the study (415 ± 15 vs. 445 ± 13 g/d ;: P 0.61) or dietary treatments (P > 0.10), but feed efficiency was worsened by the indirect immune challenge (P 0.25), but both control + antibiotics and control + lysozyme pigs had decreased accretion of whole-body lipid (P pigs reared in the clean nursery (P pigs consuming antibiotics or lysozyme had lower TNF-α, haptoglobin, and CRP compared to control pigs, regardless of nursery environment (P < 0.04). Thus, lysozyme is a suitable alternative to antibiotics in swine nursery diets, and lysozyme ameliorates the effects of a chronic indirect immune challenge.

  8. Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli


    Kim,Jong-Wan; Yoe,Jeehyun; Lee,Gil Ho; Yoe,Sung Moon


    The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by ...

  9. Laser ablation of lysozyme with UV, visible and infrared femto- and nanosecond pulses

    DEFF Research Database (Denmark)

    Schou, Jørgen; Canulescu, Stela; Matei, Andreea

    yields ever measured. The mass loss is mainly caused by fragmentation of the lysozyme into simple gases, such as H2S, H2O and CO2 , which are rapidly pumped away in the vacuum chamber. We have investigated the mass loss by ablation of lysozyme in all regimes to see whether a similar mechanism governs...... the ablation process for different wavelengths and time duration. Measurements for 6-7-ns laser ablation were carried out at DTU on Risø Campus, while measurements with pulses of 300 fs were carried out at the University of Naples in a similar setup. For all wavelengths except at nanosecond laser pulses at 355...

  10. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules


    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Rehault-Godbert, Sophie


    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ova...

  11. Atrial Fibrillation after Robotic Cardiac Surgery




    Atrial fibrillation is a common arrhythmia after conventional open heart surgery. A minimally invasive robotic approach has the potential to lower its occurrence. We sought to review the literature on the incidence of post operative atrial fibrillation in robotic heart surgery and compare it to the incidence in conventional cardiac surgery. The types of operation investigated were: coronary artery bypass surgery, mitral valve repair, atrial septal defect closure and myxoma excision. Operation...

  12. [Anticoagulation in atrial fibrillation - an update]. (United States)

    Antz, Matthias; Hullmann, Bettina; Neufert, Christian; Vocke, Wolfgang


    The correct anticoagulation regimen for prevention of thromboembolic events is essential in patients with atrial fibrillation. However, only a minority of patients receives anticoagulation according to the guidelines. The current guidelines are intended to make the indication for anticoagulation more simple and are summarized in the present article. This includes recommendations for chronic anticoagulation, prevention of thromboembolic events after cardioversion and in ablation of atrial fibrillation.

  13. Salbutamol Abuse is Associated with Ventricular Fibrillation

    Directory of Open Access Journals (Sweden)

    Emin UYSAL


    Full Text Available SUMMARY: Salbutamol-induced cardiac complications are well-established. Herein, we describe a case of a 24-year female who was admitted to the emergency department because of a suicide attempt with salbutamol (76 mg. Salbutamol abuse induced the development of supraventricular tachycardia and ventricular fibrillation. Regular sinus rhythm was restored with defibrillation. The hypokalemic patient who stayed in the intensive care unit was discharged after 48 hours of hospitalization. Key words: Salbutamol, suicide, ventricular fibrillation

  14. Different Analytical Approaches in Assessing Antibacterial Activity and the Purity of Commercial Lysozyme Preparations for Dairy Application

    Directory of Open Access Journals (Sweden)

    Luisa Pellegrino


    Full Text Available 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.

  15. Artificial chaperone-assisted refolding of denatured-reduced lysozyme: modulation of the competition between renaturation and aggregation. (United States)

    Rozema, D; Gellman, S H


    Conditions that promote renaturation of an unfolded protein also promote protein aggregation, in many cases, because these competing intramolecular and intermolecular processes are driven by similar networks of noncovalent interactions. The GroEL/GroES system and related biological chaperones facilitate the renaturation of substrate proteins by minimizing the aggregation pathway. We have devised a two-step method in which small molecules, "artificial chaperones," facilitate protein refolding from a chemically denatured state. In the first step, the protein is captured by a detergent as guanidinium chloride is diluted to a non-denaturing concentration; formation of a protein-detergent complex prevents both protein aggregation and proper refolding. In the second step, a cyclodextrin strips detergent from the protein, allowing the protein to refold. Here we describe the first application of this method to a protein that must form disulfides in the native state. Lysozyme (hen egg white) can be refolded from the Gdm-denatured, DTT-reduced state in good yields at final protein concentrations as high as 1 mg/mL with the artificial chaperone method. Several mechanistic aspects of artificial chaperone-assisted refolding have been probed, and a detailed mechanism for the kinetically controlled stripping step is proposed.

  16. Fibril Fragmentation Enhances Amyloid Cytotoxicity*♦ (United States)

    Xue, Wei-Feng; Hellewell, Andrew L.; Gosal, Walraj S.; Homans, Steve W.; Hewitt, Eric W.; Radford, Sheena E.


    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity. PMID:19808677

  17. White Rock (United States)


    (Released 19 April 2002) The Science 'White Rock' is the unofficial name for this unusual landform which was first observed during the Mariner 9 mission in the early 1970's. As later analysis of additional data sets would show, White Rock is neither white nor dense rock. Its apparent brightness arises from the fact that the material surrounding it is so dark. Images from the Mars Global Surveyor MOC camera revealed dark sand dunes surrounding White Rock and on the floor of the troughs within it. Some of these dunes are just apparent in the THEMIS image. Although there was speculation that the material composing White Rock could be salts from an ancient dry lakebed, spectral data from the MGS TES instrument did not support this claim. Instead, the White Rock deposit may be the erosional remnant of a previously more continuous occurrence of air fall sediments, either volcanic ash or windblown dust. The THEMIS image offers new evidence for the idea that the original deposit covered a larger area. Approximately 10 kilometers to the southeast of the main deposit are some tiny knobs of similarly bright material preserved on the floor of a small crater. Given that the eolian erosion of the main White Rock deposit has produced isolated knobs at its edges, it is reasonable to suspect that the more distant outliers are the remnants of a once continuous deposit that stretched at least to this location. The fact that so little remains of the larger deposit suggests that the material is very easily eroded and simply blows away. The Story Fingers of hard, white rock seem to jut out like icy daggers across a moody Martian surface, but appearances can be deceiving. These bright, jagged features are neither white, nor icy, nor even hard and rocky! So what are they, and why are they so different from the surrounding terrain? Scientists know that you can't always trust what your eyes see alone. You have to use other kinds of science instruments to measure things that our eyes can

  18. European Whiteness?

    DEFF Research Database (Denmark)

    Blaagaard, Bolette


    Born out of the United States’ (U.S.) history of slavery and segregation and intertwined with gender studies and feminism, the field of critical whiteness studies does not fit easily into a European setting and the particular historical context that entails. In order for a field of European...... critical whiteness studies to emerge, its relation to the U.S. theoretical framework, as well as the particularities of the European context need to be taken into account.. The article makes a call for a multi-layered approach to take over from the identity politics so often employed in the fields of U...

  19. Contribution of Long Fibrils and Peptides to Surface and Foaming Behavior of Soy Protein Fibril System

    NARCIS (Netherlands)

    Wan, Zhili; Yang, Xiaoquan; Sagis, L.M.C.


    When soy glycinin (11S) is heated for a prolonged time at pH 2 (20 h at 85 °C), a mixture is formed consisting of long semiflexible 11S fibrils and small peptides. The surface and foaming properties of this mixture were investigated at different pHs, and compared to the behavior of pure fibrils and

  20. Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12 Cells.

    Directory of Open Access Journals (Sweden)

    Mansooreh Mazaheri

    Full Text Available In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity.

  1. RR-Interval variance of electrocardiogram for atrial fibrillation detection (United States)

    Nuryani, N.; Solikhah, M.; Nugoho, A. S.; Afdala, A.; Anzihory, E.


    Atrial fibrillation is a serious heart problem originated from the upper chamber of the heart. The common indication of atrial fibrillation is irregularity of R peak-to-R-peak time interval, which is shortly called RR interval. The irregularity could be represented using variance or spread of RR interval. This article presents a system to detect atrial fibrillation using variances. Using clinical data of patients with atrial fibrillation attack, it is shown that the variance of electrocardiographic RR interval are higher during atrial fibrillation, compared to the normal one. Utilizing a simple detection technique and variances of RR intervals, we find a good performance of atrial fibrillation detection.

  2. Lysozyme adsorption on the colloidal chromium(III) oxide surface: Its impact on the system stability

    Energy Technology Data Exchange (ETDEWEB)

    Szewczuk-Karpisz, Katarzyna, E-mail:; Wiśniewska, Małgorzata; Myśliwiec, Dawid


    Highlights: • Lysozyme adsorption mechanism on the chromium(III) oxide surface was determined. • Surface charge density as well as zeta potential of Cr{sub 2}O{sub 3} particles were measured. • Turbidimetric method was used to estimate the suspension stability. • Depending on the pH value, lysozyme increases or decreases the system stability. - Abstract: This paper describes the lysozyme (LSZ) presence effect on the chromium(III) oxide (Cr{sub 2}O{sub 3}) suspension stability. First, the electrokinetic properties of the examined system, i.e. surface charge density and zeta potential of solid particles in the absence and presence of LSZ, were determined. The lysozyme addition reduces the metal oxide surface charge, which may be related to the interaction of the LSZ protonated amino groups with the adsorbent surface moieties. The LSZ macromolecules undergo adsorption on the Cr{sub 2}O{sub 3} surface only under electrostatic attraction. At the LSZ concentrations above 50 ppm the macromolecules cover completely the particle surface, which is evidenced by the observed zeta potential values. The LSZ influence on the Cr{sub 2}O{sub 3} suspension stability depends on the solution pH value. At pH 3, 4.6 and 7.6, the LSZ addition improves the system stability. In turn, at pH 9 it is associated with the slight suspension destabilization.

  3. Complex formation in mixtures of lysozyme-stabilized emulsions and human saliva

    NARCIS (Netherlands)

    Silletti, E.; Vingerhoeds, M.H.; Norde, W.; Aken, van G.A.


    In this paper, we studied the interaction between human unstimulated saliva and lysozyme-stabilized oil-in-water emulsions (10 wt/wt% oil phase, 10 mM NaCl, pH 6.7), to reveal the driving force for flocculation of these emulsions. Confocal scanning laser microscopy (CSLM) showed formation of

  4. Lysozyme orientation and conformation on MoS2 surface: Insights from molecular simulations. (United States)

    Fan, Hongjie; Zhao, Daohui; Li, Yingtu; Zhou, Jian


    Two-dimensional molybdenum disulfide (MoS2) has attracted intense interest owing to its unique properties and promising biosensor applications. To develop effective biocompatible platforms, it is crucial to understand the interactions between MoS2 and biological molecules such as proteins, but little knowledge exists on the orientation and conformation of proteins on the MoS2 surface at the molecular level. In this work, the lysozyme adsorption on the MoS2 surface was studied by molecular dynamics simulations, wherein six different orientations were selected based on the different faces of lysozyme. Simulation results showed that lysozyme tends to adsorb on the MoS2 surface in an "end-on" orientation, indicating that orientations within this range are favorable for stable adsorption. The end-on orientation could be further categorized into "bottom end-on" and "top end-on" orientations. The driving forces responsible for the adsorption were dominated by van der Waals interactions and supplemented by electrostatic interactions. Further, the conformations of the lysozyme adsorbed on the MoS2 surface were basically preserved. This simulation study promotes the fundamental understanding of interactions between MoS2 and proteins and can guide the development of future biomedical applications of MoS2.

  5. Papaya (Carica papaya) lysozyme is a member of the family 19 (Basic, class II) chitinases

    NARCIS (Netherlands)

    Subroto, T; Sufiati, S; Beintema, JJ


    The most comprehensive studies on a plant lysozyme (EC are those on the enzyme from papaya (Carica papaya) latex, published in 1967 and 1969. However, the N-terminal amino acid sequence of five amino acid sequence of this enzyme, determined by manual Edman degradation, did not allow

  6. Effect of Encapsulation on Antimicrobial Activity of Herbal Extracts with Lysozyme

    Directory of Open Access Journals (Sweden)

    Petra Matouskova


    Full Text Available Resistance of microorganisms to antibiotics has increased. The use of natural components with antimicrobial properties can be of great significance to reduce this problem. The presented work is focused on the study of the effect of encapsulation of selected plant and animal antimicrobial substances (herbs, spices, lysozyme and nisin on their activity and stability. Antimicrobial components were packaged into liposomes and polysaccharide particles (alginate, chitosan and starch. Antimicrobial activity was tested against two Gram-positive (Bacillus subtilis and Micrococcus luteus and two Gram-negative (Escherichia coli and Serratia marcescens bacteria. Encapsulation was successful in all types of polysaccharide particles and liposomes. The prepared particles exhibited very good long-term stability, especially in aqueous conditions. Antimicrobial activity was retained in all types of particles. Liposomes with encapsulated herb and spice extracts exhibited very good inhibitory effect against all tested bacterial strains. Most of herbal extracts had very good antimicrobial effect against the tested Gram-negative bacterial strains, while Gram-positive bacteria were more sensitive to lysozyme particles. Thus, particles with co-encapsulated herbs and lysozyme are more active against different types of bacteria, and more stable and more effective during long-term storage. Particles with encapsulated mixture of selected plant extracts and lysozyme could be used as complex antimicrobial preparation with controlled release in the production of food and food supplements, pharmaceutical and cosmetic industries.

  7. Lysozyme-magnesium aluminum silicate microparticles: Molecular interaction, bioactivity and release studies

    DEFF Research Database (Denmark)

    Kanjanakawinkul, Watchara; Medlicott, Natalie J.; Rades, Thomas


    The objectives of this study were to investigate the adsorption behavior of lysozyme (LSZ) onto magnesium aluminum silicate (MAS) at various pHs and to characterize the LSZ–MAS microparticles obtained from the molecular interaction between LSZ and MAS. The results showed that LSZ could be bound...

  8. The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability (United States)

    Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.


    The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

  9. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating

    NARCIS (Netherlands)

    Muszanska, Agnieszka K.; Busscher, Henk J.; Herrmann, Andreas; van der Mei, Henny C.; Norde, Willem

    This paper describes the preparation and characterization of polymer protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the

  10. Antimicrobial lysozyme-containing starch microgel to target and inhibit amylase-producing micro-organisms

    NARCIS (Netherlands)

    Li, Yuan; Kadam, S.; Abee, T.; Slaghek, T.M.; Timmermans, J.W.; Cohen Stuart, M.A.; Norde, W.; Kleijn, J.M.


    The aim of this study is to determine the release of lysozyme from oxidized starch microgels and subsequently test its antimicrobial activity. The gels are made of oxidized potato starch polymers, which are chemically cross-linked by sodium trimetaphosphate (STMP). The microgel is negatively charged


    NARCIS (Netherlands)



    The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a

  12. Interaction between Humic Acid and Lysozyme, Studied by Dynamic Light Scattering and Isothermal Titration Calorimetry

    NARCIS (Netherlands)

    Tan, Wen Feng; Koopal, Luuk K.; Norde, Willem


    Interactions of purified Aldrich humic acid (PAHA) with the protein lysozyme (LSZ) are studied with dynamic light scattering and isothermal titration calorimetry by mixing LSZ and PAHA at various mass ratios. In solution LSZ is positive and PAHA is negative at the investigated pH values. Up to

  13. Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

    Energy Technology Data Exchange (ETDEWEB)

    Yadav, Indresh, E-mail:; Aswal, V. K. [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, J. [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)


    The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. The magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.

  14. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Yuan; Zhang, Zeshi; van Leeuwen, Herman P.; Stuart, Martien A. Cohen; Norde, Willem; Kleijn, J. Mieke


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10-20 mu m) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3-9, the

  15. Uptake and release kinetics of lysozyme in and from an oxidized starch polymer microgel

    NARCIS (Netherlands)

    Li, Y.; Zhang, Z.; Leeuwen, van H.P.; Cohen Stuart, M.A.; Norde, W.; Kleijn, J.M.


    The kinetics of uptake and release of fluorescently labeled lysozyme by/from spherical oxidized starch polymer microgel particles (diameter 10–20 µm) was investigated using confocal laser scanning microscopy. Both the protein and the microgel have a pH dependent charge; in the pH range 3–9, the

  16. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    ... activity of lysozyme in transfected cells culture medium was 180 U/ml. To obtain the gene targeted cells line, bovine fetal fibroblasts were isolated and transfected with linear targeting vector (21.9 kb) using nucleofector device, which the transfection rate was about 25%. After seven rounds of independent cell transfection, ...

  17. Investigation of factors affecting the stability of lysozyme spray dried from ethanol-water solutions

    DEFF Research Database (Denmark)

    Ji, Shuying; Thulstrup, Peter Waaben; Mu, Huiling


    -ethanol mixtures. The effect of formulation additives (trehalose, Tween 20 and phosphate-buffered saline) and processing conditions (inlet temperature and storage time of lysozyme in the feed solution before the spray drying process) on the protein bioactivity was investigated. The results showed...

  18. Impact of tear film components on lysozyme deposition to contact lenses. (United States)

    Ng, Alan; Heynen, Miriam; Luensmann, Doerte; Jones, Lyndon


    To investigate the impact of lactoferrin and lipids on the kinetic deposition of lysozyme on silicone and conventional hydrogel lenses, using a complex artificial tear solution (ATS). Two silicone hydrogel lenses (AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A) and two conventional hydrogel lenses (ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A) were investigated. Lenses were incubated in four different solutions: a complex ATS consisting of various salts, lipids, proteins, and mucins, an ATS without lactoferrin (ATS w/o Lac), an ATS without lipids (ATS w/o Lip), and an ATS without lactoferrin and lipids (ATS w/o Lac & Lip), each containing 2% radiolabeled (125I) lysozyme (1.9 mg/ml). After each time point (4, 12 h and 1, 2, 3, 5, 7, 14, 21, 28 days), the amount of lysozyme per lens was quantified. After 28 days, lotrafilcon B lenses incubated in ATS deposited significantly less lysozyme (9.7 ± 1.4 μg) than when incubated in solutions not containing lactoferrin and lipids (more than 11.8 μg) (p lenses was higher in ATS w/o Lip (5.3 ± 0.1 μg) compared with other solutions (less than 3.9 μg) (p lenses deposited the most lysozyme in all four solutions compared with the rest of the lens types (p lenses, less lysozyme was deposited when incubated in ATS w/o Lip (588.6 ± 0.4 μg) compared with the other solutions (more than 642.6 μg) (p lenses in ATS w/o Lac accumulated significantly less lysozyme (12.8 ± 1.0 μg) compared with the other solutions (more than 14.2 μg) (p silicone and conventional hydrogel contact lenses. When performing in vitro experiments to study protein deposition on contact lenses, more complex models should be used to better mimic the human tear film.

  19. Covalent immobilization of lysozyme onto woven and knitted crimped polyethylene terephthalate grafts to minimize the adhesion of broad spectrum pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Al Meslmani, Bassam M., E-mail: [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Mahmoud, Gihan F., E-mail: [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Department of Pharmaceutics and Industrial Pharmacy, Helwan University, Ain Helwan, 11795 Cairo (Egypt); Leichtweiß, Thomas, E-mail: [Institute of Physical Chemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, 35392 Giessen (Germany); Strehlow, Boris, E-mail: [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany); Sommer, Frank O., E-mail: [Institute for Medical Microbiology and Hospital Hygiene, Marburg University, Hans Meerwein Str 2, 35032 Marburg (Germany); Lohoff, Michael D., E-mail: [Institute for Medical Microbiology and Hospital Hygiene, Marburg University, Hans Meerwein Str 2, 35032 Marburg (Germany); Bakowsky, Udo, E-mail: [Department of Pharmaceutical Technology and Biopharmaceutics, Marburg University, Ketzerbach 63, 35037 Marburg (Germany)


    Graft-associated infections entirely determine the short-term patency of polyethylene terephthalate PET cardiovascular graft. We attempted to enzymatically inhibit the initial bacterial adhesion to PET grafts using lysozyme. Lysozyme was covalently immobilized onto woven and knitted forms of crimped PET grafts by the end-point method. Our figures of merit revealed lysozyme immobilization yield of 15.7 μg/cm{sup 2}, as determined by the Bradford assay. The activity of immobilized lysozyme on woven and knitted PET manifested 58.4% and 55.87% using Micrococcus lysodeikticus cells, respectively. Noteworthy, the adhesion of vein catheter-isolated Staphylococcus epidermidis decreased by 6- to 8-folds and of Staphylococcus aureus by 11- to 12-folds, while the Gram-negative Escherichia coli showed only a decrease by 3- to 4-folds. The anti-adhesion efficiency was specific for bacterial cells and no significant effect was observed on adhesion and growth of L929 cells. In conclusion, immobilization of lysozyme onto PET grafts can inhibit the graft-associated infection. - Highlights: • Lysozyme was covalently immobilized on crimped polyethylene terephthalate (PET). • The activity of immobilized lysozyme was meaningfully reduced. • The maintained activity significantly declined the adhesion of Gram-positive stains. • The enzymatic anti-adhesion efficiency reported lesser extent against Gram-negative. • The anti-bacterial activity displayed no significant effect on cells compatibility.

  20. The Natural Antimicrobial Enzyme Lysozyme is Up-Regulated in Gastrointestinal Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    Carlos A. Rubio


    Full Text Available The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014 exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days, by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett’s oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease, collagenous colitis, lymphocytic colitis, and Crohn’s colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention.

  1. Investigation of effects of terpene skin penetration enhancers on stability and biological activity of lysozyme. (United States)

    Varman, Rahul M; Singh, Somnath


    The transport of proteins through skin can be facilitated potentially by using terpenes as chemical enhancers. However, we do not know about the effects of these enhancers on the stability and biological activity of proteins which is crucial for the development of safe and efficient formulations. Therefore, this project investigated the effects of terpene-based skin penetration enhancers which are reported as nontoxic to the skin (e.g., limonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol, carveol, carvone, fenchone, and verbenone), on the conformational stability and biological activity of a model protein lysozyme. Terpene (5% v/v) was added to lysozyme solution and kept for 24 h (the time normally a transdermal patch remains) for investigating conformational stability profiles and biological activity. Fourier transform infrared spectrophotometer was used to analyze different secondary structures, e.g., α-helix, β-sheet, β-turn, and random coil. Conformational changes were also monitored by differential scanning calorimeter by determining midpoint transition temperature (Tm) and calorimetric enthalpy (ΔH). Biological activity of lysozyme was determined by measuring decrease in A (450) when it was added to a suspension of Micrococcus lysodeikticus. The results of this study indicate that terpenes 9, 10, and 11 (carvone, L-fenchone, and L-verbenone) decreased conformational stability and biological activity of lysozyme significantly (p terpenes used in this study. It is concluded that smaller terpenes containing ketones with low lipophilicity (log K (ow) ∼2.00) would be optimal for preserving conformational stability and biological activity of lysozyme in a transdermal formulation containing terpene as permeation enhancer.

  2. Risk of atrial fibrillation and stroke in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Lindhardsen, Jesper; Ahlehoff, Ole; Gislason, Gunnar Hilmar


    To determine if patients with rheumatoid arthritis have increased risk of atrial fibrillation and stroke.......To determine if patients with rheumatoid arthritis have increased risk of atrial fibrillation and stroke....


    Directory of Open Access Journals (Sweden)

    Johnson Francis


    Full Text Available Atrial fibrillation is the most common perioperative cardiac arrhythmia. Sympathetic overactivity, inflammatory state and oxidative stress are important contributors to the genesis of postoperative atrial fibrillation. Advancing age and mitral valve disease along with left atrial size are important parameters in noted in multivariate prediction model. Genetic predisposition has also been noted. Preventive strategies tried include beta blockers, statins, posterior pericardiotomy, carperitide infusion and thoracic epidural analgesia. Treatment options include rate and rhythm control along with anticoagulation if it persists more than 48 hours with high CHADS2 score. Some of the therapeutic modalities which have been found to be NOT useful in preventing post operative atrial fibrillation are dexamethasone, magnesium infusion and concomitant pulmonary vein isolation.

  4. Antihypertensive treatment and risk of atrial fibrillation

    DEFF Research Database (Denmark)

    Marott, Sarah C W; Nielsen, Sune F; Benn, Marianne


    AIMS: To examine the associations between antihypertensive treatment with angiotensin-converting enzyme inhibitors (ACEis) or angiotensin receptor blockers (ARBs), β-blockers, diuretics, or calcium-antagonists, and risk of atrial fibrillation. We examined these associations using the entire Danish......, and hyperthyroidism at baseline and none received any other antihypertensive medication. We studied risk of atrial fibrillation, and used risk of stroke, influenced by lowering blood pressure rather than renin-angiotensin system blockade per se, as an indicator of the importance of blood pressure lowering per se...... of stroke did not differ among the five antihypertensive medications. CONCLUSION: Use of ACEis and ARBs compared with β-blockers and diuretics associates with a reduced risk of atrial fibrillation, but not stroke, within the limitations of a retrospective study reporting associations. This suggests...

  5. The state of immunological reactivity and nonspecific protection factor (lysozyme in children with reactive arthritis

    Directory of Open Access Journals (Sweden)

    Vladimir Savvo


    Full Text Available Aim. An improvement of diagnostics and prognostication of ReA clinical course in children on the base of studying the immunological reactivity and non-specific protection factor (lysozyme.Materials and methods. Examination of children took place in the municipal children’s cardiorheumatologic department of MHPI “Kharkov municipal children’s clinical hospital № 24" and municipal children’s polyclinic of the Kharkov city (№ 1, 2, 7, 12, 13, 14, 23.40 children with ReA underwent immunological examinations, detection of sIgA in the saliva and lysozyme in the blood serum in acute period and in 9–12 months after the beginning of disease. 19 children (47,5 % – 2–6 years old, 21 children (52,5 %. – 7–14 years old. Boys – 22 (55,0 %, girls – 18 (45,0 %. The mean age of children in group was 7,2±0,32. The control group included 32 healthy children. The mean age of children in group was 7,4±0,54.The ReA diagnosis was set according to the order of Ukrainian MHP of 19.07.2005 № 362 “Protocol of diagnostics and treatment of disease of musculoskeletal system and connective tissue in children ICD-D М00-М25 arthropathies”.Immunological examinations included the study of indices of cellular, humoral, monocytic-phagocytic links of immunity, content of cytokines (IL-1β, IL-6, detection of sIgA and index of nonspecific protection factor (lysozyme.Assessment of results of researches was carried out using STATISTICА program for Windows (version 10.0, Microsoft Excel 2012, MATLAB 2015a.Results. In the ReA acute period in children was observed depression of T-system on the background of activation of immunity B-system as the reliable decrease of СD8, СD25 and increase of СD21. There was revealed an increase of IL-6, increase of phagocytic number, spontaneous NBT-test and spontaneous neutrophils activity index.The sIgA level reliably exceeded the standard. At determination of lysozyme the blood serum of patients with ReA in acute

  6. Effect of age on stroke prevention therapy in patients with atrial fibrillation: the atrial fibrillation investigators

    DEFF Research Database (Denmark)

    van Walraven, Carl; Hart, Robert G; Connolly, Stuart


    on the relative efficacy of oral anticoagulants (OAC) and antiplatelet (AP) therapy (including acetylsalicylic acid and triflusal) on ischemic stroke, serious bleeding, and vascular events in patients with atrial fibrillation. METHODS: This is an analysis of the Atrial Fibrillation Investigators database, which...... contains patient level-data from randomized trials of stroke prevention in atrial fibrillation. We used Cox regression models with age as a continuous variable that controlled for sex, year of randomization, and history of cerebrovascular disease, diabetes, hypertension, and congestive heart failure...

  7. Acute Chest Pain and Broad Complex Tachycardia. A Non-typical Case of Pre-excited Atrial Fibrillation


    Arias, Ramon Suarez; Villanueva, Nuria Perez; Cubero, Gustavo Iglesias; Lopez, Jose Rubin


    Wolff-Parkinson-White syndrome is a common condition in the emergency department. A case is presented of a 76-year-old patient with acute chest pain and broad complex tachycardia. Despite the fact that previous and post cardioversion ECG tracings in sinus rhythm showed no signs of pre-excitation, the characteristic pattern of pre-excited atrial fibrillation (AF) is recognized and after successful DC cardioversion the patient is referred for catheter ablation of the accessory pathway. This cas...

  8. Mapping out the multistage fibrillation of glucagon

    DEFF Research Database (Denmark)

    Ghodke, Shirin; Nielsen, Søren B.; Christiansen, Gunna


    at 25 °C and pH 2.5. The existence of these states is supported by complementary fluorescence and electron microscopy data. This highlights a multitude of structural transitions of glucagon from unordered structure to β sheets, β turns and further tertiary‐level changes. We attribute the observed......The 29‐residue peptide hormone glucagon forms many different morphological types of amyloid‐like fibrils, depending on solvent conditions. Here, we combine time‐series far‐UV CD with singular value decomposition analysis to reveal six different conformational states populated during fibrillation...

  9. Double sequential defibrillation for refractory ventricular fibrillation. (United States)

    El Tawil, Chady; Mrad, Sandra; Khishfe, Basem F


    A 54-year-old suffered from an out-of-hospital cardiac arrest. Compressions were started within minutes and the patient was in refractory ventricular fibrillation despite multiple asynchronized shocks and maximal doses of antiarrhythmic agents. Double sequential defibrillation was attempted with successful Return Of Spontaneous Circulation (ROSC) after a total of 61min of cardiac arrest. The patient was discharged home neurologically intact. Double sequential defibrillation could be a simple effective approach to patients with refractory ventricular fibrillation. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Cryoballoon Catheter Ablation in Atrial Fibrillation

    Directory of Open Access Journals (Sweden)

    Cevher Ozcan


    Full Text Available Pulmonary vein isolation with catheter ablation is an effective treatment in patients with symptomatic atrial fibrillation refractory or intolerant to antiarrhythmic medications. The cryoballoon catheter was recently approved for this procedure. In this paper, the basics of cryothermal energy ablation are reviewed including its ability of creating homogenous lesion formation, minimal destruction to surrounding vasculature, preserved tissue integrity, and lower risk of thrombus formation. Also summarized here are the publications describing the clinical experience with the cryoballoon catheter ablation in both paroxysmal and persistent atrial fibrillation, its safety and efficacy, and discussions on the technical aspect of the cryoballoon ablation procedure.

  11. Atrial natriuretic peptide in patients with heart failure and chronic atrial fibrillation : Role of duration of at atrial fibrillation

    NARCIS (Netherlands)

    Van Den Berg, MP; Crijns, HJGM; Van Veldhuisen, DJ; Van Gelder, IC; De Kam, PJ; Lie, KI

    The purpose of this study was to analyze the determinants of atrial natriuretic peptide level in patients with congestive heart failure and atrial fibrillation. In particular, the duration of atrial fibrillation was analyzed because atrial fibrillation per se might have a specific effect on atrial

  12. [The estimation of systemic chemotherapy treatment administered in breast cancer on lysozyme activity in tears--preliminary report]. (United States)

    Wojciechowska, Katarzyna; Jurowski, Piotr; Wieckowska-Szakiel, Marzena; Rózalska, Barbara


    Estimation of cytostatics influence used in breast cancer treatment on lysozyme activity in human tears depend on time of treatment. 8 women were treated at the base of chemotherapy schema: docetaxel with doxorubicin and 4 women treated with schema CMF: cyclophosphamide, methotrexate, 5-fluorouracil. Lysozyme activity in tears was assessed by measurement of diameter zone of Micrococcus lysodeicticus growth inhibition. It was revealed that both chemotherapy schema caused statistically significant reduction of diameter zone of M. lysodeicticus growth inhibition, after first and second course of chemotherapy treatment. After second chemotherapy course CMF schema induced loss of lysozyme activity in patient's tears (zero mm of M. lysodeicticus diameter zone growth inhibition). Systemic chemotherapy administered in breast cancer induce reduction of lysozyme activity in tears, that may cause higher morbidity of ocular surface infections caused by Gram-positive bacteria.

  13. Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system. (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu


    The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Ultra-sensitive quantification of lysozyme based on element chelate labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Yang, MingWei [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Department of Chemistry, Florida International University, Miami, FL 33199 (United States); Wu, WeiHua; Ruan, YaJuan; Huang, LiMei [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Wu, Zujian [Department of Plant Protection, Fujian Agriculture and Forest University, Fuzhou 350002, Fujian (China); Cai, Yong [Department of Chemistry, Florida International University, Miami, FL 33199 (United States); Fu, FengFu, E-mail: [Key Laboratory of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, Fujian (China); Department of Plant Protection, Fujian Agriculture and Forest University, Fuzhou 350002, Fujian (China)


    Graphical abstract: An ultra-sensitive method for the determination of lysozyme was developed based on the Gd{sup 3+} chelate labeling and CE–ICP–MS. The proposed method has an extremely low detection limit of 3.89 attomole and has been successfully used to detect lysozyme in saliva sample, showing excellent reliability. The success of the present method provides a new possibility for biological assays and clinical diagnoses. -- Highlights: •An ultra-sensitive method for detecting lysozyme based on CE–ICP–MS was described. •The proposed method has an extremely low detection limit of 3.89 attomole. •It can be used to detect trace lysozyme in saliva sample with a satisfied recovery. •The method provides a new potential for sensitive detection of low-abundant proteins. -- Abstract: In this study, an ultra-sensitive method for the quantification of lysozyme based on the Gd{sup 3+} diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry (CE–ICP–MS) was described. The Gd{sup 3+}-tagged lysozyme was effectively separated by capillary electrophoresis (CE) and sensitively determined by inductively coupled plasma mass spectrometry (ICP–MS). Based on the gadolinium-tagging and CE–ICP–MS, the lysozyme was determined within 12 min with an extremely low detection limit of 3.89 attomole (3.89 × 10{sup −11} mol L{sup −1} for 100 nL of sample injection) and a RSD < 6% (n = 5). The proposed method has been successfully used to detect lysozyme in saliva samples with a recovery of 91–106%, suggesting that our method is sensitive and reliable. The success of the present method provides a new potential for the biological assays and sensitive detection of low-abundant proteins.

  15. Lysozyme Expression Can be Useful to Distinguish Mammary Analog Secretory Carcinoma from Acinic Cell Carcinoma of Salivary Glands. (United States)

    Mariano, Fernanda Viviane; Gómez, Camila Andrea Concha; de Souza do Nascimento, Juliana; Dos Santos, Harim Tavares; Egal, Erika Said; Montalli, Victor Angelo Martins; Vargas, Pablo Agustin; de Almeida, Oslei Paes; Altemani, Albina


    Lysozyme is an enzymatic marker of acinar and intercalated duct cells of normal salivary glands. The aim of this study was to verify whether lysozyme expression could be useful to distinguish acinic cell carcinoma (ACC) from its main mimic, mammary analog secretory carcinoma (MASC). For comparison, DOG1 expression was analyzed as well. Seventeen cases of ACC, 15 MASC, and 125 other salivary tumors were studied. Lysozyme expression was found in tumor cells as well as in secreted material of MASC (86.6 % of cases) and in ductal cells of epithelial-myoepithelial carcinoma (EMC-53.8 %), pleomorphic adenoma (PA-29.1 %) and polymorphous low-grade adenocarcinoma (PLGA-23.8 %). However, in ACC, lysozyme was not expressed. Three patterns of DOG1 staining were seen: apical-luminal, cytoplasmic, and mixed cytoplasmic/membranous. The apical-luminal pattern was detected in ductal cells of ACC (58.8 % of cases), EMC (38.4 %), adenoid-cystic carcinoma (AdCC-35.3 %), PA (8.3 %), and PLGA (4.8 %). These tumors also showed mixed membranous/cytoplasmic staining for DOG1. MASC, mucoepidermoid, and salivary duct carcinomas exhibited only DOG1 cytoplasmic staining. In conclusion, lysozyme cannot be used as a marker of acinar differentiation in salivary tumors. However, lysozyme expression can be helpful to distinguish MASC from ACC due to its high frequency in the former and absence in ACC. It is likely that in MASC, lysozyme expression may reflect a lactational-like secretory differentiation since lysozyme belongs to breast milk proteins. Regarding DOG1 expression, the apical-luminal pattern is related to acinar and intercalated duct differentiation whereas the cytoplasmic staining does not seem to be associated with a specific cellular phenotype.

  16. New risk factors for atrial fibrillation : causes of 'not-so-lone atrial fibrillation'

    NARCIS (Netherlands)

    Schoonderwoerd, Bas A.; Smit, Marcelle D.; Pen, Lucas; Van Gelder, Isabelle C.

    Atrial fibrillation (AF) is a prevalent arrhythmia in patients with cardiovascular disease. The classical risk factors for developing AF include hypertension, valvular disease, (ischaemic) cardiomyopathy, diabetes mellitus, and thyroid disease. In some patients with AF, no underlying

  17. Type III collagen can be present on banded collagen fibrils regardless of fibril diameter



    Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibo...

  18. Heat-denatured lysozyme could be a novel disinfectant for reducing hepatitis A virus and murine norovirus on berry fruit. (United States)

    Takahashi, Michiko; Okakura, Yumiko; Takahashi, Hajime; Imamura, Minami; Takeuchi, Akira; Shidara, Hiroyuki; Kuda, Takashi; Kimura, Bon


    Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. In situ preparation of gold nanoparticle-loaded lysozyme-dextran nanogels and applications for cell imaging and drug delivery (United States)

    Cai, Huanxin; Yao, Ping


    An effective, green, and facile approach to synthesize gold nanoparticle-loaded protein-polysaccharide nanogels was developed in this study. Biocompatible gold nanoparticle-loaded lysozyme-dextran (Au@Lys-Dex) nanogels were produced using lysozyme-dextran nanogels as reducing and stabilizing agents. Lysozyme-dextran nanogels have a size of about 200 nm and a structure of lysozyme core and dextran shell. At pH around 4, AuCl4- ions are attracted and locally enriched by lysozyme due to the electrostatic and coordination interactions. When the solution is under UV irradiation, the AuCl4- ions are reduced to gold nanoparticles in situ by solvated electrons and reactive radicals produced from aromatic amino acid residues in the lysozyme. The produced gold nanoparticles with a size of about 8 nm are trapped inside the nanogels and the Au@Lys-Dex nanogels are well dispersible by virtue of the dextran shell. Antitumor drug, doxorubicin, can be loaded effectively inside Au@Lys-Dex nanogels via diffusion. In vitro study demonstrates the doxorubicin loaded Au@Lys-Dex nanogels have the same antitumor activity as free doxorubicin. The nanogels can be used as a contrasting agent in optical cell imaging, in which direct visual images of the subcellular distributions of the gold nanoparticles and the released doxorubicin are presented synchronously. The dual functional drug loaded Au@Lys-Dex nanogels are a promising system for simultaneous drug delivery and biomedical imaging.

  20. Management and prognosis of atrial fibrillation in the diabetic patient

    DEFF Research Database (Denmark)

    Pallisgaard, Jannik Langtved; Lindhardt, Tommi Bo; Olesen, Jonas Bjerring


    The global burden of atrial fibrillation and diabetes mellitus (diabetes) is considerable, and prevalence rates are increasing. Diabetes is associated with an increased risk of developing atrial fibrillation; however, diabetes also influences the management and prognosis of atrial fibrillation...... and outcomes of heart failure and the success rates of both ablation and cardioversion in atrial fibrillation patients with diabetes. Finally, this article describes the association of HbA1c levels with the management and prognosis of atrial fibrillation patients........ In the following article, the authors describe the association between diabetes and atrial fibrillation; specifically, the significance of diabetes on the risk of atrial fibrillation, ischemic stroke and bleeding complications associated with anticoagulation. In addition, the authors evaluate the risks...

  1. Growth of (101) Faces of Tetragonal Lysozyme Crystals: Measured Growth Rate Trends (United States)

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


    Earlier extensive measurements of the growth rates of the (110) face of tetragonal lysozyme crystals had shown unexpected dependencies on the supersaturation. In this study similar growth rate measurements were done for the (101) faces of the crystals. The data show a similar dependence on the supersaturation, becoming appreciable only at high supersaturations, reaching a maximum value and then decreasing. As reported in earlier studies, the (101) growth rates are larger at low supersaturations than the (110) growth rates at the same conditions, and smaller at high supersaturations. These trends suggest that the growth mechanism of the (101) is similar to that of the (110) face, involving the addition of lysozyme aggregates formed in solution, but with a growth unit smaller than that of the (110) face.

  2. Multifunctional Biomaterial Coating Based on Bio-Inspired Polyphosphate and Lysozyme Supramolecular Nanofilm. (United States)

    Xu, Xinyuan; Zhang, Dongyue; Gao, Shangwei; Shiba, Toshikazu; Yuan, Quan; Cheng, Kai; Tan, Hong; Li, Jianshu


    Current implant materials have widespread clinical applications together with some disadvantages, the majority of which are the ease with which infections are induced and difficulty in exhibiting biocompatibility. For the efficient improvement of their properties, the development of interface multifunctional modification in a simple, universal, and environmently benign approach becomes a critical challenge and has acquired the attention of numerous scientists. In this study, a lysozyme-polyphosphate composite coating was fabricated for titanium(Ti)-based biomaterial to obtain a multifunctional surface. This coating was easily formed by sequentially soaking the substrate in reduced-lysozyme and polyphosphate solution. Such a composite coating has shown predominant antibacterial activity against Gram-negative bacteria (E. coli) and improved cell adhesion, proliferation, and differentiation, which are much better than those of the pure substrate. This facile modification endows the biomaterial with anti-infective and potential bone-regenerative performance for clinical applications of biomaterial implants.

  3. Laser ablation of the lysozyme protein: a model system for soft materials

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin

    Lysozyme is a well-known protein which is used in food processing and is also an important constituent of human secretions such as sweat and saliva. It has a well-defined mass (14307 u) and can easily be detected by mass spectrometric methods such as MALDI (Matrix-assisted laser desorption...... ionization) in contrast to many other organic materials. Also the thermal properties, including the heat-induced decomposition behavior are comparatively well-known. For laser-irradiation at wavelengths above 310 nm, no photochemical processes occur initially, but the material is ejected via photothermal...... processes. The ablation of lysozyme from a dry pressed target in vacuum was measured by weight loss for nanosecond and femtosecond laser pulses at 355 or around 532 nm with a fluence of 1 J/cm2. A typical ablation yield for a 10-mJ pulse is about 150 micrograms/pulse, corresponding to the removal of ~ 6...

  4. SAS-Based Studies of Protein Fibrillation

    DEFF Research Database (Denmark)

    Marasini, Carlotta; Vestergaard, Bente


    Protein fibrillation is associated with a number of fatal amyloid diseases (e.g. Alzheimer's and Parkinson's diseases). From a structural point of view, the aggregation process starts from an ensemble of native states that convert into transiently formed oligomers, higher order assemblies...

  5. Dynamic fibrils in Hα and C IV

    NARCIS (Netherlands)

    de Wijn, A.G.; de Pontieu, B.


    Aims. To study the interaction of the solar chromosphere with the transition region, in particular active-region jets in the transition region and their relation to chromospheric fibrils. Methods. We carefully align image sequences taken simultaneously in Civ with the Transition Region and Coronal

  6. Genetic aspects of lone atrial fibrillation

    DEFF Research Database (Denmark)

    Andreasen, Laura; Nielsen, Jonas B; Olesen, Morten S


    Atrial fibrillation (AF) is the most common cardiac arrhythmia. A subgroup of patients presents with AF without traditional risk factors and is diagnosed before the age of 60 years. Such patients are commonly referred as having "lone AF" and comprise 10-20% of all cases. A number of studies have ...

  7. Genetic Risk Prediction of Atrial Fibrillation

    NARCIS (Netherlands)

    Lubitz, Steven A; Yin, Xiaoyan; Lin, Henry; Kolek, Matthew; Smith, J Gustav; Trompet, Stella; Rienstra, Michiel; Rost, Natalia S; Teixeira, Pedro; Almgren, Peter; Anderson, Christopher D; Chen, Lin Y; Engström, Gunnar; Ford, Ian; Furie, Karen L; Guo, Xiuqing; Larson, Martin G; Lunetta, Kathryn; Macfarlane, Peter W; Psaty, Bruce M; Soliman, Elsayed Z; Sotoodehnia, Nona; Stott, David J; Taylor, Kent D; Weng, Lu-Chen; Yao, Jie; Geelhoed, Bastiaan; Verweij, Niek; Siland, Joylene E; Kathiresan, Sekar; Roselli, Carolina; Roden, Dan M; van der Harst, Pim; Darbar, Dawood; Jukema, J Wouter; Melander, Olle; Rosand, Jonathan; Rotter, Jerome I; Heckbert, Susan R; Ellinor, Patrick T; Alonso, Alvaro; Benjamin, Emelia J


    BACKGROUND: Atrial fibrillation (AF) has a substantial genetic basis. Identification of individuals at greatest AF risk could minimize the incidence of cardioembolic stroke. METHODS: To determine whether genetic data can stratify risk for development of AF, we examined associations between AF

  8. Risk of atrial fibrillation in diabetes mellitus

    DEFF Research Database (Denmark)

    Pallisgaard, Jannik L; Schjerning, Anne-Marie; Lindhardt, Tommi B


    .81-8.98) and 20.0 (19.9-20.2) in the background population and 0.13 (0.09-0.20), 2.10 (2.00-2.20), 8.41 (8.10-8.74) and 20.1 (19.4-20.8) in the diabetes group, respectively. The adjusted incidence rate ratios in the diabetes group with the background population as reference were 2.34 (1.52-3.60), 1.52 (1......AIM: Diabetes has been associated with atrial fibrillation but the current evidence is conflicting. In particular knowledge regarding young diabetes patients and the risk of developing atrial fibrillation is sparse. The aim of our study was to investigate the risk of atrial fibrillation in patients...... with diabetes compared to the background population in Denmark. METHODS AND RESULTS: Through Danish nationwide registries we included persons above 18 years of age and without prior atrial fibrillation and/or diabetes from 1996 to 2012. The study cohort was divided into a background population without diabetes...

  9. Aggregation and fibrillation of bovine serum albumin

    DEFF Research Database (Denmark)

    Holm, NK; Jespersen, SK; Thomassen, LV


    The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and cha...

  10. Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

    Directory of Open Access Journals (Sweden)

    Alexandre Wohlkönig

    Full Text Available BACKGROUND: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. RESULTS: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46 share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. CONCLUSIONS: The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.

  11. An improved 96-well turbidity assay for T4 lysozyme activity (United States)


    room temperature, according to the manufacturer’s instructions. 4. Wash resin twice with 10 bed volumes of wash buffer (30mM potassium phosphate pH...measurements for T4L. First, the 30minute time allowance between cell suspension and the start of the assay was optimal for cells to rehydrate and settle...Determination of lysozyme activities in a microplate format, Anal. Biochem. 310 (2002) 223–224. [3] D. Christybapita, M. Divyagnaneswari, R.D. Michael, Oral

  12. Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits


    Jackson, Kathryn A.; Berg, Jolene M.; Murray, James D.; Maga, Elizabeth A.


    While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their...

  13. The bulk association mechanism between lysozyme and α-lactalbumine is revealed by interfacial measurements


    Pezennec, Stephane; Bouhallab, Said; Hamon, Pascaline; Renault, A; Beaufils, S.


    Controlled interactions and assembly of proteins with one another promise to be a powerful approach for generating novel supramolecular architectures. In this study, we report on two oppositely charged proteins, Lysozyme (LYS) and alpha-Lactalbumine (ALA) which self-assemble into well-defined micrometer-sized spherical particles under specific physicochemical conditions. These particles are spontaneously built following two main steps: first the formation of oligomers constituting the element...

  14. Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates


    Vijay Kumar Ravi; Mohit Goel; Hema Chandra Kotamarthi; Sri Rama Koti Ainavarapu; Rajaram Swaminathan


    Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL) at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescenc...

  15. Effect of Cholesterol on the Properties of Spray-Dried Lysozyme-Loaded Liposomal Powders


    Charnvanich, Dusadee; Vardhanabhuti, Nontima; Kulvanich, Poj


    The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respec...

  16. The Averaged Face Growth Rates of lysozyme Crystals: The Effect of Temperature (United States)

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


    Measurements of the averaged or macroscopic face growth rates of lysozyme crystals are reported here for the (110) face of tetragonal lysozyme, at three sets of pH and salt concentrations, with temperatures over a 4-22 C range for several protein concentrations. The growth rate trends with supersaturation were similar to previous microscopic growth rate measurements. However, it was found that at high super-saturations the growth rates attain a maximum and then start decreasing. No 'dead zone' was observed but the growth rates were found to approach zero asymptotically at very low super-saturations. The growth rate data also displayed a dependence on pH and salt concentration which could not be characterized solely by the super-saturation. A complete mechanism for lysozyme crystal growth, involving the formation of an aggregate growth unit, mass transport of the growth unit to the crystal interface and faceted crystal growth by growth unit addition, is suggested. Such a mechanism may provide a more consistent explanation for the observed growth rate trends than those suggested by other investigators. The nutrient solution interactions leading to the formation of the aggregate growth unit may, thus, be as important as those occurring at the crystal interface and may account for the differences between small molecule and protein crystal growth.

  17. Lysozyme and defense peptides as suppressors of phenoloxidase activity in Galleria mellonella. (United States)

    Zdybicka-Barabas, Agnieszka; Mak, Paweł; Jakubowicz, Teresa; Cytryńska, Małgorzata


    The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response. © 2014 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Hanyu Wu

    Full Text Available 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.

  19. Crystallization of insulin and lysozyme under reduced convection condition in a large gradient magnetic field (United States)

    Yin, D. C.; Wakayama, N. I.; Fujiwara, M.; Harata, K.; Xue, X. P.; Fu, Z. X.; Zhang, S. W.; Shang, P.; Tanimoto, Y.

    The crystallization of protein from solution is governed by the process of transport phenomenon Any reason affecting the process of solute transport will impose effects on the crystallization process thus further affects the crystal quality Recent advancement in superconducting magnet technology makes it possible to provide a low cost long-time durable low effective gravity environment for the control of convection which is similar to the environment in the space As an ideal means to damp natural convection in a non-conductive solution on the Earth it may find applications in the field of protein crystallization In this presentation the authors investigated the crystallization of orthorhombic lysozyme crystals tetragonal lysozyme crystals and insulin crystals in a large gradient magnetic field Three effective gravity levels were used milli-gravity around 0G normal gravity 1G and hypergravity 1 8G Comparisons of the crystal quality obtained inside and outside the magnetic field showed that both the magnetic field and the effective gravity could affect the crystal quality But the effect also depends on the crystal and protein type For lysozyme crystals in tetragonal form the magnetic field and effective gravity showed no obvious effect on the quality whereas for the crystals in orthorhombic form both the magnetic field and effective gravity improved the crystal quality For insulin crystal which is highly symmetrical magnetic field and effective gravity showed no strong effect on the crystal quality It is well known that

  20. Cis-elements required for the demethylation of the mouse M-lysozyme downstream enhancer. (United States)

    Schmitz, A; Short, M; Ammerpohl, O; Asbrand, C; Nickel, J; Renkawitz, R


    The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.

  1. Methylation of the mouse M-lysozyme downstream enhancer inhibits heterotetrameric GABP binding. (United States)

    Nickel, J; Short, M L; Schmitz, A; Eggert, M; Renkawitz, R


    Expression of the mouse M-lysozyme gene is a specific marker for the differentiation of macrophage/granulocyte cell lineages. Analysis of the mechanisms regulating M-lysozyme gene expression revealed an enhancer element in the 3'-flanking region of the gene, termed the M-lysozyme downstream enhancer (MLDE). Here we demonstrate that the nuclear factors binding to MLDE are present in all tested myeloid and non-myeloid mouse cell lines. Sequence analysis of MLDE identified two different sequences, CAGGAAGT and CCGGAAGT, which match the consensus binding sequences for proteins of the ets gene superfamily. The two sites are oriented palindromicly and separated by 10 bp. DMS/DEPC interference assays revealed different patterns of DNA-protein contacts on the two sites. Mutation of each consensus sequence leads to an individual change in protein binding in vitro. Despite these differences, both sequences are bound by GABP, forming a heterotetrameric complex. Tissue specificity is correlated with demethylation of a single CpG dinucleotide located in one of the two Ets motifs. This site when methylated inhibits GABP binding to both sequences in non-macrophage cell types.

  2. Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method. (United States)

    Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X


    Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

  3. Lysozyme complexes with thermo- and pH-responsive PNIPAM-b-PAA block copolymer

    Energy Technology Data Exchange (ETDEWEB)

    Pippa, Natassa [National and Kapodistrian University of Athens, Department of Pharmaceutical Technology, Faculty of Pharmacy (Greece); Meristoudi, Anastasia; Pispas, Stergios, E-mail: [National Hellenic Research Foundation, Theoretical and Physical Chemistry Institute (Greece); Demetzos, Costas [National and Kapodistrian University of Athens, Department of Pharmaceutical Technology, Faculty of Pharmacy (Greece)


    Lysozyme is an enzyme responsible for the damage of bacterial cell walls and is abundant in a number of secretions such as tears and human milk. In the present study, we investigated the structure, the physicochemical characteristics, and the temperature-responsiveness of lysozyme complexes with poly(N-isopropylacrylamide)-b-poly(acrylic acid) block polyelectrolyte in aqueous media. A gamut of light-scattering techniques and fluorescence spectroscopy were used in order to examine the complexation process, as well as the structure, solution behavior, and temperature response of the nanosized complexes. The concentration of copolymer polyelectrolyte was kept constant. The values of the scattering intensity, I{sub 90}, which is proportional to the mass of the species in solution, increased gradually as a function of C{sub LYS,} providing proof of the occurring complexation, while the size of the nanostructures decreased. The structure of the complexes became more open as the C{sub LYS} increased. The increase of the salinity did not affect the structural characteristics of the supramolecular nanoparticulate aggregates. On the other hand, the physicochemical and structural characteristics of the complexes changed upon increasing temperature, and the changes depended on the initial ratio block polyelectrolyte/lysozyme. The knowledge on developing block polyelectrolyte/protein complexes through electrostatic interactions, obtained from this investigation, may be applied to the design of nutraceuticals.

  4. Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils. (United States)

    Ragland, Stephanie A; Schaub, Ryan E; Hackett, Kathleen T; Dillard, Joseph P; Criss, Alison K


    Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil-rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram-negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation. © 2016 John Wiley & Sons Ltd.

  5. Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2, 4-pentanediol

    Energy Technology Data Exchange (ETDEWEB)

    Stauber, Mark [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Jakoncic, Jean [Brookhaven National Laboratory, Building 725D, Upton, NY 11973-5000 (United States); Berger, Jacob; Karp, Jerome M.; Axelbaum, Ariel; Sastow, Dahniel [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Buldyrev, Sergey V. [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Hrnjez, Bruce J. [Collegiate School, 260 West 78th Street, New York, NY 10024-6559 (United States); Asherie, Neer, E-mail: [Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States); Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312 (United States)


    Crystallization of lysozyme with (R)-2-methyl-2, 4-pentanediol produces more ordered crystals and a higher resolution protein structure than crystallization with (S)-2-methyl-2, 4-pentanediol. The results suggest that chiral interactions with chiral additives are important in protein crystal formation. Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2, 4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

  6. First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2. (United States)

    Ghasemi, Seyedhadi; Ahmadian, Gholamreza; Sadeghi, Mehdi; Zeigler, Daniel R; Rahimian, Heshmatollah; Ghandili, Soheila; Naghibzadeh, Neda; Dehestani, Ali


    Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Adsorption and conformations of lysozyme and α-lactalbumin at a water-octane interface (United States)

    Cheung, David L.


    As proteins contain both hydrophobic and hydrophilic amino acids, they will readily adsorb onto interfaces between water and hydrophobic fluids such as oil. This adsorption normally causes changes in the protein structure, which can result in loss of protein function and irreversible adsorption, leading to the formation of protein interfacial films. While this can be advantageous in some applications (e.g., food technology), in most cases it limits our ability to exploit protein functionality at interfaces. To understand and control protein interfacial adsorption and function, it is necessary to understand the microscopic conformation of proteins at liquid interfaces. In this paper, molecular dynamics simulations are used to investigate the adsorption and conformation of two similar proteins, lysozyme and α-lactalbumin, at a water-octane interface. While they both adsorb onto the interface, α-lactalbumin does so in a specific orientation, mediated by two amphipathic helices, while lysozyme adsorbs in a non-specific manner. Using replica exchange simulations, both proteins are found to possess a number of distinct interfacial conformations, with compact states similar to the solution conformation being most common for both proteins. Decomposing the different contributions to the protein energy at oil-water interfaces suggests that conformational change for α-lactalbumin, unlike lysozyme, is driven by favourable protein-oil interactions. Revealing these differences between the factors that govern the conformational change at interfaces in otherwise similar proteins can give insight into the control of protein interfacial adsorption, aggregation, and function.

  8. Uniform spatial distribution of collagen fibril radii within tendon implies local activation of pC-collagen at individual fibrils

    CERN Document Server

    Rutenberg, Andrew D; Kreplak, Laurent


    Collagen fibril cross-sectional radii show no systematic variation between the interior and the periphery of fibril bundles, indicating an effectively constant rate of collagen incorporation into fibrils throughout the bundle. Such spatially homogeneous incorporation constrains the extracellular diffusion of collagen precursors from sources at the bundle boundary to sinks at the growing fibrils. With a coarse-grained diffusion equation we determine stringent bounds, using parameters extracted from published experimental measurements of tendon development. From the lack of new fibril formation after birth, we further require that the concentration of diffusing precursors stays below the critical concentration for fibril nucleation. We find that the combination of the diffusive bound, which requires larger concentrations to ensure homogeneous fibril radii, and lack of nucleation, which requires lower concentrations, is only marginally consistent with fully-processed collagen using conservative bounds. More real...

  9. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow. (United States)

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A


    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  10. Stroke as the First Manifestation of Atrial Fibrillation. (United States)

    Jaakkola, Jussi; Mustonen, Pirjo; Kiviniemi, Tuomas; Hartikainen, Juha E K; Palomäki, Antti; Hartikainen, Päivi; Nuotio, Ilpo; Ylitalo, Antti; Airaksinen, K E Juhani


    Atrial fibrillation may remain undiagnosed until an ischemic stroke occurs. In this retrospective cohort study we assessed the prevalence of ischemic stroke or transient ischemic attack as the first manifestation of atrial fibrillation in 3,623 patients treated for their first ever stroke or transient ischemic attack during 2003-2012. Two groups were formed: patients with a history of atrial fibrillation and patients with new atrial fibrillation diagnosed during hospitalization for stroke or transient ischemic attack. A control group of 781 patients with intracranial hemorrhage was compiled similarly to explore causality between new atrial fibrillation and stroke. The median age of the patients was 78.3 [13.0] years and 2,009 (55.5%) were women. New atrial fibrillation was diagnosed in 753 (20.8%) patients with stroke or transient ischemic attack, compared to 15 (1.9%) with intracranial hemorrhage. Younger age and no history of coronary artery disease or other vascular diseases, heart failure, or hypertension were the independent predictors of new atrial fibrillation detected concomitantly with an ischemic event. Thus, ischemic stroke was the first clinical manifestation of atrial fibrillation in 37% of younger (<75 years) patients with no history of cardiovascular diseases. In conclusion, atrial fibrillation is too often diagnosed only after an ischemic stroke has occurred, especially in middle-aged healthy individuals. New atrial fibrillation seems to be predominantly the cause of the ischemic stroke and not triggered by the acute cerebrovascular event.

  11. Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods: e0142095

    National Research Council Canada - National Science Library

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


    .... 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...

  12. The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook. (United States)

    Primo, Emiliano D; Otero, Lisandro H; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter


    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, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  13. HPLC of the Polypeptides in a Hydrolyzate of Egg-White Lysozyme. An Experiment for the Undergraduate Biochemistry Laboratory. (United States)

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


    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)

  14. Evidence of structurally continuous collagen fibrils in tendon

    DEFF Research Database (Denmark)

    Svensson, Rene B; Herchenhan, Andreas; Starborg, Tobias


    in this structure-function relationship is the collagen fibril length. During embryogenesis short fibrils are produced but they grow rapidly with maturation. There is some controversy regarding fibril length in adult tendon, with mechanical data generally supporting discontinuity while structural investigations...... favor continuity. This study initially set out to trace the full length of individual fibrils in adult human tendons, using serial block face-scanning electron microscopy. But even with this advanced technique the required length could not be covered. Instead a statistical approach was used on a large...... fibrils was confirmed. In light of these results, possible mechanisms that could reconcile the opposing findings on fibril continuity are discussed. STATEMENT OF SIGNIFICANCE: Connective tissues hold all parts of the body together and are mostly constructed from thin threads of the protein collagen...

  15. Atrial fibrillation: non cardiologist physicians approach

    Directory of Open Access Journals (Sweden)

    Forero-Gómez, Julián Eduardo


    Full Text Available Atrial fibrillation is the most frequent arrhythmia. Its classification according to pattern and clinical type allows to decide the therapeutic strategy to use, that most include control of symptoms and prevention of cardioembolic events. The election of the treatment depends on the presence of triggering events, risk factors for thromboembolism, risk factors for bleeding, cardiac function, patient funcionality, medication costoefectiveness and health care access. The type of anticoagulant has to be supported on the type of atrial fibrillation and the presence of contraindications, documented ineffective anticoagulation or high risk of failure to warfarin. In case of contraindications for anticoagulation this could still be used in high bleeding risk patients, when risk factors are controllable or corrected; leaving left atrial appendage closure as an option for patients that remain in high risk for bleeding events.

  16. Aggregation and fibrillation of bovine serum albumin

    DEFF Research Database (Denmark)

    Holm, NK; Jespersen, SK; Thomassen, LV


    The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology...... and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a lag phase, is independent of seeding and shows only moderate concentration dependence, suggesting intramolecular aggregation nuclei. Nevertheless, multi-exponential increases in dye-binding signal...... and changes in morphology suggest the existence of different aggregate species. Although beta-sheet content increases from 0 to ca. 40% upon aggregation, the aggregates retain significant amounts of alpha-helix structure, and lack a protease-resistant core. Thus BSA is able to form well-ordered beta...

  17. Deglutition induced atrial tachycardia and atrial fibrillation. (United States)

    Kanjwal, Yousuf; Imran, Naser; Grubb, Blair


    Deglutition induced supraventricular tachycardia is an uncommon condition postulated to be a vagally mediated phenomenon due to mechanical stimulation. Patients usually present with mild symptoms or may have severe debilitating symptoms. Treatment with Class I agents, beta blockers, calcium channel blockers, amiodarone and radiofrquency catheter ablation has shown to be successful in the majority of reported cases. We report the case of a 46-year-old healthy woman presenting with palpitations on swallowing that was documented to be transient atrial tachycardia with aberrant ventricular conduction as well as transient atrial fibrillation. She was successfully treated with propafenone with no induction of swallowing-induced tachycardia after treatment. This is also the first case to show swallowing-induced atrial tachycardia and atrial fibrillation in the same patient.

  18. Sequence determinants of amyloid fibril formation


    López de la Paz, Manuela; Serrano, Luis


    The establishment of rules that link sequence and amyloid feature is critical for our understanding of misfolding diseases. To this end, we have performed a saturation mutagenesis analysis on the de novo-designed amyloid peptide STVIIE (1). The positional scanning mutagenesis has revealed that there is a position dependence on mutation of amyloid fibril formation and that both very tolerant and restrictive positions to mutation can be found within an amyloid sequence. In this system, mutation...

  19. Atrial Fibrillation During an Exploration Class Mission (United States)

    Lipset, Mark A.; Lemery, Jay; Polk, J. D.; Hamilton, Douglas R.


    Background: A long-duration exploration class mission is fraught with numerous medical contingency plans. Herein, we explore the challenges of symptomatic atrial fibrillation (AF) occurring during an exploration class mission. The actions and resources required to ameliorate the situation, including the availability of appropriate pharmaceuticals, monitoring devices, treatment modalities, and communication protocols will be investigated. Challenges of Atrial Fibrillation during an Exploration Mission: Numerous etiologies are responsible for the initiation of AF. On Earth, we have the time and medical resources to evaluate and determine the causative situation for most cases of AF and initiate therapy accordingly. During a long-duration exploration class mission resources will be severely restricted. How is one to determine if new onset AF is due to recent myocardial infarction, pulmonary embolism, fluid overload, thyrotoxicosis, cardiac structural abnormalities, or CO poisoning? Which pharmaceutical therapy should be initiated and what potential side effects can be expected? Should anti-coagulation therapy be initiated? How would one monitor the therapeutic treatment of AF in microgravity? What training would medical officers require, and which communication strategies should be developed to enable the best, safest therapeutic options for treatment of AF during a long-duration exploration class mission? Summary: These questions will be investigated with expert opinion on disease elucidation, efficient pharmacology, therapeutic monitoring, telecommunication strategies, and mission cost parameters with emphasis on atrial fibrillation being just one illustration of the tremendous challenges that face a long-duration exploration mission. The limited crew training time, medical hardware, and drugs manifested to deal with such an event predicate that aggressive primary and secondary prevention strategies be developed to protect a multibillion-dollar asset like the

  20. Acute atrial fibrillation during dengue hemorrhagic fever

    Directory of Open Access Journals (Sweden)

    Veloso Henrique Horta


    Full Text Available Dengue fever is a viral infection transmitted by the mosquito, Aedes aegypti. Cardiac rhythm disorders, such as atrioventricular blocks and ventricular ectopic beats, appear during infection and are attributed to viral myocarditis. However, supraventricular arrhythmias have not been reported. We present a case of acute atrial fibrillation, with a rapid ventricular rate, successfully treated with intravenous amiodarone, in a 62-year-old man with dengue hemorrhagic fever, who had no structural heart disease.

  1. Cardiac fibrillation risk of Taser weapons. (United States)

    Leitgeb, Norbert


    The debate on potential health hazards associated with delivering electric discharges to incapacitated subjects, in particular on whether electric discharge weapons are lethal, less lethal or non-lethal, is still controversial. The cardiac fibrillation risks of Taser weapons X26 and X3 have been investigated by measuring the delivered high-tension pulses in dependence on load impedance. Excitation thresholds and sinus-to-Taser conversion factors have been determined by numerical modeling of endocardial, myocardial, and epicardial cells. Detailed quantitative assessment of cardiac electric exposure has been performed by numerical simulation at the normal-weighted anatomical model NORMAN. The impact of anatomical variation has been quantified at an overweight model (Visible Man), both with a spatial resolution of 2 × 2 × 2 mm voxels. Spacing and location of dart electrodes were systematically varied and the worst-case position determined. Based on volume-weighted cardiac exposure assessment, the fibrillation probability of the worst-case hit was determined to 30% (Taser X26) and 9% (Taser X3). The overall risk assessment of Taser application accounting for realistic spatial hit distributions was derived from training sessions of police officers under realistic scenarios and by accounting for the influence of body (over-)weight as well as gender. The analysis of the results showed that the overall fibrillation risk of Taser use is not negligible. It is higher at Taser X26 than at Taser X3 and amounts to about 1% for Europeans with an about 20% higher risk for Asians. Results demonstrate that enhancement as well as further reduction of fibrillation risk depends on responsible use or abuse of Taser weapons.

  2. Aspects epidemiologiques et etiologiques de la fibrillation ...

    African Journals Online (AJOL)

    Les étiologies les plus fréquentes étaient représentées par les cardiomyopathies hypertensives (34,3%), les cardiomyopathies dilatées d'allure primitives (23,7%) et les valvulopathies rhumatismales (7%) Conclusion : La fibrillation auriculaire est fréquente chez l'adulte noir togolais. Ses étiologies sont dominées par ...

  3. Formation of hydrogels based on chitosan/alginate for the delivery of lysozyme and their antibacterial activity. (United States)

    Wu, Tiantian; Huang, Jiaqi; Jiang, Yangyang; Hu, Yaqin; Ye, Xingqian; Liu, Donghong; Chen, Jianchu


    Novel hydrogels based on chitosan/sodium alginate (CS-ALG) were prepared to deliver and protect lysozyme while eliminating food-borne microorganisms. These hydrogels were characterized according to the zeta potential, optical microscopy, scanning electron microscopy (SEM), UV-visible spectroscopy (UV-vis), fourier transform infrared (FT-IR), and small-angle X-ray scattering (SAXS). The results demonstrated that the resultant hydrogels were negatively charged and spherical in shape. In addition, the maximum swelling ratio was 45.66±7.62 for CS-ALG hydrogels loaded with lysozyme. The relative activity of the released lysozyme was 87.72±3.96%, indicating that CS-ALG hydrogels are promising matrices for enzyme loading and adsorption. Furthermore, a 100% bacterial clearance rate of CS/ALG loaded with lysozyme was observed to correspond to the superposition effect stimulated by CS and lysozyme, which improved the antibacterial activity against E. coli and S. aureus compared to CS/ALG, suggesting its potential use in the food industry as well as other applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Data supporting the effects of lysozyme on mRNA and protein expression in a colonic epithelial scratch wound model

    Directory of Open Access Journals (Sweden)

    Sarah K. Abey


    Full Text Available Colonic epithelial health is implicated in a host of gastrointestinal (GI diseases and disorders. Lysozyme is suspected to play a role in the ability of the epithelium to recover from injury (Abey et al., in press; Gallo, 2012; Rubio, 2014 [1–3]. Disrupted repair mechanisms may lead to delayed or ineffective recovery and disruptions to epithelial biology resulting in GI symptoms and altered barrier function (Peterson and Artis, 2014 [4]. The effect of lysozyme on the transcriptomic and proteomic profile of healthy colonic epithelial cells was investigated. Epithelial cells in culture were scratch wounded and treated with lysozyme. mRNA and protein profiles were simultaneously quantified in the same sample using a digital counting technology. Gene and protein expressions altered by the presence or absence of lysozyme are described in this article. Extensive statistical and bioinformatic analysis, and interpretation of the results can be found in “Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress” (Abey et al., in press [1].

  5. A Single-Step Purification of Cauliflower Lysozyme and Its Dual Role Against Bacterial and Fungal Plant Pathogens. (United States)

    Manikandan, Muthu; Balasubramaniam, R; Chun, Se-Chul


    A novel lysozyme from cauliflower was purified in a single step, for the first time, using Sephadex G100 column chromatography. The purified lysozyme exhibited a homogenized single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its molecular mass was calculated to be 22.0 kDa. The purified lysozyme showed activity between 30 to 60 °C with 40 °C as the optimum temperature for its maximal activity. Although the purified lysozyme was functional at pH ranges between 3.0 and 9.0, the optimum pH for the enzyme activity was 8.0. By Michaelis-Menten equation, the threshold substrate concentration for the optimal enzyme activity was calculated to be 133.0 μg. The purified lysozyme showed extraordinary activity against plant pathogenic bacteria and fungi. At 10-μg concentrations, it inhibited the growth of plant pathogenic bacteria such as Pseudomonas syringae, Xanthomonas campestris, and Erwinia carotovora exhibiting 4.28, 5.90, and 3.88-fold inhibition, respectively. Further, it also completely inhibited the conidial germination of Archemonium obclavatum and, to a very large extent, other fungal species such as Fusarium solani (79.3 %), Leptosphaeria maculans (88.6 %), Botrytis cinera (73.3 %), Curvularia lunata (68 %), Rhizoctonia solani (79.6 %), and Alternaria alternata (83.6 %).

  6. Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity.

    Directory of Open Access Journals (Sweden)

    Camille Detree

    Full Text Available The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR. They harbour in specialized gill cells two types of endosymbiont (gram-bacteria: sulphide oxidizing bacteria (SOX and methanotrophic bacteria (MOX. This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike--1700 m, Mid-Atlantic Ridge, and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.

  7. Sequence determinants of amyloid fibril formation. (United States)

    López de la Paz, Manuela; Serrano, Luis


    The establishment of rules that link sequence and amyloid feature is critical for our understanding of misfolding diseases. To this end, we have performed a saturation mutagenesis analysis on the de novo-designed amyloid peptide STVIIE (1). The positional scanning mutagenesis has revealed that there is a position dependence on mutation of amyloid fibril formation and that both very tolerant and restrictive positions to mutation can be found within an amyloid sequence. In this system, mutations that accelerate beta-sheet polymerization do not always lead to an increase of amyloid products. On the contrary, abundant fibrils are typically found for mutants that polymerize slowly. From these experiments, we have extracted a sequence pattern to identify amyloidogenic stretches in proteins. The pattern has been validated experimentally. In silico sequence scanning of amyloid proteins also supports the pattern. Analysis of protein databases has shown that highly amyloidogenic sequences matching the pattern are less frequent in proteins than innocuous amino acid combinations and that, if present, they are surrounded by amino acids that disrupt their aggregating capability (amyloid breakers). This study provides the potential for a proteome-wide scanning to detect fibril-forming regions in proteins, from which molecules can be designed to prevent and/or disrupt this process.

  8. Atrial Fibrillation in Eight New World Camelids. (United States)

    Bozorgmanesh, R; Magdesian, K G; Estell, K E; Stern, J A; Swain, E A; Griffiths, L G


    There is limited information on the incidence of clinical signs, concurrent illness and treatment options for atrial fibrillation (AF) in New World Camelids (NWC). Describe clinical signs and outcome of AF in NWC. Eight New World Camelids admitted with AF. A retrospective observational study of camelids diagnosed with AF based on characteristic findings on electrocardiogram (ECG). All animals had an irregularly irregular heart rhythm detected on physical examination and 4 cases had obtunded mentation on admission. Three camelids were diagnosed with AF secondary to oleander intoxication, 3 animals had underlying cardiovascular disease, 1 was diagnosed with lone AF and 1 had AF diagnosed on examination for a urethral obstruction. Five of eight animals survived to discharge and nonsurvivors consisted of animals which died or were euthanized as a result of cardiovascular disease (2/8) or extra-cardiac disease unrelated to the AF (1/8). Atrial fibrillation occurs in NWC in association with cardiovascular disease, extra-cardiac disease or as lone AF. Amiodarone and transthoracic cardioversion were attempted in one llama with lone AF, but were unsuccessful. Atrial fibrillation was recorded in 0.1% of admissions. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  9. Depth Profile Assessment of the Early Phase Deposition of Lysozyme on Soft Contact Lens Materials Using a Novel In Vitro Eye Model. (United States)

    Qiao, Han; Phan, Chau-Minh; Walther, Hendrik; Subbaraman, Lakshman N; Jones, Lyndon


    To characterize the location of fluorescently labeled lysozyme on commercial contact lenses (CLs) using an in vitro eye model that simulates tear volume, tear flow, air exposure, and mechanical wear. One commercially available conventional hydrogel CL material (etafilcon A) and three silicone hydrogel CL materials (balafilcon A, lotrafilcon B, and senofilcon A) were evaluated in this study. The CLs were mounted on the in vitro eye model and exposed to artificial tear fluid containing fluorescein isothiocyanate (FITC)-labeled lysozyme for 2 and 10 hrs. After these short incubation periods, circular discs were punched from the CLs at the center and periphery and were prepared for confocal laser scanning microscopy (CLSM). The CLSM captured a series of consecutive images spaced 5 μm apart, and the resulting images were rendered into two dimensional cross-sectional views of the CL. The mean fluorescence at each 5 μm slice was used to generate a histogram depicting the penetration of FITC-lysozyme into CLs. For both incubation periods, the CLSM images and histogram of etafilcon A showed that FITC-lysozyme is more concentrated at the lens surface, with a moderate amount of deposition in the lens matrix. For balafilcon A, FITC-lysozyme was evenly distributed throughout the lens. For lotrafilcon B, there was a greater amount of FITC-lysozyme deposition on the surfaces of the lens versus the matrix. Senofilcon A had differential FITC-lysozyme distribution profiles depending on the location of the lens. At the lens periphery, FITC-lysozyme primarily deposited on the surface, whereas FITC-lysozyme was uniformly distributed at the center of the lens. With the use of a sophisticated in vitro eye model, the study revealed a complex deposition pattern of FITC-labeled lysozyme on various CL materials after short periods of exposure. An understanding of the early deposition pattern of lysozyme on different CL material may elucidate new insights into the processes behind CL

  10. Toxic species in amyloid disorders: Oligomers or mature fibrils

    Directory of Open Access Journals (Sweden)

    Meenakshi Verma


    Full Text Available Protein aggregation is the hallmark of several neurodegenerative disorders. These protein aggregation (fibrillization disorders are also known as amyloid disorders. The mechanism of protein aggregation involves conformation switch of the native protein, oligomer formation leading to protofibrils and finally mature fibrils. Mature fibrils have long been considered as the cause of disease pathogenesis; however, recent evidences suggest oligomeric intermediates formed during fibrillization to be toxic. In this review, we have tried to address the ongoing debate for these toxic amyloid species. We did an extensive literature search and collated information from Pubmed ( and Google search using various permutations and combinations of the following keywords: Neurodegeneration, amyloid disorders, protein aggregation, fibrils, oligomers, toxicity, Alzheimer′s Disease, Parkinson′s Disease. We describe different instances showing the toxicity of mature fibrils as well as oligomers in Alzheimer′s Disease and Parkinson′s Disease. Distinct structural framework and morphology of amyloid oligomers suggests difference in toxic effect between oligomers and fibrils. We highlight the difference in structure and proposed toxicity pathways for fibrils and oligomers. We also highlight the evidences indicating that intermediary oligomeric species can act as potential diagnostic biomarker. Since the formation of these toxic species follow a common structural switch among various amyloid disorders, the protein aggregation events can be targeted for developing broad-range therapeutics. The therapeutic trials based on the understanding of different protein conformers (monomers, oligomers, protofibrils and fibrils in amyloid cascade are also described.

  11. The use of warfarin in veterans with atrial fibrillation

    Directory of Open Access Journals (Sweden)

    Rosenbeck Karen


    Full Text Available Abstract Background Warfarin therapy is effective for the prevention of stroke in patients with atrial fibrillation. However, warfarin therapy is underutilized even among ideal anticoagulation candidates. The purpose of this study was to examine the use of warfarin in both inpatients and outpatients with atrial fibrillation within a Veterans Affairs (VA hospital system. Methods This retrospective medical record review included outpatients and inpatients with atrial fibrillation. The outpatient cohort included all patients seen in the outpatient clinics of the VA Connecticut Healthcare System during June 2000 with a diagnosis of atrial fibrillation. The inpatient cohort included all patients discharged from the VA Connecticut Healthcare System West Haven Medical Center with a diagnosis of atrial fibrillation during October 1999 – March 2000. The outcome measure was the rate of warfarin prescription in patients with atrial fibrillation. Results A total of 538 outpatients had a diagnosis of atrial fibrillation and 73 of these had a documented contraindication to anticoagulation. Among the 465 eligible outpatients, 455 (98% were prescribed warfarin. For the inpatients, a total of 212 individual patients were discharged with a diagnosis of atrial fibrillation and 97 were not eligible for warfarin therapy. Among the 115 eligible inpatients, 106 (92% were discharged on warfarin. Conclusions Ideal anticoagulation candidates with atrial fibrillation are being prescribed warfarin at very high rates within one VA system, in both the inpatient and outpatient settings; we found warfarin use within our VA was much higher than that observed for Medicare beneficiaries in our state.

  12. Hybrid ablation for atrial fibrillation: current approaches and future directions. (United States)

    Bisleri, Gianluigi; Glover, Benedict


    Catheter ablation of atrial fibrillation has rapidly evolved during the past decade: although the treatment of paroxysmal atrial fibrillation via a transcatheter approach has been consistently successful, persistent and long-standing atrial fibrillation still represents a major clinical challenge with less favorable outcomes to date. Because novel, minimally invasive surgical approaches have been developed for atrial fibrillation ablation, the aim of the present review is to analyze the current evidence surrounding the integration of surgical and transcatheter strategies in a hybrid fashion for the treatment of atrial fibrillation. Long-standing persistent, atrial fibrillation requires further understanding. Wide antral circumferential ablation of the pulmonary veins represents the cornerstone of any ablation therapy. Additional linear lesions and/or targeting complex fractionated atrial electrograms may also be considered. One of the limitations is achieving a transmural lesion. The combined endocardial and epicardial approach may represent a superior approach. Hybrid ablation of atrial fibrillation represents a novel therapeutic strategy for the treatment of complex scenarios, such as long-standing persistent atrial fibrillation. A specialized team including dedicated surgeons and cardiologists appears to be crucial in order to achieve durable and satisfactory outcomes following hybrid ablation of atrial fibrillation.


    Andryuschenko, S V; Perunova, N B


    Determination of modulating effect of acid-base state of medium for cultivation of enterobacteria on activity of C-type lysozyme. Escherichia coli BL21 (DE3) strain for protein expression, Escherichia coli K12 MG1655 model strain, Escherichia coli No. 242 strain, isolated from intestine biotope; 2 Klebsiella pneumoniae strains, one of those contained plasmid homologue of periplasmatic lysozyme inhibitor gene pliC; 1 typical Salmonella enterica ATCC 14028 strain and a Micrococcus luteus ATCC 15307 strain as a control--served as material for the study. The bacteria were cultivated for 24 hours in 2 ml of liquid medium LB at 37 degrees C, 250 rpm. Determination of antilysozyme activity (ALA) was carried out by a photonepehlometrical method according to O.V. Bukharin et al. (1999) with alterations. All the studied microorganisms, including Micrococcus luteus, at the specified conditions 24 hours after cultivation were established to change the pH of the liquid nutrient medium LB from the initial value of 6.6 ± 0.1 to 8.2 ± 0.2 units. ALA determination in the cultivation medium without buffer correction was accompanied by a decline of lysozyme activity at an order of magnitude. The effect was absent during ALA measurement by a standard technique. The local shift of acid-base state of biotope under the conditions of buffer system insufficiency results in a reversible alteration of antimicrobial activity of muramidase, that among other non-specific factors of the environment determines the background of interactions on the level of associative symbiosis. This aspect should be taken into consideration during development of models, that are close to real conditions of microsymbiocenotical interactions.

  14. Binding patterns and structure-affinity relationships of food azo dyes with lysozyme: a multitechnique approach. (United States)

    Peng, Wei; Ding, Fei; Peng, Yu-Kui; Jiang, Yu-Ting; Zhang, Li


    Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the

  15. Lysozyme- and chitinase activity in latex bearing plants of genus Euphorbia--A contribution to plant defense mechanism. (United States)

    Sytwala, Sonja; Günther, Florian; Melzig, Matthias F


    Occurrence of latices in plants is widespread, there are 40 families of plants characterized to establish lactiferous structures. Latices exhibit a constitutive part of plant defense due to the stickiness. The appearance of proteins incorporated in latices is well characterized, and hydrolytic active proteins are considerable. A lot of plants constitute so-called pathogenesis-related (PR) proteins, to overcome stressful conditions. In our investigation we are focused on latex bearing plants of Euphorbiaceae Juss., and investigated the appearance of chitinase- and lysozyme activity in particular. The present outcomes represent a comprehensive study, relating to the occurrence of lysozyme and chitinase activity of genus Euphorbia at the first time. 110 different species of genus Euphorbia L. were tested, and the appearance of chitinase and lysozyme were determined in different quantities. The appearance itself, and the physicochemical properties of latices indicate an efficient interaction for plant defense against pathogen attack. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  16. Lysozyme/EDTA disodium salt and modified-atmosphere packaging to prolong the shelf life of burrata cheese. (United States)

    Conte, A; Brescia, I; Del Nobile, M A


    To prolong the shelf life of burrata cheese, we evaluated the effects of lysozyme and EDTA disodium salt (Na(2)-EDTA) with or without modified-atmosphere packaging (MAP) conditions. In particular, 3 concentrations of enzyme were combined with packaging in air and under MAP (95:5 CO(2):N(2)). The decline in quality of burrata cheese stored at 8°C was assessed by monitoring microbiological and sensory quality, in addition to pH and headspace composition. The combination of lysozyme/Na(2)-EDTA and MAP prolonged cheese shelf life, especially at the highest lysozyme concentration. In particular, the tested strategy was effective against microbial spoilage phenomena that appeared to be the quality factor that determine product unacceptability. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Production of Antimicrobial Films by Incorporation of Partially Purified Lysozyme into Biodegradable Films of Crude Exopolysaccharides Obtained from Aureobasidium pullulans Fermentation

    Directory of Open Access Journals (Sweden)

    Nilay Kandemir


    Full Text Available Antimicrobial films were produced by incorporating partially purified lysozyme into films of crude exopolysaccharides (59 % pullulan obtained from Aureobasidium pullulans fermentation. After film making, the films containing lysozyme at 100, 260, 520 and 780 μg/cm2 showed 23 to 70 % of their expected enzyme activities. The highest recovery of enzyme activity (65–70 % after the film making was obtained in films prepared by incorporating lysozyme at 260 μg/cm2 (1409 U/cm2. The incorporation of disodium EDTA×2H2O and sucrose did not affect the initial lysozyme activity of the films significantly. With or without the presence of disodium EDTA×2H2O at 52 or 520 μg/cm2, lysozyme activity showed sufficient stability in the films during 21 days of cold storage. However, the presence of sucrose at 10 mg/cm2 in the films caused the destabilization of part of enzyme activity (almost 35 % at the end of storage. The combinational incorporation of lysozyme at 780 μg/cm2 (4227 U/cm2 and disodium EDTA×2H2O at 520 μg/cm2 gave antimicrobial films effective on Escherichia coli. However, in the studied lysozyme concentration range the films did not show any antimicrobial activity against Lactobacillus plantarum. This study clearly showed that the partially purified lysozyme and crude exopolysaccharides from Aureobasidium pullulans may be used to obtain antimicrobial films to increase the safety of foods.

  18. Modeling growth of Alicyclobacillus acidoterrestris DSM 3922 type strain vegetative cells in the apple juice with nisin and lysozyme

    Directory of Open Access Journals (Sweden)

    Celenk Molva


    Full Text Available In the present study, the effect of storage temperature on A. acidoterrestris DSM 3922 cells (105 CFU/mL was examined during growth in reconstituted apple juice (pH 3.8, °Brix 11.3 containing nisin (0–100 IU/mL and lysozyme (0–100 mg/L. The growth curves were obtained at three temperatures of 27, 35 and 43 °C using absorbance data (OD600 nm. Based on the results, the minimal inhibitory concentrations (MICs of nisin were found as 10 IU/mL at all tested temperatures. On the other hand, increasing the temperature decreased the amount of lysozyme for growth inhibition. The MICs of lysozyme were found as 10, 2.5 and 1.25 mg/L at 27, 35 and 43 °C, respectively. At selected non-inhibitory doses, nisin (1.25–5 IU/mL and lysozyme (0.3–2.5 mg/L prolonged the lag time compared to the controls at the corresponding temperatures. In addition, there was a strong linear relationship between the lag time and lysozyme concentrations at 27 and 35 °C (R2 > 0.98. The results of this study demonstrated that both nisin and lysozyme could be used to inhibit the growth of A. acidoterrestris cells in the apple juice. The results also indicated that the growth parameters were variable depending on the storage temperature and the type of the antimicrobial agent used in the apple juice.

  19. Immunohistochemical study on the secretory host defense system with lysozyme and secretory phospholipase A2 throughout rat respiratory tract. (United States)

    Masuda, Natsumi; Mantani, Youhei; Yoshitomi, Chiaki; Yuasa, Hideto; Nishida, Miho; Arai, Masaya; Kawano, Junichi; Yokoyama, Toshifumi; Hoshi, Nobuhiko; Kitagawa, Hiroshi


    The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

  20. Localization of lysozyme mRNA in the labial salivary glands by in situ hybridization in Sjoegren's syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Konttinen, Y.T.; Segerberg-Konttinen, M.; Nordstroem, D.; Groenblad, M. (Fourth Department of Medicine, Helsinki University Central Hospital, Helsinki (Finland)); Kulomaa, M.; Keinaenen, R. (Department of Biochemical Sciences, University of Tampere, Tampere (Finland)); Malmstroem, M. (Institute of Dentistry, Department of Oral Surgery, University of Helsinki, Helsinki (Finland))


    In this study, lysozyme mRNA in labial salivary glands has been localized with in situ hybridization technique using 35S-labeled hen lysozyme cDNA (cDNALZM) as a hybridization probe in normals and in patients with Sjoegren's syndrome. 35S-DNALZM:mRNA hybrids were detected only in acinar serous cells, although lysozyme was identified in ductal cells using immunohistochemical techniques. Our results suggest that the serous acinar cells are the only site of lysozyme synthesis in small salivary glands. The presence of lysozyme in ductal cells may be a result of reabsorption from the saliva or concentration from the blood or surrounding tissues. (author).

  1. Influence of particle size and antigen binding on effectiveness of aluminum salt adjuvants in a model lysozyme vaccine. (United States)

    Clausi, Amber; Cummiskey, Jessica; Merkley, Scott; Carpenter, John F; Braun, Latoya Jones; Randolph, Theodore W


    It has been suggested that agglomeration of aluminum salt adjuvant particles during freezing and drying can cause loss of immunogenicity of vaccines formulated with such adjuvants. In this study, we tested this hypothesis and examined the immune response in a murine model to various liquid, freeze-thawed, and lyophilized vaccine formulations, using lysozyme as a model antigen. The various processing techniques and excipient levels resulted in a wide range of particle size distributions (PSDs) and antigen-adjuvant binding levels. Anti-lysozyme titers were independent of the PSD for vaccines adjuvanted with either aluminum hydroxide or aluminum phosphate and also were unaffected by the level of antigen binding to the adjuvant.

  2. Acute phase proteins, C9, factor B, and lysozyme in recurrent oral ulceration and Behçet's syndrome.


    Lehner, T; Adinolfi, M


    The concentrations and sequential changes of some acute phase proteins, factor B, and lysozyme have been assayed in recurrent oral ulceration and Behçet's syndrome. C9 was elevated in both groups of patients and was the sensitive index of disease activity; however, it failed to discriminate between the three types of recurrent oral ulcers and four types of Behçet's syndrome. The level of alpha 1 acid glycoprotein and lysozyme were significantly increased predominantly in the ocular type, wher...

  3. Does Myocardial Infarction Beget Atrial Fibrillation and Atrial Fibrillation Beget Myocardial Infarction?

    NARCIS (Netherlands)

    Vermond, Rob A.; Van Gelder, Isabelle C.; Crijns, Harry J.; Rienstra, Michiel


    Atrial fibrillation (AF) affects millions of people worldwide.(1) It is already known several decades that AF is not a benign condition, and it's associated with a 5-fold increased risk of stroke, 3-fold increased risk of heart failure, and doubling of risk of dementia and death.(2-4) Myocardial

  4. Sucrose modulates insulin amyloid-like fibril formation: effect on the aggregation mechanism and fibril morphology

    DEFF Research Database (Denmark)

    Marasini, Carlotta; Foderà, Vito; Vestergaard, Bente


    the protein self-assembly pathways. Using a combination of fluorescence spectroscopy, synchrotron radiation circular dichroism and transmission electron microscopy, we study the kinetics of formation and structural properties of human insulin fibrils in the presence of sucrose. The presence of sucrose results...

  5. Critical contribution of VH–VL interaction to reshaping of an antibody: The case of humanization of anti-lysozyme antibody, HyHEL-10 (United States)

    Nakanishi, Takeshi; Tsumoto, Kouhei; Yokota, Akiko; Kondo, Hidemasa; Kumagai, Izumi


    To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function. PMID:18227432

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

    Energy Technology Data Exchange (ETDEWEB)

    Ghattyvenkatakrishna, Pavan K [ORNL; Carri, Gustavo A. [University of Akron


    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, 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.

  7. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Helen P McWilliams-Koeppen

    Full Text Available Light chain (AL amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(PH-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  8. Effect of Stirring and Seeding on Whey Protein Fibril Formation

    NARCIS (Netherlands)

    Bolder, S.G.; Sagis, L.M.C.; Venema, P.; Linden, van der E.


    The effect of stirring and seeding on the formation of fibrils in whey protein isolate (WPI) solutions was studied. More fibrils of a similar length are formed when WPI is stirred during heating at pH 2 and 80 C compared to samples that were heated at rest. Addition of seeds did not show an

  9. Atrial fibrillation in an adolescent--the agony of ecstasy. (United States)

    Madhok, Ashish; Boxer, Robert; Chowdhury, Devyani


    Ecstasy (MDMA), a popular drug of abuse among teenagers, is thought to be "relatively" safe. A case of atrial fibrillation following the ingestion of ecstasy in a previously well adolescent is presented. Emergency room physicians should consider ecstasy abuse in the differential diagnosis of young patients presenting with atrial fibrillation.

  10. Oral antiarrhythmic drugs in converting recent onset atrial fibrillation

    NARCIS (Netherlands)

    Deneer, Vera H. M.; Borgh, Marieke B. I.; Kingma, J. Herre; Lie-A-Huen, Loraine; Brouwers, Jacobus R. B. J.


    AIM: This article reviews clinical studies on oral antiarrhythmic drugs in converting recent onset atrial fibrillation. An oral loading dose of an antiarrhythmic drug for cardioversion of atrial fibrillation could be an option, due to its simplicity, both for patients admitted to outpatient

  11. Multifaceted fibrils: self-assembly, polymorphism and functionalization

    NARCIS (Netherlands)

    Sidhu, A.


    In vitro fibrillization of proteins into amyloid fibrils provides critical insights into the factors influencing protein aggregation and has a key role in understanding the molecular basis of several neurodegenerative diseases caused by amyloids. α-synuclein (αSyn), an intrinsically disordered

  12. La fibrillation auriculaire chez le noir Africain: Aspects ...

    African Journals Online (AJOL)

    Rheumatic valvulopathies (especially mitral valvulopathies), where the most frequent etiology (39%), followed by cardiomyopathies (33%). External electrical cardioversion had the best rate of atrial fibrillation reduction (81.8%), in comparison with anti-arythmic drugs (45.1%). However, atrial fibrillation arise the problem of it ...

  13. Radiofrequency ablation as initial therapy in paroxysmal atrial fibrillation

    DEFF Research Database (Denmark)

    Cosedis Nielsen, Jens; Johannessen, Arne; Raatikainen, Pekka


    There are limited data comparing radiofrequency catheter ablation with antiarrhythmic drug therapy as first-line treatment in patients with paroxysmal atrial fibrillation.......There are limited data comparing radiofrequency catheter ablation with antiarrhythmic drug therapy as first-line treatment in patients with paroxysmal atrial fibrillation....

  14. Shear flow suppresses the volume of the nucleation precursor clusters in lysozyme solutions (United States)

    Byington, Michael C.; Safari, Mohammad S.; Conrad, Jacinta C.; Vekilov, Peter G.


    Shear flow alters the rate at which crystals nucleate from solution, yet the underlying mechanisms remain poorly understood. To fill this knowledge gap, we explore the response to shear of dense liquid clusters, which may serve as crystal nucleation precursors. Solutions of the protein lysozyme were sheared in a Couette cell at rates from 0.3 to 200 s-1 for up to seven hours. The cluster size and total population volume were characterized by dynamic light scattering. We demonstrate that shear rates greater than 10 s-1 applied for longer than one hour reduce the volume of the cluster population. The likely mechanism of the observed response involves enhanced partial unfolding of the lysozyme molecules, which exposes hydrophobic surfaces between the constituent domains to the aqueous solution. We show that disruption of the intramolecular S-S bridges does not contribute to the mechanism of response to shear. The decrease of the cluster population volume with increasing shear rate or shear time implies that nucleation could be inhibited at moderate shear rates.

  15. Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Dionisia Ortiz-Aguayo


    Full Text Available This research develops a label-free aptamer biosensor (aptasensor based on graphite-epoxy composite electrodes (GECs for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN6]3−/[Fe(CN6]4− as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM−1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis.

  16. Chaperonin-Inspired pH Protection by Mesoporous Silica SBA-15 on Myoglobin and Lysozyme. (United States)

    Lynch, Michele M; Liu, Jichuan; Nigra, Michael; Coppens, Marc-Olivier


    While enzymes are valuable tools in many fields of biotechnology, they are fragile and must be protected against denaturing conditions such as unfavorable solution pH. Within living organisms, chaperonins help enzymes fold into their native shape and protect them from damage. Inspired by this natural solution, mesoporous silica SBA-15 with different pore diameters is synthesized as a support material for immobilizing and protecting enzymes. In separate experiments, the model enzymes myoglobin and lysozyme are physically adsorbed to SBA-15 and exposed to a range of buffered pH conditions. The immobilized enzymes' biocatalytic activities are quantified and compared to the activities of nonimmobilized enzymes in the same solution conditions. It has been observed that myoglobin immobilized on SBA-15 is protected from acidic denaturation from pH 3.6 to 5.1, exhibiting relative activity of up to 350%. Immobilized lysozyme is protected from unfavorable conditions from pH 6.6 to 7.6, with relative activity of up to 200%. These results indicate that the protective effects conferred to enzymes immobilized by physical adsorption to SBA-15 are driven by the enzymes' electrostatic attraction to the material's surface. The pore diameter of SBA-15 affects the quality of protection given to immobilized enzymes, but the contribution of this effect at different pH values remains unclear.

  17. Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland. (United States)

    Maga, E A; Shoemaker, C F; Rowe, J D; Bondurant, R H; Anderson, G B; Murray, J D


    The potential for applying biotechnology to benefit animal agriculture and food production has long been speculated. The addition of human milk components with intrinsic antimicrobial activity and positive charge to livestock milk by genetic engineering has the potential to benefit animal health, as well as food safety and production. We generated one line of transgenic goats as a model for the dairy cow designed to express human lysozyme in the mammary gland. Here we report the characterization of the milk from 5 transgenic females of this line expressing human lysozyme in their milk at 270 microg/mL or 68% of the level found in human milk. Milk from transgenic animals had a lower somatic cell count, but the overall component composition of the milk and milk production were not different from controls. Milk from transgenic animals had a shorter rennet clotting time and increased curd strength. Milk of such nature may be of benefit to the producer by influencing udder health and milk processing.

  18. Lysozyme Solubility and Conformation in Neat Ionic Liquids and Their Mixtures with Water. (United States)

    Strassburg, Stephen; Bermudez, Harry; Hoagland, David


    The room temperature solubility of a number of model proteins is assessed for a diverse set of neat ionic liquids (ILs). For two soluble protein-IL pairs, lysozyme in [C2MIM][EtSO4] (1-ethyl-3-methylimidazolium ethylsulfate) and in [C2,4,4,4P][Et2PO4] (tributyl(ethyl)phosphonium diethylphosphate), protein solubility and structure at various temperatures are probed by dynamic light scattering (assessing dissolved molecular size), turbidimetry (reflecting degree of solubility), and Fourier transform infrared spectroscopy (uncovering helical secondary structure). As compared to aqueous environments, [C2,4,4,4P][Et2PO4] thermally stabilizes protein size and secondary structure while [C2MIM][EtSO4] does the opposite. Lysozyme denatured in [C2MIM][EtSO4] does not aggregate, presumably due to an absence of hydrophobic interactions, and the denaturation appears thermally reversible. Both ILs at room temperature are miscible with water in all proportions, but to create the corresponding ternary mixtures with protein, the order of mixing is important. Mixed to avoid additions of water to IL-dissolved protein, stable solutions are obtained with [C2MIM][EtSO4] at all solvent compositions. When water is added to IL-rich solutions, liquid-liquid demixing is noted.

  19. Nuclease-hypersensitive sites in the chromatin domain of the chicken lysozyme gene. (United States)

    Fritton, H P; Sippel, A E; Igo-Kemenes, T


    We have examined the chromatin structure of a 22 kilobase-pair chromosomal region containing the lysozyme gene in laying hen. Nuclease-hypersensitive sites were probed with DNAase I by using an indirect end-labeling technique. Eight DNAase I-hypersensitive sites could be mapped in the flanking regions of the gene in oviduct cells, in which the gene is expressed. The same sensitive sites were detected by utilization of an endogenous nuclease activity present in oviduct nuclei. Only one hypersensitive site was detected in the chromatin from erythrocytes, in which the gene is not expressed. The 3'-terminus of the lysozyme gene is highly exposed in nuclei from both tissues. Of special interest is the hypersensitive site at the 5'-terminus of the actively transcribed gene since it maps at the region of multiple initiation sites of transcription and the putative control regions of steroid hormones. DNAase I-hypersensitive chromatin structures also at a greater distance from the gene may take part in the control of gene expression. Images PMID:6304632

  20. Self-Assembled Monolayers of an Azobenzene Derivative on Silica and Their Interactions with Lysozyme. (United States)

    Wei, Tao; Sajib, Md Symon Jahan; Samieegohar, Mohammadreza; Ma, Heng; Shing, Katherine


    The capability of the photoresponsive isomerization of azobenzene derivatives in self-assembled monolayer (SAM) surfaces to control protein adsorption behavior has very promising applications in antifouling materials and biotechnology. In this study, we performed an atomistic molecular dynamics (MD) simulation in combination with free-energy calculations to study the morphology of azobenzene-terminated SAMs (Azo-SAMs) grafted on a silica substrate and their interactions with lysozyme. Results show that the Azo-SAM surface morphology and the terminal benzene rings' packing are highly correlated with the surface density and the isomer state. Higher surface coverage and the trans-isomer state lead to a more ordered polycrystalline backbone as well as more ordered local packing of benzene rings. On the Azo-SAM surface, water retains a high interfacial diffusivity, whereas the adsorbed lysozyme is found to have extremely low mobility but a relative stable secondary structure. The moderate desorption free energy (∼60 kT) from the trans-Azo-SAM surface was estimated by using both the nonequilibrium-theorem-based Jarzynski's equality and equilibrium umbrella sampling.

  1. In situ chitin isolation from body parts of a centipede and lysozyme adsorption studies. (United States)

    Bulut, Esra; Sargin, Idris; Arslan, Ozlem; Odabasi, Mehmet; Akyuz, Bahar; Kaya, Murat


    Isolation of structurally intact chitin samples for biotechnological applications has gained much recent attention. So far, three-dimensional chitin isolates have been obtained from only diplopods and sponges. In this study, three-dimensional chitin isolates were obtained from the body parts of centipede Scolopendra sp. (antennae, head, forcipule, collum, trunk, trunk legs and last pair of legs) without leading to structural failure. FT-IR spectra of chitin isolates confirmed that chitin samples are in α allomorph. TGA, XRD and SEM analyses and lysozyme adsorption studies revealed that each chitin isolate had different thermal stability, crystallinity and surface characteristics. Among the chitin isolates, Cu(II)-immobilized forcipule chitin showed the highest affinity for lysozyme (54.1mg/g), whereas chitin from last pair of legs exhibited the lowest affinity (3.7mg/g). This study demonstrated that structurally intact chitin isolates can be obtained from the body parts of centipede Scolopendra sp. (antennae, head, forcipule, collum, trunk, trunk legs and last pair of legs) by using a simple chemical procedure. Also, it gives a biotechnological perspective to the organisms in the group of Chilipoda. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Diamond Nanogel-Embedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release (United States)


    Temporarily implanted devices, such as drug-loaded contact lenses, are emerging as the preferred treatment method for ocular diseases like glaucoma. Localizing the delivery of glaucoma drugs, such as timolol maleate (TM), can minimize adverse effects caused by systemic administration. Although eye drops and drug-soaked lenses allow for local treatment, their utility is limited by burst release and a lack of sustained therapeutic delivery. Additionally, wet transportation and storage of drug-soaked lenses result in drug loss due to elution from the lenses. Here we present a nanodiamond (ND)-embedded contact lens capable of lysozyme-triggered release of TM for sustained therapy. We find that ND-embedded lenses composed of enzyme-cleavable polymers allow for controlled and sustained release of TM in the presence of lysozyme. Retention of drug activity is verified in primary human trabecular meshwork cells. These results demonstrate the translational potential of an ND-embedded lens capable of drug sequestration and enzyme activation. PMID:24506583

  3. L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains

    Directory of Open Access Journals (Sweden)

    Nagai Kazuo


    Full Text Available Abstract Background A non-pathogenic species of coryneform bacteria, Corynebacterium glutamicum, was originally isolated as an L-glutamate producing bacterium and is now used for fermentative production of various amino acids. A mutation in the C. glutamicum ltsA gene caused susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production. Results The characteristics of eight lysozyme-sensitive mutants which had been isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis were examined. Complementation analysis with the cloned wild-type ltsA gene and DNA sequencing of the ItsA region revealed that four mutants had a mutation in the ltsA gene. Among them, two mutants showed temperature-sensitive growth and overproduced L-glutamate at higher temperatures, as well as the previously reported ltsA mutant. Other two showed temperature-resistant growth: one missense mutant produced L-glutamate to some extent but the other nonsense mutant did not. These two mutants remained temperature-resistant in spite of introduction of ltsA::kan mutation that causes temperature-sensitive growth in the wild-type background. Conclusions These results indicate that a defect caused by the ltsA mutations is responsible for temperature-sensitive growth and L-glutamate overproduction by C. glutamicum. The two temperature-resistant mutants seem to carry suppressor mutations that rendered cells temperature-resistance and abolished L-glutamate overproduction.

  4. An investigation of magnetic field effects on the dissolution of lysozyme crystal and related phenomena. (United States)

    Yin, D C; Inatomi, Y; Wakayama, N I; Huang, W D; Kuribayashi, K


    It is now widely known that a magnetic field, either homogeneous or inhomogeneous, depresses the growth process of protein crystals. In this report, the dissolution process of tetragonal lysozyme crystals is also confirmed to be depressed by a homogeneous magnetic field (inhomogeneity dissolution process was monitored using a Mach-Zehnder interferometer. The results showed that the concentration change during the dissolution process was slowed in a magnetic field compared with that in the absence of a magnetic field. It was concluded that the diffusion coefficient of the lysozyme molecules in the solution was decreased by the magnetic field. The decrease in the diffusion coefficient may contribute to the slowed growth process. The changes in the spatial concentration distribution under a vertical temperature gradient before crystallization in the absence of a magnetic field was also studied. The concentration in the lower, colder part of the cell increased, while it decreased in the upper, hotter part, a similar phenomenon to that discovered by previous investigators in an isothermal supersaturated solution system. Aggregated domain formation is proposed to explain the concentration redistribution before crystal growth and a suspended crystal model is proposed to explain the decrease of diffusivity in a magnetic field.

  5. Effect of cholesterol on the properties of spray-dried lysozyme-loaded liposomal powders. (United States)

    Charnvanich, Dusadee; Vardhanabhuti, Nontima; Kulvanich, Poj


    The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respect to morphology, thermal property, and crystallinity using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction, respectively. Most formulations gave slightly aggregated, spherical particles, and percentage yields of the spray-dried powders decreased with increasing Chol content. Degree of particle aggregation depended on the powder composition. The powders spontaneously formed liposomes which efficiently entrapped LSZ after reconstitution with HEPES buffered saline (HBS) at 37 degrees C. Lysozyme entrapment efficiency and size distribution of the reconstituted liposomes were evaluated after the powders were reconstituted with HBS. Increasing Chol content resulted in a decrease in size of the reconstituted liposomes and an increase in entrapment efficiency of LSZ. These results correlated with thermal behaviors of the reconstituted liposomes. Biological activity of LSZ was not affected by the spray-drying process. It was also demonstrated that LSZ-loaded liposomal powders could be produced without the need to preload the LSZ into liposomes prior to spray-drying process.

  6. Analysis of raw goat milk microbiota: impact of stage of lactation and lysozyme on microbial diversity. (United States)

    McInnis, Elizabeth A; Kalanetra, Karen M; Mills, David A; Maga, Elizabeth A


    To protect infants from infection, human milk contains high levels of the enzyme lysozyme, unlike the milk of dairy animals. We have genetically engineered goats to express human lysozyme (hLZ milk) in their milk at 68% the amount found in human milk to help extend this protection. This study looked at the effect of hLZ on bacteria in raw milk over time. As the microbial diversity of goats' milk has yet to be investigated in depth using next-generation sequencing (NGS) technologies, we applied NGS and clone library sequencing (CLS) to determine the microbiota of raw goat milk (WT milk) and hLZ milk at early, mid and late lactation. Overall, in WT milk, the bacterial populations in milk at early and mid lactation were similar to each other with a shift occurring at late lactation. Both methods found Proteobacteria as the dominant bacteria at early and mid lactation, while Actinobacteria surged at late lactation. These changes were related to decreases in Pseudomonas and increases in Micrococcus. The bacterial populations in hLZ milk were similar to WT milk at early and mid lactation with the only significant differences occurring at late lactation with the elevation of Bacillaceae, Alicyclobacillaceae, Clostridiaceae and Halomonadaceae. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Concentration effects on adsorption of bacteriophage T4 lysozyme stability variants to silica. (United States)

    Lee, Woo-Kul; McGuire, Joseph; Bothwell, Michelle K


    The adsorption kinetics and dodeceyltrimethylammonium bromide-mediated elution of the wild type and two structural stability mutants of bacteriophage T4 lysozyme were recorded in situ, at silica surfaces. Experiments were performed at different solution concentrations, ranging from 0.01 to 1.0 mg/ml. Plateau values of adsorbed mass generally increased with increasing solution concentration, with the adsorbed layer being only partially eluted by buffer. Treatment with surfactant removed more of the adsorbed protein in each case, with the remaining adsorbed mass varying little with concentration. Comparison of the data to an adsorption mechanism allowing for three adsorbed states, distinguished by binding strength, showed that the fraction of adsorbed molecules present in the most tightly bound state (state 3) decreased as adsorption occurred from solutions of increasing concentration. However, the absolute amounts of state 3 molecules present in each case were less dependent on solution concentration. Adsorption of T4 lysozyme into state 3 is suggested to occur early in the adsorption process and continue until some critical surface concentration is reached. Beyond this critical value of adsorbed mass, adsorption is suggested to progress with adoption of more loosely bound states.

  8. SDS-Induced Fibrillation of α-Synuclein

    DEFF Research Database (Denmark)

    Giehm, L.; Oliveira, Cristiano Luis Pinto De; Pedersen, J.S.


    A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of α-synuclein (αSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering......, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per αSN molecule, which is also...... the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of α-helical structure...

  9. Carbosilane dendrimers affect the fibrillation of α-synuclein (United States)

    Milowska, Katarzyna; Gomez-Ramirez, Rafael; de la Mata, Francisco Javier; Gabryelak, Teresa; Bryszewska, Maria


    Participation of α-synuclein (ASN) in the pathogenesis of Parkinson's disease is undeniable. This protein is important for functioning of neurons. Conformational changes in ASN and its aggregation result in neurodegeneration. Therefore, the factors preventing aggregation need to be identified. The search for the potential agents preventing fibrillation of proteins in neurodegenerative diseases has also involved polymers such as dendrimers. The aim of this study was to examine the role of carbosilane dendrimers (CBS) in α-synuclein fibrillation process and to assess the structural changes in α-synuclein under the influence of dendrimers. ASN interactions with carbosilane dendrimers were examined by measuring the zeta potential. The fibrillation and structural changes were examined using CD spectroscopy. The results obtained in this study suggest that carbosilane dendrimers can be potential inhibitors of ASN fibril formation. The fact that dendrimers can prevent ASN fibrillation in suspension is important for further research because it may lead to the design of effective pharmacological strategies.

  10. Atrial fibrillation and vascular disease-a bad combination

    DEFF Research Database (Denmark)

    Bjerring Olesen, Jonas; Gislason, Gunnar Hilmar; Torp-Pedersen, Christian


    This article provides an overview of (i) the risk of stroke associated with vascular disease (acute coronary syndromes and peripheral artery disease) in patients with atrial fibrillation, (ii) the frequent coexistence of vascular disease in patients with atrial fibrillation and, (iii......) the cardiovascular risk associated with the coexisting of the two diseases. The literature on this topic is relatively sparse, and we discuss results from both clinical trials and observational studies. There is a clear indication of an increased stroke risk associated with vascular disease in patients with atrial...... fibrillation. Indeed, patients with atrial fibrillation often had coexisting vascular disease (around 18%), and the combination of the two diseases substantially increases the risk of future cardiovascular events. The increased risk associated with peripheral artery disease in atrial fibrillation is even more...

  11. Cardiac fibrillation risk of TASER X-26 dart mode application. (United States)

    Leitgeb, Norbert; Niedermayr, Florian; Loos, Gerhart; Neubauer, Robert


    In view of reported fatalities there are still controversial discussions on whether electronic stun law enforcement weapons can cause cardiac fibrillation. Experimental data are contradictory. Simplified theoretical estimations led to a negligible low risk of 8.10(-7). With a detailed numerical-anatomical model of an adult man (NORMAN) cardiac exposure to Taser X26 high-tension pulses was quantitatively assessed and the fibrillation risk estimated by accounting for its dependence on excited volume based on 3D cardiac exposure patterns. For distance mode and worst case dart hits it could be demonstrated that cardiac exposure can reach the 30% fibrillation risk level. Risk reduces considerably if direct current flow across the heart is prevented. The overall fibrillation risk of Taser application is further reduced by the limited probability of critical hits. However, in agreement with experimental findings it is demonstrated that cardiac fibrillation risk of Taser X26 dart mode application is small, however, not negligible.

  12. Multimorbidity in Older Adults with Atrial Fibrillation. (United States)

    Chen, Michael A


    Older adults with atrial fibrillation often have multiple comorbid conditions, including common geriatric syndromes. Pharmacologic therapy, whether for rate control or rhythm control, can result in complications related to polypharmacy in patients who are often on multiple medications for other conditions. Because of uncertainty about the relative risks and benefits of rate versus rhythm control (including antiarrhythmic or ablation therapy), anticoagulation, and procedural treatments (eg, ablation, left atrial appendage closure, pacemaker placement) in older patients with multimorbidity, shared decision-making is essential. However, this may be challenging in patients with cognitive dysfunction, high fall risk, or advanced comorbidity. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Integrating new approaches to atrial fibrillation management

    DEFF Research Database (Denmark)

    Kotecha, Dipak; Breithardt, Günter; Camm, A John


    cardiovascular events. New approaches to AF management, including the use of novel technologies and structured, integrated care, have the potential to enhance clinical phenotyping or result in better treatment selection and stratified therapy. Here, we report the outcomes of the 6th Consensus Conference...... of the Atrial Fibrillation Network (AFNET) and the European Heart Rhythm Association (EHRA), held at the European Society of Cardiology Heart House in Sophia Antipolis, France, 17-19 January 2017. Sixty-two global specialists in AF and 13 industry partners met to develop innovative solutions based on new...

  14. Concomitant atrial fibrillation surgery: worth the effort? (United States)

    Nashef, Samer A M; Abu-Omar, Yasir


    Concomitant surgery for atrial fibrillation is a conceptually and clinically difficult area of cardiac surgical decision making. This review introduces the pathophysiological background, provides insight and guidance for cardiac surgeons on some of the conflicting evidence and claims, and explores the fields in which further research may help elucidate a cardiac surgical clinical strategy for tackling this common and potentially lethal form of arrhythmia. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

  15. Current approaches in atrial fibrillation treatment

    Directory of Open Access Journals (Sweden)

    Cenk Sarı


    Full Text Available Atrial fibrillation (AF is the most common sustained arrhythmia encountered in clinical practice. Its incidence increases with age. AF is classified into subtypes according to the duration and/or able to provide sinus rhytym. İnitially, patients should be evaluated for rhythm or rate control for appropriate treatment. Second stage of strategy aimed to investigate the feasibility of anticoagulation therapy. Recently, due to the progress made in treatment with rhythm control and anticoagulation therapy, either American or European guidelines have been renovated. These developments have taken place in the newly published guide. In this article, the current change in the management of AF is discussed.

  16. Potassium channel gene mutations rarely cause atrial fibrillation

    Directory of Open Access Journals (Sweden)

    Nam Edwin G


    Full Text Available Abstract Background Mutations in several potassium channel subunits have been associated with rare forms of atrial fibrillation. In order to explore the role of potassium channels in inherited typical forms of the arrhythmia, we have screened a cohort of patients from a referral clinic for mutations in the channel subunit genes implicated in the arrhythmia. We sought to determine if mutations in KCNJ2 and KCNE1-5 are a common cause of atrial fibrillation. Methods Serial patients with lone atrial fibrillation or atrial fibrillation with hypertension were enrolled between June 1, 2001 and January 6, 2005. Each patient underwent a standardized interview and physical examination. An electrocardiogram, echocardiogram and blood sample for genetic analysis were also obtained. Patients with a family history of AF were screened for mutations in KCNJ2 and KCNE1-5 using automated sequencing. Results 96 patients with familial atrial fibrillation were enrolled. Eighty-three patients had lone atrial fibrillation and 13 had atrial fibrillation and hypertension. Patients had a mean age of 56 years at enrollment and 46 years at onset of atrial fibrillation. Eighty-one percent of patients had paroxysmal atrial fibrillation at enrollment. Unlike patients with an activating mutation in KCNQ1, the patients had a normal QTc interval with a mean of 412 ± 42 ms. Echocardiography revealed a normal mean ejection fraction of 62.0 ± 7.2 % and mean left atrial dimension of 39.9 ± 7.0 mm. A number of common polymorphisms in KCNJ2 and KCNE1-5 were identified, but no mutations were detected. Conclusion Mutations in KCNJ2 and KCNE1-5 rarely cause typical atrial fibrillation in a referral clinic population.

  17. Progression of atrial fibrillation in the REgistry on Cardiac rhythm disORDers assessing the control of Atrial Fibrillation cohort

    DEFF Research Database (Denmark)

    De Vos, Cees B; Breithardt, Günter; Camm, A John


    Paroxysmal atrial fibrillation (AF) may progress to persistent AF. We studied the clinical correlates and the effect of rhythm-control strategy on AF progression.......Paroxysmal atrial fibrillation (AF) may progress to persistent AF. We studied the clinical correlates and the effect of rhythm-control strategy on AF progression....

  18. Validation of the GROMOS force-field parameter set 45A3 against nuclear magnetic resonance data of hen egg lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Soares, T. A. [ETH Hoenggerberg Zuerich, Laboratory of Physical Chemistry (Switzerland); Daura, X. [Universitat Autonoma de Barcelona, InstitucioCatalana de Recerca i Estudis Avancats and Institut de Biotecnologia i Biomedicina (Spain); Oostenbrink, C. [ETH Hoenggerberg Zuerich, Laboratory of Physical Chemistry (Switzerland); Smith, L. J. [University of Oxford, Oxford Centre for Molecular Sciences, Central Chemistry Laboratory (United Kingdom); Gunsteren, W. F. van [ETH Hoenggerberg Zuerich, Laboratory of Physical Chemistry (Switzerland)], E-mail:


    The quality of molecular dynamics (MD) simulations of proteins depends critically on the biomolecular force field that is used. Such force fields are defined by force-field parameter sets, which are generally determined and improved through calibration of properties of small molecules against experimental or theoretical data. By application to large molecules such as proteins, a new force-field parameter set can be validated. We report two 3.5 ns molecular dynamics simulations of hen egg white lysozyme in water applying the widely used GROMOS force-field parameter set 43A1 and a new set 45A3. The two MD ensembles are evaluated against NMR spectroscopic data NOE atom-atom distance bounds, {sup 3}J{sub NH{alpha}} and {sup 3}J{sub {alpha}}{sub {beta}} coupling constants, and {sup 1}5N relaxation data. It is shown that the two sets reproduce structural properties about equally well. The 45A3 ensemble fulfills the atom-atom distance bounds derived from NMR spectroscopy slightly less well than the 43A1 ensemble, with most of the NOE distance violations in both ensembles involving residues located in loops or flexible regions of the protein. Convergence patterns are very similar in both simulations atom-positional root-mean-square differences (RMSD) with respect to the X-ray and NMR model structures and NOE inter-proton distances converge within 1.0-1.5 ns while backbone {sup 3}J{sub HN{alpha}}-coupling constants and {sup 1}H- {sup 1}5N order parameters take slightly longer, 1.0-2.0 ns. As expected, side-chain {sup 3}J{sub {alpha}}{sub {beta}}-coupling constants and {sup 1}H- {sup 1}5N order parameters do not reach full convergence for all residues in the time period simulated. This is particularly noticeable for side chains which display rare structural transitions. When comparing each simulation trajectory with an older and a newer set of experimental NOE data on lysozyme, it is found that the newer, larger, set of experimental data agrees as well with each of the

  19. Wolff-Parkinson-White Syndrome: Electrocardiogram

    Directory of Open Access Journals (Sweden)

    Brianna Miner


    Full Text Available History of present illness: A 26-year-old male with no significant past medical history presented to the emergency department with palpitations. The patient experienced these symptoms five times before in his life, but they had self-resolved with squatting or raising his arms. He denied chest pain, shortness of breath, dizziness, syncope, or pre-syncope symptoms. He denied any recent illnesses, cough, chest pain, drug use, or infections. Significant findings: The initial EKG showed wide complex, irregular tachycardia > 200 bpm (EKG 1. Given the possibility of Wolff-Parkinson-White (WPW, procainamide was given to the patient. The patient’s heart rate responded and decreased to 120-140 bpm with narrowing of the QRS complex. A repeat EKG showed narrow complex tachycardia without P waves approximately 120 bpm (EKG 2. Once the procainamide infusion was complete, the patient had converted to sinus rhythm with a delta wave now apparent, consistent with WPW (EKG 3. Discussion: Wolff-Parkinson-White syndrome is a combination of the presence of a certain congenital accessory pathway (Bundle of Kent and episodes of tachyarrhythmia.1 The incidence is less than one percent of the general population,2 but it is associated with the risk of sudden cardiac death, making the diagnosis imperative. When examining a wide-based tachycardia, it is important to consider WPW when choosing medications for rate control or chemical cardioversion. The first EKG shows an example of atrial fibrillation with WPW. While procainamide blocks the accessory pathway, other drugs such as beta blockers and calcium channel blockers can inhibit the AV node resulting in increased use of the accessory pathway. This can worsen the tachyarrhythmia and potentially lead to ventricular fibrillation, ventricular tachycardia, or asystole.

  20. Increased amount of atrial fibrosis in patients with atrial fibrillation secondary to mitral valve disease

    NARCIS (Netherlands)

    Geuzebroek, Guillaume S. C.; van Amersfoorth, Shirley C. M.; Hoogendijk, Mark G.; Kelder, Johannes C.; van Hemel, Norbert M.; de Bakker, Jacques M. T.; Coronel, Ruben


    Objective: Atrial fibrosis is related to atrial fibrillation but may differ in patients with mitral valve disease or lone atrial fibrillation. Therefore, we studied atrial fibrosis in patients with atrial fibrillation + mitral valve disease or with lone atrial fibrillation and compared it with


    NARCIS (Netherlands)



    Background: Hevamine is a member of one of several families of plant chitinases and lysozymes that are important for plant defence against pathogenic bacteria and fungi. The enzyme can hydrolyze the linear polysaccharide chains of chitin and peptidoglycan. A full understanding of the

  2. Interaction between lysozyme and humic acid in layer-by-layer assemblies : Effects of pH and ionic strength

    NARCIS (Netherlands)

    Tan, Wenfeng; Norde, Willem; Koopal, Luuk K.


    The interaction between protein and soluble organic matter is studied through layer-by-layer assembly of lysozyme (LSZ) and purified Aldrich humic acid (PAHA) at a solid surface (2-D) and in solution (3-D). By bringing a silica surface in alternating contact with solutions of LSZ and PAHA a

  3. Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment. (United States)

    Ooi, Beng Guat; Branning, Sharon Alyssa


    Chlorine dioxide (ClO2) is a potent oxidizing agent used for the treatment of drinking water and decontamination of facilities and equipment. The purpose of this research is to elucidate the manner in which ClO2 destroys proteins by studying the effects of ClO2 on lysozyme. The degree of enzyme activity lost can be correlated to the treatment time and levels of the ClO2 used. Lysozyme activity was drastically reduced to 45.3% of original enzyme activity when exposed to 4.3 mM ClO2 in the sample after 3 h. Almost all activities were lost in 3 h after exposure to higher ClO2 concentrations of up to 16.8 and 21.9 mM. Changes in protein conformation and amount as a result of ClO2 treatment were determined using the Raman spectroscopy and gel electrophoresis. Raman shifts and the alteration of spectral features observed in the ClO2-treated lysozyme samples are associated with loss of the α-helix secondary structure, tertiary structure, and disulfide bond. Progressive degradation of the denatured lysozyme by increasing levels of chlorine dioxide was also observed in gel electrophoresis. Hence, ClO2 can effectively cause protein denaturation and degradation resulting in loss of enzyme activity.

  4. Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor

    NARCIS (Netherlands)

    Terwisscha van Scheltinga, Anke C.; Kalk, Kor H.; Beintema, Jaap J.; Dijkstra, Bauke W.


    Background: Hevamine is a member of one of several families of plant chitinases and lysozymes that are important for plant defence against pathogenic bacteria and fungi. The enzyme can hydrolyze the linear polysaccharide chains of chitin and peptidoglycan. A full understanding of the

  5. The effects of hyaluronic acid incorporated as a wetting agent on lysozyme denaturation in model contact lens materials. (United States)

    Weeks, Andrea; Boone, Adrienne; Luensmann, Doerte; Jones, Lyndon; Sheardown, Heather


    Conventional and silicone hydrogels as models for contact lenses were prepared to determine the effect of the presence of hyaluronic acid on lysozyme sorption and denaturation. Hyaluronic acid was loaded into poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) hydrogels, which served as models for conventional and silicone hydrogel contact lens materials. The hyaluronic acid was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of dendrimers. Active lysozyme was quantified using a Micrococcus lysodeikticus assay while total lysozyme was determined using 125-I radiolabeled protein. To examine the location of hyaluronic acid in the gels, 6-aminofluorescein labeled hyaluronic acid was incorporated into the gels using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry and the gels were examined using confocal laser scanning microscopy. Hyaluronic acid incorporation significantly reduced lysozyme sorption in poly(2-hydroxyethyl methacrylate) (p denaturation in poly(2-hydroxyethyl methacrylate) (p denaturation in contact lens applications. The distribution of hyaluronic acid within hydrogels appears to affect denaturation, with more surface mobile, lower molecular weight hyaluronic acid being more effective in preventing denaturation.

  6. The interaction of equine lysozyme:oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism

    DEFF Research Database (Denmark)

    Nielsen, Søren Bang; Wilhelm, Kristina; Vad, Brian


    The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complex...

  7. Impact of salivary flow and lysozyme content and output on the oral health of rheumatoid arthritis patients

    Directory of Open Access Journals (Sweden)

    Anna Zalewska


    Full Text Available Purpose:The aim of the study was to examine salivary flow rate, DMF index, lysozyme concentration and its output in two groups of rheumatoid patients and to compare the results with those of healthy controls.Material/Methods:Rheumatoid arthritis (RA patients were divided into two study groups: with reduced salivary flow rate ≤0.15 ml/min (RA HS, hyposalivation and with normal salivary secretion rate >0.2 ml/min (RA NS, normal salivation. The healthy control group (C was recruited from the Department of Conservative Dentistry. Salivary lysozyme concentration was determined by radial immunodiffusion. ANOVA followed by LSD test were used for the statistical analysis.Results:We found that lysozyme concentration was higher and lysozyme output and salivary flow rate were statistically lower in the RA HS group in comparison to the RA NS and C groups. The DMF index was statistically higher in both RA groups in comparison to the control group.Conclusions:RA disease impacts negatively on oral health and salivary parameters. Hyposalivation of RA patients increases the negative influence of RA on oral health. RA patients should receive more stomatological attention.

  8. Metal ions-binding T4 lysozyme as an intramolecular protein purification tag compatible with X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Bäumlová, Adriana; Chalupská, Dominika; Dubánková, Anna; Klíma, Martin


    Roč. 26, č. 6 (2017), s. 1116-1123 ISSN 0961-8368 R&D Projects: GA ČR(CZ) GJ17-07058Y; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : phage T4 * lysozyme * endolysin * histidine tag * protein purification * crystal structure Subject RIV: CE - Biochemistry Impact factor: 2.523, year: 2016

  9. Destruction of single-species biofilms of Escherichia coli or Klebsiella pneumoniae subsp. pneumoniae by dextranase, lactoferrin, and lysozyme (United States)

    The activity of dextranase, lactoferrin, lysozyme, and nisin against biofilms composed of either Klebsiella pneumonia or Escherichia coli was examined using the MBEC Assay™. Mature biofilms were treated and then sonicated to remove the adherent biofilm. This material was quantified using a lumines...

  10. Drug-targeting to the kidney : Renal delivery and degradation of a naproxen-lysozyme conjugate in vivo

    NARCIS (Netherlands)

    Haas, M; Kluppel, ACA; Wartna, ES; Moolenaar, F; Meijer, DKF; deJong, PE; deZeeuw, D


    A renal-specific controlled release of an active drug may enable a reduction of the required dose and may provide a reduction of extra-renal toxicity. To achieve renal specific targeting of the NSAID naproxen, the low-molecular-weight protein (LMWP) lysozyme was employed as carrier since it is

  11. Chronic kidney disease and prevalent atrial fibrillation: the Chronic Renal Insufficiency Cohort (CRIC). (United States)

    Soliman, Elsayed Z; Prineas, Ronald J; Go, Alan S; Xie, Dawei; Lash, James P; Rahman, Mahboob; Ojo, Akinlolu; Teal, Val L; Jensvold, Nancy G; Robinson, Nancy L; Dries, Daniel L; Bazzano, Lydia; Mohler, Emile R; Wright, Jackson T; Feldman, Harold I


    The epidemiology of atrial fibrillation (AF) has been mainly investigated in patients with end-stage renal disease, with limited data on less advanced chronic kidney disease (CKD) stages. A total of 3,267 adult participants (50% non-Hispanic blacks, 46% women) with CKD from the Chronic Renal Insufficiency Cohort were included in this study. None of the study participants had been on dialysis. Those with self-identified race/ethnicity other than non-Hispanic black or white (n = 323) or those without electrocardiographic data (n = 22) were excluded. Atrial fibrillation was ascertained by a 12-lead electrocardiogram and self-report. Age-, sex-, and race/ethnicity-specific prevalence rates of AF were estimated and compared between subgroups. Cross-sectional associations and correlates with prevalent AF were examined using unadjusted and multivariable-adjusted logistic regression analysis. The mean estimated glomerular filtration rate was 43.6 (+/-13.0) mL/(min 1.73 m(2)). Atrial fibrillation was present in 18% of the study population and in >25% of those > or =70 years old. In multivariable-adjusted models, 1-SD increase in age (11 years) (odds ratio 1.27, CI 95% 1.13-1.43, P Insufficiency Cohort, a national study of CKD, had evidence of AF at study entry, a prevalence similar to that reported among patients with end-stage renal disease and 2 to 3 times of that reported in the general population. Risk factors for AF in this CKD population do not mirror those reported in the general population. Copyright 2010 Mosby, Inc. All rights reserved.

  12. C-reactive protein and risk of atrial fibrillation in East Asians. (United States)

    Kwon, Chang Hee; Kang, Jung Gyu; Lee, Hyun Jong; Kim, Nan Hee; Sung, Joo-Wook; Cheong, EunSun; Sung, Ki-Chul


    Inflammation has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). It is uncertain whether C-reactive protein, a robust inflammatory marker, is associated with AF incidence in Asians with lower levels of C-reactive protein compared with western population. This study aimed to determine the association between C-reactive protein and risk of AF in a large population of Koreans. A total of 402 946 Koreans were enrolled in a health screening programme from January 2002 to December 2013. Among them, 210 208 subjects were analysed during the mean follow-up of 4.59 years (1 062 513 person-years). Atrial fibrillation was identified by electrocardiography at every visits. Atrial fibrillation was identified in 561 subjects (0.1%) at baseline. The median (inter-quartile) baseline C-reactive protein levels were higher in subject with AF than in those without AF [0.9 mg/L (0.4-0.9) vs. 0.4 mg/L (0.2-1.0), P C-reactive protein had more AF than those in the lowest quartile [adjusted odds ratio (OR) 2.02, 95% confidence interval (CI) 1.45-2.81; PC-reactive protein had a 1.68-fold (95% CI 1.06-2.67) increased risk of AF than the lowest quartile in multivariate Cox regression analysis. Baseline C-reactive protein levels are significantly associated with the prevalence of AF and the risk of AF in Korean populations even C-reactive protein concentrations are substantially lower than reported in white populations.

  13. Amyloid fibrils: formation, replication, and physics behind them (United States)

    Saric, Andela

    The assembly of normally soluble proteins into long fibrils, known as amyloids, is associated with a range of pathologies, including Alzheimer's and Parkinson's diseases. A large number of structurally unrelated proteins form this type of fibrils, and we are in a pursuit of physical principles that underlie the amyloid formation and propagation. We show that small disorders oligomers, which are increasingly believed to be the prime cause for cellular toxicity, serve as nucleation centers for the fibril formation. We then relate experimentally measurable kinetic descriptors of amyloid aggregation to the microscopic mechanisms of the process. Once formed, amyloid fibrils can catalyse the formation of new oligomers and fibrils in a process that resembles self-replication. By combining simulations with biosensing and kinetic measurements of the aggregation of Alzheimer's A β peptide, we propose a mechanistic explanation for the self-replication of protein fibrils, and discuss its thermodynamic signature. Finally, we consider the design of possible inhibitors of the fibril self-replication process. Mechanistic understandings provided here not only have implications for future efforts to control pathological protein aggregation, but are also of interest for the rational assembly of bionanomaterials, where achieving and controlling self-replication is one of the unfulfilled goals.

  14. [Identification of patients with atrial fibrillation using HRV parameters]. (United States)

    Kikillus, Nicole; Hammer, Gerd; Bolz, Armin


    Atrial fibrillation is the most common sustained cardiac rhythm disturbance. One of the most drastic complications is embolism, particularly stroke. Patients with atrial fibrillation have to be identified. This can lead to early therapy and thus avoiding strokes. The algorithm presented here detects atrial fibrillation securely and reliably. It is based on a single-channel ECG, which takes 60 min. First, the R-peaks are detected from the ECG and the RR interval is calculated. To be independent from pulse variations, the RR interval is normalized to 60 bpm. A parameter of heart rate variability is calculated in time domain (SDSD) and the so-called Poincaré plot is generated. The image analysis of the figures of the Poincaré plot is made automatically. The results from analysis in time domain, as well as image analysis, yield a risk level, which indicates the probability for the occurrence of atrial fibrillation. Even if there is no atrial fibrillation in the ECG while analyzing, it is possible to identify patients with atrial fibrillation. The sensitivity depends on the burden of atrial fibrillation. Even if a burden of 0% is assumed, the results still prove satisfactory (sensitivity of nearly 83%).

  15. Cold denaturation of α-synuclein amyloid fibrils. (United States)

    Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Saiki, Miyu; Yagi, Hisashi; Kawata, Yasushi; Goto, Yuji


    Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer's Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Replica Exchange Simulations of the Thermodynamics of Aβ Fibril Growth (United States)

    Takeda, Takako; Klimov, Dmitri K.


    Abstract Replica exchange molecular dynamics and an all-atom implicit solvent model are used to probe the thermodynamics of deposition of Alzheimer's Aβ monomers on preformed amyloid fibrils. Consistent with the experiments, two deposition stages have been identified. The docking stage occurs over a wide temperature range, starting with the formation of the first peptide-fibril interactions at 500 K. Docking is completed when a peptide fully adsorbs on the fibril edge at the temperature of 380 K. The docking transition appears to be continuous, and occurs without free energy barriers or intermediates. During docking, incoming Aβ monomer adopts a disordered structure on the fibril edge. The locking stage occurs at the temperature of ≈360 K and is characterized by the rugged free energy landscape. Locking takes place when incoming Aβ peptide forms a parallel β-sheet structure on the fibril edge. Because the β-sheets formed by locked Aβ peptides are typically off-registry, the structure of the locked phase differs from the structure of the fibril interior. The study also reports that binding affinities of two distinct fibril edges with respect to incoming Aβ peptides are different. The peptides bound to the concave edge have significantly lower free energy compared to those bound on the convex edge. Comparison with the available experimental data is discussed. PMID:19167295

  17. Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum. (United States)

    Yang, Dinglong; Wang, Qing; Cao, Ruiwen; Chen, Lizhu; Liu, Yongliang; Cong, Ming; Wu, Huifeng; Li, Fei; Ji, Chenglong; Zhao, Jianmin


    Lysozymes play an important role in the innate immune responses with which mollusks respond to bacterial invasion through its lytic activity. In the present study, two c-type lysozymes (designed as VpCLYZ-1 and VpCLYZ-2, respectively) were identified and characterized from the manila clam Venerupis philippinarum. The full-length cDNA of VpCLYZ-1 and VpCLYZ-2 was of 629 and 736 bp, encoding a polypeptide of 156 and153 amino acid residues, respectively. The deduced amino acid sequences of VpCLYZs showed high similarity to other known invertebrate c-type lysozymes. Multiple alignments and phylogenetic relationship strongly suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family. Both VpCLYZ-1 and VpCLYZ-2 transcripts were constitutively expressed in a wide range of tissues with different levels. The VpCLYZ-1 transcript was dominantly expressed in hepatopancreas and hemocytes, while VpCLYZ-2 transcript was mainly expressed in the tissues of hepatopancreas and gills. Both the mRNA expression of VpCLYZ-1 and VpCLYZ-2 was significantly up-regulated at 12 h post Vibrio anguillarum challenge. The recombinant VpCLYZ-1 and VpCLYZ-2 (designed as rVpCLYZ-1 and rVpCLYZ-2) exhibited lytic activity against all tested bacteria, and rVpCLYZ-1 showed higher activities than rVpCLYZ-2 in killing Micrococcus luteus and V. anguillarum. Overall, our results suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of pathogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Vanishing White Matter Disease (United States)

    ... In Memory Of Obituaries Contact Us Donate Vanishing White Matter Disease What is Vanishing White Matter Disease? ... of the genetic basis of VWM was a great step forward. First of all, it allows genetic ...

  19. Growth of (101) faces of tetragonal lysozyme crystals: measured growth-rate trends (United States)

    Forsythe, E. L.; Nadarajah, A.; Pusey, M. L.


    Previous extensive measurements of the growth rates of the (110) face of tetragonal lysozyme crystals have shown unexpected dependencies on the supersaturation. In this study, similar growth-rate measurements were performed for the (101) faces of the crystals. The data show a similar dependence on the supersaturation, becoming appreciable only at high supersaturations, reaching a maximum value and then decreasing. The (101) growth rates are larger at low supersaturations than the (110) growth rates under the same conditions and are smaller at high supersaturations. These trends suggest that the growth mechanism of the (101) face is similar to that of the (110) face: both processes involve the addition of multimeric growth units formed in solution, but the average size of the units for the (101) face is likely to be smaller than for the (110) face.

  20. Combined effect of hydrodynamic and interfacial flow parameters on lysozyme deactivation in a stirred tank bioreactor. (United States)

    Ghadge, Rajaram S; Patwardhan, Ashwin W; Joshi, Jyeshtharaj B


    The dynamic environment within a bioreactor and in the purification equipment is known to affect the activity and yield of enzyme production. The present research focuses on the effect of hydrodynamic flow parameters (average energy dissipation rate, maximum energy dissipation rate, average shear rate, and average normal stress) and the interfacial flow parameters (specific interfacial area and mass transfer coefficient) on the activity of lysozyme. Flow parameters were estimated using CFD simulation based on the k-epsilon approach. Enzyme deactivation was investigated in 0.1, 0.3, 0.57, and 1 m i.d. vessels. Enzyme solution was subjected to hydrodynamic stress using various types of impellers and impeller combinations over a wide range of power consumption (0.03 mass transfer coefficient.