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Sample records for activity serum lysozyme

  1. A novel anti-inflammatory activity of lysozyme: modulation of serum complement activation.

    PubMed Central

    Ogundele, M O

    1998-01-01

    Lysozyme is an ubiquitous enzyme found in most biological secretions and leukocytes. This study was aimed at investigating its interaction with other inflammatory mediators on mucosa surfaces, particularly the complement system. Lysozyme has been shown in our present study, to inhibit the haemolytic activity of serum complement in a dose-dependent fashion, when tested within the levels present in normal and inflamed breast-milk samples, and other mucosal secretions. This represents a new anti-inflammatory action of lysozyme in relation to the serum complement, and the exact mode of the interaction need further studies. PMID:9883972

  2. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes: Enzyme assay for immunotoxicity of xenobiotics

    SciTech Connect

    Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C. . Dept. of Biological Sciences); Venables, B.J. . Dept. of Biological Sciences TRAC Laboratories Inc., Denton, TX )

    1994-04-01

    Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes appears sufficiently sensitive for use as a nonmammalian biomarker to detect toxic effects of sublethal body burdens of Cu[sup 2+]. Lysozyme, a phylogenetically conserved enzyme, is capable of bactericidal activity via action on peptidoglycan of gram-positive bacterial cell walls and functions as a component of an organism's innate antimicrobial defense mechanism. Coelomic fluid and coelomocyte lysozyme activities, which exhibit temperature-response patterns similar to those of human saliva, plasma, serum and leukocyte extracts, were sensitive to Cu[sup 2+] exposure. Lysozyme activity of coelomic fluid and coelomocyte extracts from earthworms exposed for 5 d to CuSO[sub 4], using filter paper contact exposure, decreased with increasing sublethal Cu[sup 2+] concentrations of 0.05 and 0.1 [mu]g/cm[sup 2]. Compared to controls, coelomic fluid lysozyme activity was suppressed significantly at both exposure concentrations, whereas coelomocyte extract lysozyme activity was suppressed significantly at the 0.1-[mu]g/cm[sup 2] exposure concentration. Low inherent natural variability and sensitivity to sublethal Cu[sup 2+] body burdens indicate that lysozyme activity has potential as a biomarker for assaying immunotoxicity of metals.

  3. Lysozyme activity of the Ruminococcus champanellensis cellulosome.

    PubMed

    Moraïs, Sarah; Cockburn, Darrell W; Ben-David, Yonit; Koropatkin, Nicole M; Martens, Eric C; Duncan, Sylvia H; Flint, Harry J; Mizrahi, Itzhak; Bayer, Edward A

    2016-12-01

    Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

  4. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  5. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  6. Immobilization of lysozyme on cotton fabrics; synthesis, characterication, and activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antimicrobial activity of lysozyme derives from the hydrolysis of the bacterial cell wall polysaccharide at the glycosidic bond that links N-acetyl-glucosamine and N-acetyl-muramic acid. Maintaining the activity of lysozyme while bound to a cellulose substrate is a goal toward developing enzyme...

  7. Immobilization of lysozyme on polyvinylalcohol films for active packaging applications.

    PubMed

    Conte, A; Buonocore, G G; Bevilacqua, A; Sinigaglia, M; Del Nobile, M A

    2006-04-01

    A new technique for the immobilization of lysozyme onto the surface of polyvinylalcohol films is presented. The active compound was sprayed along with a suitable bonding agent onto the surface of the cross-linked polymeric matrix. Active compound release tests determined the amount of lysozyme immobilized on the film surface. With the use of Micrococcus lysodeikticus, the antimicrobial activity of the films was determined and the results correlated with the amount of immobilized lysozyme. This new technique was effective for immobilizing the enzyme, and the developed films were active against the test microorganism. Results were compared with those obtained with a different immobilizing technique, in which the active compound was bound into the bulk of the polymeric film. As expected, the surface-immobilized lysozyme films have a higher antimicrobial activity than bulk-bound films.

  8. Interrelationship between serum beta-lysin, lysozyme, and the antibody-complement system in killing Escherichia coli.

    PubMed

    Donaldson, D M; Roberts, R R; Larsen, H S; Tew, J G

    1974-09-01

    The effects of different serum components alone and in conjunction with each other on Escherichia coli B were investigated. In general, the viability, turbidity, and electron microscope results were compatible with the following conclusions. The most efficient killing and destruction of E. coli B occurred when beta-lysin, lysozyme, and the antibody-complement system functioned in cooperation with each other at the serum concentration in isotonic solutions. The addition of sucrose protected the bacteria from the lethal and lytic action of these agents. Elimination of lysozyme from serum had the least effect on bactericidal activity, even though lysozyme treatment caused the cell wall to separate from the cytoplasmic membrane and caused clear areas to appear in the inner granular layer of the cell wall. Beta-lysin removal had an intermediate effect on the serum bactericidal activity. Beta-lysin treatment caused cell walls to collapse, allowed cytoplasmic contents to leak out of the cells, and stopped the separation of cell wall and cytoplasmic membrane, which normally takes place in 0.5 M sucrose solution. Inactivation of the complement eliminated the serum bactericidal activity against E. coli B. After treatment with antibody and complement, the cell walls became thick and indistinct, a portion of the cytoplasmic contents escaped, and patches of the middle layer of the cell wall appeared in freeze-etch preparations. Beta-lysin damaged the cytoplasmic membrane, lysozyme damaged the inner peptidoglycan layer of the cell wall, and the antibody-complement system damaged both the middle lipopolysaccharide layer of the cell wall and the cytoplasmic membrane.

  9. Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces.

    PubMed

    Vasilescu, Alina; Gáspár, Szilveszter; Gheorghiu, Mihaela; David, Sorin; Dinca, Valentina; Peteu, Serban; Wang, Qian; Li, Musen; Boukherroub, Rabah; Szunerits, Sabine

    2017-03-15

    Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples.

  10. Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity.

    PubMed

    Mori, Tsukasa; Hiraka, Ikuei; Kurata, Youichi; Kawachi, Hiroko; Mano, Nobuhiro; Devlin, Robert H; Nagoya, Hiroyuki; Araki, Kazuo

    2007-03-01

    immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F(2) and F(3) by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter alpha-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Delta6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F(2) and immature F(3) salmon. Further, serum lysozyme activity, one of innate immunity, was significantly (p<0.05) decreased in both F(2) and F(3) GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth.

  11. An improved 96-well turbidity assay for T4 lysozyme activity

    PubMed Central

    Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

  12. Size-dependent interaction of silica nanoparticles with lysozyme and bovine serum albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, Vinod K.; Kohlbrecher, Joachim

    2016-05-01

    The interaction of three different sized (diameter 10, 18, and 28 nm) anionic silica nanoparticles with two model proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa)] and anionic bovine serum albumin (BSA) (MW 66.4 kDa) has been studied by UV-vis spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The adsorption behavior of proteins on the nanoparticles, measured by UV-vis spectroscopy, is found to be very different for lysozyme and BSA. Lysozyme adsorbs strongly on the nanoparticles and shows exponential behavior as a function of lysozyme concentration irrespective of the nanoparticle size. The total amount of adsorbed lysozyme, as governed by the surface-to-volume ratio, increases on lowering the size of the nanoparticles for a fixed volume fraction of the nanoparticles. On the other hand, BSA does not show any adsorption for all the different sizes of the nanoparticles. Despite having different interactions, both proteins induce similar phase behavior where the nanoparticle-protein system transforms from one phase (clear) to two phase (turbid) as a function of protein concentration. The phase behavior is modified towards the lower concentrations for both proteins with increasing the nanoparticle size. DLS suggests that the phase behavior arises as a result of the nanoparticles' aggregation on the addition of proteins. The size-dependent modifications in the interaction potential, responsible for the phase behavior, have been determined by SANS data as modeled using the two-Yukawa potential accounting for the repulsive and attractive interactions in the systems. The protein-induced interaction between the nanoparticles is found to be short-range attraction for lysozyme and long-range attraction for BSA. The magnitude of attractive interaction irrespective of protein type is enhanced with increase in the size of the nanoparticles. The total (attractive+repulsive) potential leading to two-phase formation is found to be

  13. Mixed-mode chromatography integrated with high-performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target.

    PubMed

    Xia, Hai-Feng; Don, Bin-Bin; Zheng, Meng-Jie

    2016-05-01

    A type of mixed-mode chromatography was integrated with high-performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed-mode column, the silica beads were activated with γ-(2,3-epoxypropoxy)-propytrimethoxysilane and coupled with 4-mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.

  14. Antibacterial activity of hen egg white lysozyme against Listeria monocytogenes Scott A in foods.

    PubMed Central

    Hughey, V L; Wilger, P A; Johnson, E A

    1989-01-01

    Egg white lysozyme killed or prevented growth of Listeria monocytogenes Scott A in several foods. Lysozyme was more active in vegetables than in animal-derived foods that we tested. For maximum activity in certain foods, EDTA was required in addition to lysozyme. Lysozyme with EDTA effectively killed inoculated populations of 10(4) L. monocytogenes per g in fresh corn, fresh green beans, shredded cabbage, shredded lettuce, and carrots during storage at 5 degrees C. Control incubations without lysozyme supported growth of L. monocytogenes to 10(6) to 10(7)/g. Lysozyme had less activity in animal-derived foods, including fresh pork sausage (bratwurst) and Camembert cheese. In bratwurst, lysozyme with EDTA prevented L. monocytogenes from growing for 2 to 3 weeks but did not kill significant numbers of cells and did not prevent eventual growth. The control sausages not containing lysozyme supported rapid and heavy growth, which indicated that lysozyme was bacteriostatic for 2 to 3 weeks in fresh pork sausage. We also prepared Camembert cheese containing 10(4) L. monocytogenes cells per g and investigated the changes during ripening in cheeses supplemented with lysozyme and EDTA. Cheeses with lysozyme by itself or together with EDTA reduced the L. monocytogenes population by approximately 10-fold over the first 3 to 4 weeks of ripening. In the same period, the control cheese wheels without added lysozyme with and without chelator slowly started to grown and eventually reached 10(6) to 10(7) CFU/g after 55 days of ripening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2494938

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

    PubMed

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

    2011-08-01

    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.

  16. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

    PubMed

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-10-01

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  17. Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in young pigs

    PubMed Central

    Brundige, Dottie R.; Maga, Elizabeth A.; Klasing, Kirk C.

    2009-01-01

    Nutrition, bacterial composition of the gastrointestinal tract, and general health status can all influence the metabolic profile of an organism. We previously demonstrated that feeding pasteurized transgenic goats’ milk expressing human lysozyme (hLZ) can positively impact intestinal morphology and modulate intestinal microbiota composition in young pigs. The objective of this study was to further examine the effect of consuming hLZ-containing milk on young pigs by profiling serum metabolites. Pigs were placed into two groups and fed a diet of solid food and either control (non-transgenic) goats’ milk or milk from hLZ-transgenic goats for 6 weeks. Serum samples were collected at the end of the feeding period and global metabolite profiling was performed. For a total of 225 metabolites (160 known, 65 unknown) semi-quantitative data was obtained. Levels of 18 known and 4 unknown metabolites differed significantly between the two groups with the direction of change in 13 of the 18 known metabolites being almost entirely congruent with improved health status, particularly in terms of the gastrointestinal tract health and immune response, with the effects of the other five being neutral or unknown. These results further support our hypothesis that consumption of hLZ-containing milk is beneficial to health. PMID:19847666

  18. Effects of metal oxide nanoparticles on the structure and activity of lysozyme.

    PubMed

    Cheng, Yu-Hong; Lai, Chia-Min; Lin, Kuen-Song; Wang, Steven S-S

    2017-03-01

    We investigated the effects of nanoparticles (NPs) on the structure and activity of hen egg-white lysozyme (HEWL) using CeO2 and ZnO NPs. Our results showed that CeO2 NPs triggered the transition of lysozyme secondary structure from α-helix to β-sheet. CeO2 NPs also induced the hydrophobic region of lysozyme to become exposed to the solvent. In contrast, the secondary structure content and hydrophobic region of lysozyme were only slightly changed in the case of ZnO NPs. In addition, the activity of the lysozyme was observed to decrease upon adsorption on CeO2 NPs, whereas the effect of ZnO NPs on activity was negligible. The glutaraldehyde crosslinking results indicated that the percentage of the dimeric form of lysozyme was greatly enhanced by the addition of both NPs. Furthermore, the adsorption capacity, degree of favorability of adsorption, and surface heterogeneity for CeO2 NPs were found to be greater than those on ZnO NPs. Given that CeO2 NPs exhibit a higher surface area/mass than ZnO NPs, the surface concentration of lysozyme on CeO2 NPs was lower than that on ZnO NPs. This result suggested that more direct interactions were involved between CeO2 NPs and lysozyme, thereby leading to a more significant effect. Moreover, higher surface curvatures may also cause destruction of lysozyme's structure and thus affect its activity. In addition, taking into account the surface properties and protein properties, the Toth adsorption model along with the generated site energy distribution was further used to exaplain the difference between the results (e.g., structure, stability, and activity) of lysozyme adsorption on CeO2 and ZnO NPs. The results reported here may aid in better understanding the beneficial or harmful impacts of nanoparticles on the biological systems.

  19. Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.

    PubMed

    Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui

    2014-10-01

    The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates.

  20. The amino acid sequence of monal pheasant lysozyme and its activity.

    PubMed

    Araki, T; Matsumoto, T; Torikata, T

    1998-10-01

    The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).

  1. Performance of the lysozyme for promoting the waste activated sludge biodegradability.

    PubMed

    He, Jun-Guo; Xin, Xiao-Dong; Qiu, Wei; Zhang, Jie; Wen, Zhi-Dan; Tang, Jian

    2014-10-01

    The fresh waste activated sludge (WAS) from a lab-scale sequencing batch reactor was used to determine the performance of the lysozyme for promoting its biodegradability. The results showed that a strict linear relationship presented between the degree of disintegration (DDM) of WAS and the lysozyme incubation time from 0 to 240min (R(2) was 0.992, 0.995 and 0.999 in accordance with the corresponding lysozyme/TS, respectively). Ratio of net SCOD increase augmented significantly by lysozyme digestion for evaluating the sludge biodegradability changes. Moreover, the protein dominated both in the EPS and SMP. In addition, the logarithm of SMP contents in supernatant presented an increasing trend similar with the ascending logarithmic relation with the lysozyme incubation time from 0 to 240min (R(2) was 0.960, 0.959 and 0.947, respectively). The SMP, especially the soluble protein, had an important contribution to the improvement of WAS biodegradability.

  2. Influence of graphene oxide and reduced graphene oxide on the activity and conformation of lysozyme.

    PubMed

    Bai, Yitong; Ming, Zhu; Cao, Yuye; Feng, Shicheng; Yang, Hua; Chen, Lingyun; Yang, Sheng-Tao

    2017-03-08

    The dramatically different bio-effects of graphene and graphene oxide (GO) have been widely observed in diverse biological systems, which determine the applications and toxicity of graphene materials. To elucidate the mechanism at molecular level, it is urgent to investigate the enzyme-graphene interaction and its consequences. In this study, we comparatively studied the influence of GO and reduced GO (RGO) on the activity and conformation of lysozyme to provide better understandings of their different bio-effects. Both GO and RGO adsorbed large quantities of lysozyme after incubation. GO inhibited lysozyme activity seriously, while RGO nearly had no influence on the enzyme activity. The different inhibitions of enzyme activity could be explained by the lysozyme conformational changes, where GO induced more changes to the protein conformation according to UV-vis absorbance, far-UV circular dichroism spectra, intrinsic fluorescence quenching, and infrared spectra. Based on the spectroscopic changes of lysozyme, GO induced the loss of secondary structure and exposed the active site of lysozyme more to the aqueous environment. In addition, neither GO nor RGO induced the fibrillation of lysozyme after 12d incubation. The results collectively indicated that the oxidation degree significantly impacted the enzyme-graphene interaction. The implications to the designs of enzyme-graphene system for bio-related applications and the toxicological effects of graphene materials are discussed.

  3. Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus.

    PubMed

    Stabili, L; Pagliara, P

    2009-03-01

    Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In this study we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialis mucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cell walls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lysozyme activity to monitoring environmental pollution are highlighted.

  4. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kopcansky, Peter; Siposova, Katarina; Melnikova, Lucia; Bednarikova, Zuzana; Timko, Milan; Mitroova, Zuzana; Antosova, Andrea; Garamus, Vasil M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Gazova, Zuzana

    2015-03-01

    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.

  5. Investigation on the interactions of clenbuterol to bovine serum albumin and lysozyme by molecular fluorescence technique.

    PubMed

    Bi, Shuyun; Pang, Bo; Wang, Tianjiao; Zhao, Tingting; Yu, Wang

    2014-01-01

    Clenbuterol interacting with bovine serum albumin (BSA) or lysozyme (LYS) in physiological buffer (pH 7.4) was investigated by the fluorescence spectroscopy and UV-vis absorption spectroscopy. The results indicated that clenbuterol quenched the intrinsic fluorescence of BSA and LYS via a static quenching procedure. The binding constants of clenbuterol with BSA and LYS were 1.16×10(3) and 1.49×10(3) L mol(-1) at 291 K. The values of ΔH and ΔS implied that hydrophobic and electrostatic interaction played a major role in stabilizing the complex (clenbuterol-BSA or clenbuterol-LYS). In the presence of Fe2+, Fe3+, Cu2+, Mg2+, Ca2+, or Zn2+, the binding constants of clenbuterol to BSA or LYS had no significant differences. The distances between the donor (BSA or LYS) and acceptor (clenbuterol) were 2.61 and 2.19 nm for clenbuterol-BSA and clenbuterol-LYS respectively. Furthermore, synchronous fluorescence spectrometry was used to analyze the conformational changes of BSA and LYS.

  6. Human Interleukin-2 and Hen Egg White Lysozyme: Screening for Bacteriolytic Activity against Various Bacterial Cells

    PubMed Central

    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.

    2016-01-01

    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

  7. Lysozyme activity and L(+)-lactic acid production in saliva in schoolchildren with high Lactobacillus counts.

    PubMed

    Twetman, S; Dahllöf, G; Wikner, S

    1987-04-01

    Out of 374 schoolchildren, aged 13-15 yr, 42 with high counts of salivary lactobacilli (greater than or equal to 10(5] were selected for this study. Lysozyme activity in saliva and L(+)-lactic acid (LA) production after addition of glucose were determined. The mean values of lysozyme activity and LA concentration were 19.4 micrograms/ml and 1.4 mmol/l respectively. The levels of LA produced without addition of glucose were less than 0.2 mmol/l. The results showed a statistically significant (P less than 0.05) negative correlation between lysozyme activity and the levels of LA produced. The findings of this study suggest that lysozyme may be of importance in limiting acid production in saliva.

  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

    PubMed Central

    Houtman, Jon C.

    2016-01-01

    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. PMID:27602573

  9. Expression and purification of active recombinant equine lysozyme in Escherichia coli.

    PubMed

    Casaite, Vida; Bruzyte, Simona; Bukauskas, Virginijus; Setkus, Arunas; Morozova-Roche, Ludmilla A; Meskys, Rolandas

    2009-11-01

    Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.

  10. Serum soluble interleukin-2 receptor level is more sensitive than angiotensin-converting enzyme or lysozyme for diagnosis of sarcoidosis and may be a marker of multiple organ involvement.

    PubMed

    Thi Hong Nguyen, Chuyen; Kambe, Naotomo; Kishimoto, Izumi; Ueda-Hayakawa, Ikuko; Okamoto, Hiroyuki

    2017-03-11

    Skin lesions in sarcoidosis are often the initial symptoms that enable the dermatologist to be the first to diagnose this granulomatosis. However, diagnosis is sometimes very problematic. In 2015, the diagnostic criteria for sarcoidosis were updated in Japan, with elevated serum soluble interleukin-2 receptor (sIL-2R) replacing negative tuberculin reaction. Therefore, we assessed the clinical utility of sIL-2R compared with two other common markers, angiotensin-converting enzyme (ACE) and lysozyme, in patients who visited the dermatology clinic. Data from 72 patients showed that sIL-2R was more sensitive than both ACE and lysozyme in supporting a diagnosis of sarcoidosis (52.8%) compared with ACE (29%) and lysozyme (26.4%). Additionally, the sIL-2R level was significantly higher in patients with multiple organ involvement and parenchymal infiltration. Patients with elevated sIL-2R levels had higher serum ACE and lysozyme levels, a higher incidence of pulmonary involvement, more severe chest radiographic stage and a high incidence of expression-specific signs by imaging analysis. Receiver-operator curve analysis showed that sIL-2R was a better marker at the threshold cut-off point compared with ACE and lysozyme for identifying patients with multiple organ involvement, detecting patients with pulmonary disease and parenchymal infiltration as well as predicting the presence of specific signs in the diagnosis of sarcoidosis. Moreover, the kinetics of sIL-2R levels correlated closely with clinical manifestations, in contrast to the modest changes of ACE and lysozyme levels during the follow-up period. In conclusion, sIL-2R may be considered a good marker for diagnosis and a potential indicator of disease activity.

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

    PubMed

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

    2011-02-01

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

  12. Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization

    PubMed Central

    1991-01-01

    This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations. PMID:1940787

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

    PubMed Central

    2016-01-01

    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. PMID:27428539

  14. Lysozyme Activity in the Plasma of Rodents Infected With Their Homologous Trypanosomes

    PubMed Central

    Maraghi, S; Molyneux, DH; Wallbanks, KR

    2012-01-01

    Background In this study the concentration of lysozyme in blood plasma of Microtus agrestis, Clethrinomys glareolus, Apodemus sylvaticus, BK rats and outbred white mice before and after infection with culture forms of Trypanosoma microti, T, evotomys, T. grosi, T. lewisi and T. musculi respectively was measured. Methods Blood samples of rodents, Microtus agrestis, Clethrionomys glareolus, Apodemus sylvaticus, BK rats and outbred mice infected with T. microti, T. evotomys, T. grosi, T. lewisi and T. musculi respectively were collected in heparinized micro- tubes immediately before inoculation and 3, 6, 12, 24, 48, 96 and more than 400 days after intra- perituneal inoculation with 5×105of their homologous trypanosome parasites of which more than half were metacyclic trypomastigote in 0.2 ml of culture medium. Micro- tubes were centrifuged and plasma samples were separated and the lysozyme activity was measured by the agar method. Results Levels of lysozyme rose rapidly three to six days after the inoculation to ten to twenty than their pre- infection levels. They then gradually decreased, although after more than one year they were still two to ten folds higher than controls. The highest level measured occurred in rats infected with T. lewisi and the lowest in A. sylvaticus infected with T. grosi. After one year the highest concentration of lysozyme was in mice infected with T. musculi and lowest in A. sylvaticus. Conclusion Persistent enhanced lysozyme levels may prevent re- infection with trypanosomes. PMID:23323096

  15. Oxalic acid-induced modifications of postglycation activity of lysozyme and its glycoforms.

    PubMed

    Gao, Hong Ying; Yaylayan, Varoujan A; Yeboah, Faustinus

    2010-05-26

    The role of selected carboxylic acids and their potential to influence the glycation pattern and the enzymatic activity of lysozyme using glucose and ribose were investigated independently of the pH of the reaction medium. The model systems were incubated with and without selected carboxylic acids (maleic, acetic, oxalic, and citraconic) at 50 degrees C for 12 or 24 and 48 h at constant pH of 6.5. The effect of carboxylic acids on the glycation of lysozyme was studied by electrospray ionization mass spectrometry (ESI-MS) and by the measurement of the residual enzyme activity of lysozyme in the glycated samples. Of the carboxylic acids evaluated, oxalic acid showed the highest antiglycation activity. The residual lysozyme activity in both oxalic acid-glucose and oxalic acid-ribose systems was >80% compared with 46 and 36% activity in the controls of glucose and ribose systems, respectively. On the other hand, maleic, acetic, and citraconic acid containing systems with both sugars did not exhibit any enhanced enzyme activity relative to the controls. The results of this study show that oxalic acid was unique among the carboxylic acids evaluated with respect to its ability to interact with sugars and inhibit glycation.

  16. The interaction of flavonoid-lysozyme and the relationship between molecular structure of flavonoids and their binding activity to lysozyme.

    PubMed

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

    2012-11-01

    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.

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

    PubMed Central

    Matouskova, Petra; Bokrova, Jitka; Benesova, Pavla

    2016-01-01

    Summary 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. PMID:27956862

  18. Lysozyme-mediated biomineralization of titanium-tungsten oxide hybrid nanoparticles with high photocatalytic activity.

    PubMed

    Kim, Jung Kyu; Jang, Ji-ryang; Choi, Noori; Hong, Dahyun; Nam, Chang-Hoon; Yoo, Pil J; Park, Jong Hyeok; Choe, Woo-Seok

    2014-10-21

    Titanium-tungsten oxide composites with greatly enhanced photocatalytic activity were synthesized by lysozyme-mediated biomineralization. It was shown for the first time that simple control of the onset of biomineralization could enable fine tuning of the composition and crystallinity of the composites to determine their photocatalytic performance.

  19. Investigation of effects of terpene skin penetration enhancers on stability and biological activity of lysozyme.

    PubMed

    Varman, Rahul M; Singh, Somnath

    2012-12-01

    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 < 0.05) less than other 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.

  20. Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin.

    PubMed

    Branen, Jill K; Davidson, P Michael

    2004-01-01

    A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.

  1. Extrusion of pea starch containing lysozyme and determination of antimicrobial activity.

    PubMed

    Nam, S; Scanlon, M G; Han, J H; Izydorczyk, M S

    2007-11-01

    Pea starch, which has inherently good gel strength, was used as the source material for manufacturing a biodegradable and bioactive packaging material. Extrudates containing 99% pea starch and 1% lysozyme were produced under various extrusion conditions (high and low shear screw configurations, 30% to 40% moisture contents, 70 to 150 degrees C die temperatures). The physical and mechanical properties of the extrudates were determined through various expansion indices, piece and cell wall solid density, compression, and 3-point bending tests. The expansion of extrudates increased with an increase in die temperature, whereas increasing moisture content had the opposite effect. Extrudate densities decreased as extrusion temperature increased, whereas lower moisture content in the extrudate dough decreased extrudate densities. The elastic modulus and fracture strengths were highly correlated in a power-law fashion to relative density, showing that the mechanical properties of extrudates were dependent on solid density and foam structure. Up to 48% of the initial lysozyme activity was recovered from the extruded pea starch matrix. The lysozyme released from extrudates showed an inhibition zone against Brochotrix thermosphacta B2. Extruded pea starch matrix containing lysozyme has potential application as an edible and biodegradable packaging material with antimicrobial activity.

  2. Chicken-type lysozyme in channel catfish: expression analysis, lysozyme activity, and efficacy as immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...

  3. Chicken-type lysozyme in channel catfish: Expression analysis, lysozyme activity and efficacy as immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...

  4. Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity.

    PubMed

    Anand, N N; Stephen, E R; Narang, S A

    1988-06-16

    The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.

  5. Lysozyme and defense peptides as suppressors of phenoloxidase activity in Galleria mellonella.

    PubMed

    Zdybicka-Barabas, Agnieszka; Mak, Paweł; Jakubowicz, Teresa; Cytryńska, Małgorzata

    2014-09-01

    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.

  6. Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase.

    PubMed

    Vannini, L; Lanciotti, R; Baldi, D; Guerzoni, M E

    2004-07-15

    It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

  7. Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process

    SciTech Connect

    Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu; Rossmann, Michael G.; Arisaka, Fumio

    2010-07-19

    Bacteriophage T4 has an efficient mechanism for injecting the host Escherichia coli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E. coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E. coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.

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

    PubMed Central

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

    2006-01-01

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

  9. Blood monocytes and tumor-infiltrating macrophages in human breast cancer: differences in activation level as assessed by lysozyme content.

    PubMed

    Steele, R J; Eremin, O; Brown, M

    1983-11-01

    The lysozyme content of tumor-infiltrating macrophages (TIM) from human breast carcinomas has been compared with that of blood monocytes both from breast cancer patients and tumor-free controls. Cells were identified as macrophages or monocytes with the use of rosetting reactions to detect receptors for the Fc portion of IgG and differentiation antigens, and lysozyme was detected by an immunoperoxidase technique on cytocentrifuge preparations of rosetted cells. Significantly more monocytes from patients with breast cancer contained lysozyme than monocytes from comparable controls, suggesting the presence of activated circulating blood monocytes. Conversely, TIM were virtually devoid of lysozyme. This lack of enzyme was not due to methodologic factors and may represent defective antitumor activity.

  10. Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.

    PubMed

    Nakimbugwe, Dorothy; Masschalck, Barbara; Atanassova, Miroslava; Zewdie-Bosüner, Abebetch; Michiels, Chris W

    2006-05-01

    The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130-300 MPa, 25 degrees C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed.

  11. Electrolyte effect on the phase behavior of silica nanoparticles with lysozyme and bovine-serum-albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2015-05-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) studies have been carried out to investigate the effect of an electrolyte on the phase behavior of anionic silica nanoparticles with two globular proteins—cationic lysozyme [molecular weight (MW) 14.7 kDa] and anionic bovine serum albumin (MW 66.4 kDa). The results are compared with our earlier published work on similar systems without any electrolyte [I. Yadav, S. Kumar, V. K. Aswal, and J. Kohlbrecher, Phys. Rev. E 89, 032304 (2014), 10.1103/PhysRevE.89.032304]. Both the nanoparticle-protein systems transform to two phase at lower concentration of protein in the presence of an electrolyte. The autocorrelation function in DLS suggests that the diffusion coefficient (D) of a nanoparticle-protein system decreases in approaching two phase with the increase in protein concentration. This variation in D can be attributed to increase in attractive interaction and/or overall increase in the size. Further, these two contributions (interaction and structure) are determined from the SANS data. The changes in the phase behavior of nanoparticle-protein systems in the presence of an electrolyte are explained in terms of modifications in both the repulsive and attractive components of interaction between nanoparticles. In a two-phase system individual silica nanoparticles coexist along with their fractal aggregates.

  12. Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt-water systems.

    PubMed

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

    2016-09-21

    Improving protein stabilisation is important for the further development of many applications in the pharmaceutical, specialty chemical, consumer product and agricultural sectors. However, protein stabilization is highly dependent on the solvent environment and, hence, it is very complex to tailor protein-solvent combinations for stable protein maintenance. Understanding solvent features that govern protein stabilization will enable selection or design of suitable media with favourable solution environments to retain protein native conformation. In this work the structural conformation and activity of lysozyme in 29 solvent systems were investigated to determine the role of various solvent features on the stability of the enzyme. The solvent systems consisted of 19 low molecular weight polar solvents and 4 protic ionic liquids (PILs), both at different water content levels, and 6 aqueous salt solutions. Small angle X-ray scattering, Fourier transform infrared spectroscopy and UV-vis spectroscopy were used to investigate the tertiary and secondary structure of lysozyme along with the corresponding activity in various solvation systems. At low non-aqueous solvent concentrations (high water content), the presence of solvents and salts generally maintained lysozyme in its native structure and enhanced its activity. Due to the presence of a net surface charge on lysozyme, electrostatic interactions in PIL-water systems and salt solutions enhanced lysozyme activity more than the specific hydrogen-bond interactions present in non-ionic molecular solvents. At higher solvent concentrations (lower water content), solvents with a propensity to exhibit the solvophobic effect, analogous to the hydrophobic effect in water, retained lysozyme native conformation and activity. This solvophobic effect was observed particularly for solvents which contained hydroxyl moieties. Preferential solvophobic effects along with bulky chemical structures were postulated to result in less

  13. A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

    PubMed

    Acharya, A S; Taniuchi, H

    1976-11-25

    The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure

  14. Lysozyme- and chitinase activity in latex bearing plants of genus Euphorbia--A contribution to plant defense mechanism.

    PubMed

    Sytwala, Sonja; Günther, Florian; Melzig, Matthias F

    2015-10-01

    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.

  15. Antiangiogenic activity of low-temperature lysozyme from a marine bacterium in vivo and in vitro

    NASA Astrophysics Data System (ADS)

    Wang, Zhenhua; Liu, Jincheng; Su, Ai; Sun, Mi; Wang, Chunbo

    2009-11-01

    We extracted marine low-temperature lysozyme (MLTL), a novel lysozyme, from a marine microorganism through fermentation. Our previous study suggested that a low molecular weight (16 kDa) may exert anti-tumor activity through antiangiogenesis. In this study, we extracted a high weight (39 kDa) and investigated its antiangiogenic activity in vivo and in vitro. Using zebrafish embryos as an in vivo study model, we found that treatment with MLTL significantly inhibited the growth of subintestinal vessels (SIVs) in a dose-dependent manner and that 400 µg/ml MLTL was sufficient to block the growth of SIVs. An in vitro study conducted using human umbilical vein endothelial cells (HUVECs) revealed that MLTL suppressed the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. Interestingly, assays by flow cytometry and DNA electrophoresis indicated that MLTL was able to induce apoptosis of HUVECs. Moreover, further study demonstrated that the disruption of intracellular Ca2+ homeostasis may play an important role in MLTL induced apoptosis of HUVECs. Taken together, the results of this study demonstrate for the first time that MLTL inhibits angiogenesis through its pleiotropic effects on vascular endothelial cells and induces apoptosis through regulation of cellular Ca2+ levels. The results of this study also revealed a possible mechanism underlying the antiangiogenic effect of MLTL and suggested that MLTL may be a promising new antiangiogenic agent for use in cancer therapy.

  16. [Features of lysozyme content in cods (order Gadiformes) and flatfish (order Pleuronectiformes)].

    PubMed

    Subbotkina, T A; Subbotkin, M F

    2013-01-01

    The content of lysozyme as an indicator of nonspecific immunity in kidney, liver, spleen, and blood serum in 8 fish species of the order Gadiformes and the order Pleuronectiformes was analyzed. The Gadiformes are characterized by a very low level of enzyme or the absence of its activity in individual organs and serum, regardless of the fish habitat location. The Pleuronectiformes demonstrated diversity in content and distribution of lysozyme in the body of various fish species. Two plaice species with the unique distribution inhabiting the Sea of Okhotsk were found. This feature is due, first, to the highest lysozyme content in serum of the previously studied fish; second, to the 2-3-fold excess of the enzyme content in serum as compared with the studied immunocompetent organs. The obtained results confirm the opinion about the existence of various related groups among fishes which essentially differ in the lysozyme role in nonspecific immunity.

  17. In vitro Effects of Selected Saponins on the Production and Release of Lysozyme Activity of Human Monocytic and Epithelial Cell Lines

    PubMed Central

    Helal, Racha; Melzig, Matthias F.

    2011-01-01

    Lysozyme is one of the most important factors of innate immunity and a unique enzybiotic in that it exerts not only antibacterial activity, but also antiviral, anti-inflammatory, anticancer and immunomodulatory activities. The purpose of the present study was to investigate whether in vitro exposure to saponins can affect the release and production of lysozyme activity in human monocytic cells THP-1, and in human epithelial cells HT-29. Lysozyme activity levels in cell culture fluids were measured using highly sensitive fluorescence-based lysozyme activity assay. Majority of the examined saponins were demonstrated to stimulate significantly the release of lysozyme activity of monocytes and epithelial cells after one hour treatment at non-toxic concentrations. On the contrary, cells treated with saponins for longer periods up to 72 hours showed tendency to decrease in the secretion and production of lysozyme activity. However, these inhibitory effects of saponins observed with long-term treatment periods were mostly associated with toxic effects of saponins to cells. The results suggested positive contribution of some saponins to lysozyme release of monocytes and epithelial cells upon short exposure. Furthermore, demonstrated ability of these saponins to enhance the release of lysozyme activity can present a new mechanism contribute to explaining important biological characteristics of saponins, including the antibacterial, antiviral, anti-inflammatory or immune-stimulating properties. PMID:21773070

  18. Engineering Escherichia coli for Soluble Expression and Single Step Purification of Active Human Lysozyme

    PubMed Central

    Lamppa, John W.; Tanyos, Sam A.; Griswold, Karl E.

    2012-01-01

    Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50 – 80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli’s ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21 mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications. PMID:23220215

  19. Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species.

    PubMed

    Hemalatha, K; Madhumitha, G; Ravi, Lokesh; Khanna, V Gopiesh; Al-Dhabi, Naif Abdullah; Arasu, Mariadhas Valan

    2016-08-01

    Aspergillosis is one of the infectious fungal diseases affecting mainly the immunocompromised patients. The scarcity of the antifungal targets has identified the importance of N-myristoyl transferase (NMT) in the regulation of fungal pathway. The dihydroquinazolinone molecules were designed on the basis of fragments responsible for binding with the target enzyme. The aryl halide, 1(a-g), aryl boronic acid and potassium carbonate were heated together in water and dioxane mixture to yield new CC bond formation in dihydroquinazolinone. The bis(triphenylphosphine)palladium(II) dichloride was used as catalyst for the CC bond formation. The synthesized series were screened for their in vitro antifungal activity against Aspergillus niger and Aspergillus fumigatus. The binding interactions of the active compound with lysozyme were explored using multiple spectroscopic studies. Molecular docking study of dihydroquinazolinones with the enzyme revealed the information regarding various binding forces involved in the interaction.

  20. Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment.

    PubMed

    Ooi, Beng Guat; Branning, Sharon Alyssa

    2016-12-13

    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.

  1. Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum.

    PubMed

    Yang, Dinglong; Wang, Qing; Cao, Ruiwen; Chen, Lizhu; Liu, Yongliang; Cong, Ming; Wu, Huifeng; Li, Fei; Ji, Chenglong; Zhao, Jianmin

    2017-03-24

    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.

  2. Influence of core and maltose surface modification of PEIs on their interaction with plasma proteins-Human serum albumin and lysozyme.

    PubMed

    Wrobel, Dominika; Marcinkowska, Monika; Janaszewska, Anna; Appelhans, Dietmar; Voit, Brigitte; Klajnert-Maculewicz, Barbara; Bryszewska, Maria; Štofik, Marcel; Herma, Regina; Duchnowicz, Piotr; Maly, Jan

    2017-04-01

    Regardless of the route of administration, some or all of a therapeutic agent will appear in the blood stream, where it can act on blood cells and other components of the plasma. Recently we have shown that poly(ethylene imines) (PEIs) which interact with plasma proteins are taken up into erythrocyte membranes. These observations led us to investigate the interactions between maltose functionalized hyperbranched PEIs (PEI-Mal) and plasma proteins. Two model proteins were chosen - human serum albumin (HSA) (albumins constitute ∼60% of all plasma proteins), and lysozyme. HSA is a negatively charged 66kDa protein at neutral pH, whereas lysozyme is a positively charged 14kDa protein. Fluorescence quenching and changes in the conformation of the amino acid tryptophan, diameter and zeta potential of proteins were investigated to evaluate the interaction of PEI-Mal with proteins. PEI-Mal interacts with both types of proteins. The strength of dendritic glycopolymer interactions was generally weak, especially with lysozyme. Greater changes were found with HSA, mainly triggered by hydrogen bonds and the electrostatic interaction properties of dendritic glycopolymers. Moreover, the structure and the size of PEI-Mal macromolecules affected these interactions; larger macromolecules with more sugar groups (95% maltose units) interacted more strongly with proteins than smaller ones with lower sugar modification (33% maltose units). Due to (i) the proven overall low toxicity of sugar-modified PEIs and, (ii) their ability to interact preferentially through hydrogen bonds with proteins of human plasma or possibly with other interesting protein targets, PEI-Mal is a good candidate for creating therapeutic nanoparticles in the fast developing field of nanomedicine.

  3. Effects of ions on partitioning of serum albumin and lysozyme in aqueous two-phase systems containing ethylene oxide/propylene oxide co-polymers.

    PubMed

    Johansson, H O; Lundh, G; Karlström, G; Tjerneld, F

    1996-08-13

    Aqueous two-phase systems composed of ethylene oxide/propylene oxide random co-polymers, EO30/PO70 or Ucon (EO50/PO50), in the top phase and dextran T500 in the bottom phase, have been studied. The cloud point diagram for EO30/PO70 in water solution was determined. EO30/PO70 has a cloud point of 32 degrees C at a concentration of 10% (w/w). The phase diagram for the system EO30/PO70-dextran T500-water was determined. Salt effects have been studied on the partitioning of two model proteins, bovine serum albumin and hen egg white lysozyme, in EO30/PO70-dextran and Ucon-dextran systems. Ions with different hydrophobicity, i.e., with different position in the Hofmeister or lyotropic series, were investigated with reference to their effect on protein partition. The counterion hydrophobicity was shown to have a strong influence on the partitioning of BSA and lysozyme. Most extreme partitioning was obtained with hydrophobic (chaotropic) ions like CIO4- and I-. A comparison of protein partitioning between PEG-dextran and EO30/PO70-dextran has been done. A more extreme protein partitioning was obtained in the EO30/PO70-dextran containing system. Temperature-induced phase separation was studied with EO30/PO70 at 45 degrees C. Both BSA and lysozyme were completely partitioned to the water phase formed above the cloud point of EO30/PO70. Model calculations, based on Flory-Huggins theory of polymer solutions, have been done which could reproduce the salt effect on the protein partitioning in aqueous-two phase system.

  4. Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine.

    PubMed

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

    2015-08-01

    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.

  5. Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.).

    PubMed

    Buonocore, Francesco; Randelli, Elisa; Trisolino, Pamela; Facchiano, Angelo; de Pascale, Donatella; Scapigliati, Giuseppe

    2014-11-01

    In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742 bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu(71), Asp(84) and Asp(95) in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal

  6. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    NASA Astrophysics Data System (ADS)

    Ashraf, Sumaira; Chatha, Mariyam Asghar; Ejaz, Wardah; Janjua, Hussnain Ahmed; Hussain, Irshad

    2014-10-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests.

  7. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    PubMed Central

    2014-01-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests. PMID:25435831

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

    SciTech Connect

    Held, Jeanette Smaalen, Sander van

    2014-04-01

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

  9. Venom of Parasitoid Pteromalus puparum Impairs Host Humoral Antimicrobial Activity by Decreasing Host Cecropin and Lysozyme Gene Expression

    PubMed Central

    Fang, Qi; Wang, Bei-Bei; Ye, Xin-Hai; Wang, Fei; Ye, Gong-Yin

    2016-01-01

    Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Here, we report that Pteromalus puparum venom impairs the antimicrobial activity of its host Pieris rapae. Inhibition zone results showed that bead injection induced the antimicrobial activity of the host hemolymph but that venom inhibited it. The cDNAs encoding cecropin and lysozyme were screened. Relative quantitative PCR results indicated that all of the microorganisms and bead injections up-regulated the transcript levels of the two genes but that venom down-regulated them. At 8 h post bead challenge, there was a peak in the transcript level of the cecropin gene, whereas the peak of lysozyme gene occurred at 24 h. The transcripts levels of the two genes were higher in the granulocytes and fat body than in other tissues. RNA interference decreased the transcript levels of the two genes and the antimicrobial activity of the pupal hemolymph. Venom injections similarly silenced the expression of the two genes during the first 8 h post-treatment in time- and dose-dependent manners, after which the silence effects abated. Additionally, recombinant cecropin and lysozyme had no significant effect on the emergence rate of pupae that were parasitized by P. puparum females. These findings suggest one mechanism of impairing host antimicrobial activity by parasitoid venom. PMID:26907346

  10. Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein.

    PubMed

    Yin, Zhi-Xin; He, Jian-Guo; Deng, Wei-Xin; Chan, Siu-Ming

    2003-07-08

    Lysozyme acts as a non-specific innate-immunity molecule against the invasion of bacteria pathogens. A leukocyte cDNA library of orange-spotted grouper Epinephelus coioides was constructed and the goose-type (g-type) lysozyme cDNA was isolated. The complete cDNA consists of an open reading frame of 585 bp encoding a protein of 194 amino acids. This protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme. Similar to most other species, the glu catalytic residue in g-type lysozymes of the grouper is conserved. Furthermore, like the flounder and carp, the 4 conserved cysteine residues identified in avian and mammalian g-type lysozymes were also absent from the grouper. Northern blot analysis indicated that the g-type lysozyme was expressed in intestine, liver, spleen, anterior kidney, posterior kidney, heart, gill, muscle and leukocytes. In addition, RT-PCR analysis detected the g-type lysozyme transcripts in the stomach, brain and ovary. When an orange-spotted grouper was injected with Vibrio alginolyticus, the number of lysozyme mRNA transcripts detected in the stomach, spleen, anterior kidney, posterior kidney, heart, brain and leucocytes increased 72 h after injection. Recombinant grouper g-type lysozyme produced in the Escherichia coli expression system showed lytic activity against Micrococcus lysodeikticus, V. alginolyticus from Epinephelus fario, V. vulnificus from culture water, Aeromonas hydrophila from soft-shell turtle, A. hydrophila from goldfish and V. parahaemolyticus, Pseudomonas fluorescens and V. fluvialis from culture water.

  11. The level of some acute phase proteins, total protein, gamma-globulins and activity of lysozyme in blood plasma of rats supplemented with vitamin E and exposed to ozone.

    PubMed

    Jakubowski, K; Jedlińska-Krakowska, M; Siwicki, A K

    2004-01-01

    In rats exposed for 28 days (5 hours a day) to ozone at a concentration of 0.5 ppm and receiving alpha-tocopherol at doses of 4.5 mg/rat and 15 mg/rat, levels of acute phase proteins (APP)--C-reactive protein (CRP), ceruloplasmin (Cp), total protein, gamma-globulins, and activity of lysozyme in blood serum were studied. The assays were performed in the presence of respective control groups, i.e. rats receiving the same doses of alpha-tocopherol but not exposed to ozone, a group of animals not supplemented with vitamin but exposed to ozone, a group of animals injected with physiological fluid and a control group not subjected to any of the treatments. The study revealed that the ozone-exposed animals had an increased lysozyme activity and a decreased total protein level. However, in rats protected by alpha-tocopherol and exposed to ozone, the concentration of APP, lysozyme activity and total protein were found to be decreased. Similar relationships also occurred in animals receiving alpha-tocopherol and not exposed to ozone.

  12. Evidence of a bacterial receptor for lysozyme: binding of lysozyme to the anti-σ factor RsiV controls activation of the ecf σ factor σV.

    PubMed

    Hastie, Jessica L; Williams, Kyle B; Sepúlveda, Carolina; Houtman, Jon C; Forest, Katrina T; Ellermeier, Craig D

    2014-10-01

    σ factors endow RNA polymerase with promoter specificity in bacteria. Extra-Cytoplasmic Function (ECF) σ factors represent the largest and most diverse family of σ factors. Most ECF σ factors must be activated in response to an external signal. One mechanism of activation is the stepwise proteolytic destruction of an anti-σ factor via Regulated Intramembrane Proteolysis (RIP). In most cases, the site-1 protease required to initiate the RIP process directly senses the signal. Here we report a new mechanism in which the anti-σ factor rather than the site-1 protease is the sensor. We provide evidence suggesting that the anti-σ factor RsiV is the bacterial receptor for the innate immune defense enzyme, lysozyme. The site-1 cleavage site is similar to the recognition site of signal peptidase and cleavage at this site is required for σV activation in Bacillus subtilis. We reconstitute site-1 cleavage in vitro and demonstrate that it requires both signal peptidase and lysozyme. We demonstrate that the anti-σ factor RsiV directly binds to lysozyme and muramidase activity is not required for σV activation. We propose a model in which the binding of lysozyme to RsiV activates RsiV for signal peptidase cleavage at site-1, initiating proteolytic destruction of RsiV and activation of σV. This suggests a novel mechanism in which conformational change in a substrate controls the cleavage susceptibility for signal peptidase. Thus, unlike other ECF σ factors which require regulated intramembrane proteolysis for activation, the sensor for σV activation is not the site-1 protease but the anti-σ factor.

  13. Human lung lysozyme: sources and properties.

    PubMed

    Konstan, M W; Chen, P W; Sherman, J M; Thomassen, M J; Wood, R E; Boat, T F

    1981-01-01

    Lysozyme in human airway secretions is thought to defend the lung against airborne bacteria. Although lysozyme has been purified and characterized from human tears, milk, saliva, and other sources (1-5), human lung lysozyme has received little attention except for measurements of concentrations in sputum (6, 7), immunocytochemical and histochemical localization (8-12),and studies of secretion by alveolar macrophages (13). This study was designed to identify the sources of secreted lung lysozyme, to quantitate the secretory activities of the various sources,and to compare the properties of lysozyme from lung cells with those from other tissues.

  14. Foam fractionation of binary mixtures of lysozyme and albumin.

    PubMed

    Lockwood, C E; Jay, M; Bummer, P M

    2000-06-01

    A nitrogen gas-based foam fractionation method was employed to separate model proteins, bovine serum albumin (BSA) and hen egg white lysozyme, from each other. Fractionation was characterized by the separation ratio and by recovery of proteins in the retentate as a function of the nominal pore size of the gas dispersion frit and solution conditions (pH and ionic strength). For binary mixtures of the proteins at pH 7.4, and ionic strength (mu) of 0.18 M, the recovery of lysozyme and the separation ratio were both dependent on the frit size employed to generate the foam. At low ionic strength (mu = 0.01 M), separation was only somewhat greater with the small pore size frits, although at values significantly lower than those found for high ionic strength. The diminished separations appear to be due to the only slight changes in recoveries observed for BSA and lysozyme.%Separation ratios of lysozyme from BSA in solutions either of high or low ionic strength were maximal at pH values equal to or less than the isoelectric point (pI) of BSA. Separation ratios were lower when foaming was carried out under low compared with high ionic strength. The recovery of lysozyme was enhanced by foaming from solutions of low pH and high ionic strength. Recoveries of BSA were greatest when the molecule was negatively charged. Electrical interactions between the positively charged lysozyme and negatively charged BSA may explain the diminished separation ratios and enhanced recoveries. Enzyme activity studies of lysozyme remaining in the retentate showed no change from prefoam activity.

  15. Effects of mercuric chloride on chemiluminescent response of phagocytes and tissue lysozyme activity in Tilapia, Oreochromis aureus

    SciTech Connect

    Low, K.W.; Sin, Y.M.

    1995-02-01

    Phagocytosis is an important defense mechanism against foreign pathogenic organisms. The cells involved are phagocytes which are comprised of peripheral blood monocytes (tissue macrophages) and polymorphonuclear (PMN) leucocytes. These cells can be activated by either particulate or soluble stimuli and undergo a respiratory burst from which several reactive oxygen species (ROS) can be formed. The reactive oxygen species and some hydrolases generated in the cells are the major antibacterial agents released during phagocytosis. Chemiluminescence (CL) is emitted, in vitro, from phagocytizing human PMN neutrophils. A similar CL response was also encountered in fish phagocytes. ROS was the causative agent of the CL emitted during in vitro phagocytosis. Phagocytic activity can be monitored by measuring the CL response of the phagocytes. Lysozyme is one of the potent hydrolases which are involved in the destruction of pathogens during phagocytosis. In fish, it was found predominantly in haematopoietic tissues, PMN leucocytes and moncytes. This enzyme has been shown to have antibacterial activity against several pathogens in fish. A combined oxidative and hydrolytic attack upon the engulfed pathogens allow phagocytes to kill infectious agents effectively. However, severe suppression or enhancement of these two functions caused by some exogenous factors may be detrimental to the host tissues. It has been reported that inorganic mercury could inhibit, in vitro, the respiratory burst and the microbicidal activities of human PMN leucocytes. It was also reported that increased in vitro release of lysozyme was found in mercury-treated human PMN leucocytes. However, such work has not been reported in fish. The aim of this research was to examine whether mercury could exert similar effects on the CL response in phagocytes and tissue lysozyme activity in fish after they were exposed to different concentrations of mercuric chloride over a period of 3 wks. 17 refs., 1 fig., 1 tab.

  16. Enhancing the antimicrobial activity of Sus scrofa lysozyme by N-terminal fusion of a sextuple unique homologous peptide.

    PubMed

    Zhu, Dewei; Cai, Guolin; Li, Xiaomin; Lu, Jian; Zhang, Liang

    2017-02-10

    Sus scrofa lysozyme (SSL), an important component of the pig immune system, is a potential candidate to replace antibiotics in feed. However, there is little antimicrobial activity of natural SSL against gram-negative bacteria, which limits its application. In this study, a unique peptide (A-W-V-A-W-K) with antimicrobial activity against gram-negative bacteria was discovered and purified from trypsin hydrolysate of natural SSL. This unique peptide was fused to natural SSL and the recombinant fused SSL exhibited improved activity against gram-negative bacteria. The N-terminal fusion likely increased the membrane penetrability and induced programmed bacterial cell death. The recombinant fused SSL also showed higher activity against some gram-positive bacteria with O-acetylation. By N-terminal fusion of the sextuple peptide, the anti-microbial activity, either to gram-positive or negative bacteria, of the recombinant SSL was higher than the fusion of only one copy of the peptide. This study provides a general, feasible, and highly useful strategy to enhance the antimicrobial activity of lysozyme.

  17. Effects of cortisol and stress on channel catfish (Ictalurus punctatus) pathogen susceptibility and lysozyme activity following exposure to Edwardsiella ictaluri.

    PubMed

    Small, Brian C; Bilodeau, A Lelania

    2005-05-15

    Periods of stress are often associated with disease outbreaks in cultured fish, and stress is often characterized by the secretion of cortisol. Although stress and cortisol secretion are highly correlated in fish, the role of cortisol in affecting channel catfish (Ictalurus punctatus) pathogen susceptibility is unclear. The effects of short-term stress and exogenous cortisol administration on channel catfish susceptibility to Edwardsiella ictaluri, the etiologic agent of enteric septicemia of catfish (ESC), were investigated. Channel catfish were exposed to virulent E. ictaluri following a standardized 30-min low-water stress or administration of dietary cortisol (100 mg/kg feed) and compared to a pathogen-challenged control group of catfish. Pathogen susceptibility increased in stressed catfish (43.3% mortality) when compared to cortisol-fed catfish (26.7%) and controls (26.7%). A greater (P<0.05) percentage of stressed catfish (25.9%) tested positive for E. ictaluri relative to cortisol-fed catfish (13.0%) over the course of the study, however, average levels of circulating bacteria were not different (P>0.05) among the treatments. Catfish challenged by the low-water stress event had elevated (P<0.05) circulating levels of cortisol 1-day post-pathogen exposure and elevated (P<0.05) lysozyme activity 4 and 14 days post-pathogen exposure when compared to cortisol-fed and control-challenged catfish. Cortisol concentrations were not correlated (P>0.05) to either lysozyme activity or bacterial levels; however, lysozyme activity was positively correlated (P=0.0197) to blood bacterial concentrations. These results implicate other stress factors or pathways, separate from or possibly in conjunction with cortisol, in the stress-associated immunosuppression of channel catfish as it relates to ESC susceptibility.

  18. Lysozyme Crystal

    NASA Technical Reports Server (NTRS)

    2004-01-01

    To the crystallographer, this may not be a diamond but it is just as priceless. A Lysozyme crystal grown in orbit looks great under a microscope, but the real test is X-ray crystallography. The colors are caused by polarizing filters. Proteins can form crystals generated by rows and columns of molecules that form up like soldiers on a parade ground. Shining X-rays through a crystal will produce a pattern of dots that can be decoded to reveal the arrangement of the atoms in the molecules making up the crystal. Like the troops in formation, uniformity and order are everything in X-ray crystallography. X-rays have much shorter wavelengths than visible light, so the best looking crystals under the microscope won't necessarily pass muster under the X-rays. In order to have crystals to use for X-ray diffraction studies, crystals need to be fairly large and well ordered. Scientists also need lots of crystals since exposure to air, the process of X-raying them, and other factors destroy them. Growing protein crystals in space has yielded striking results. Lysozyme's structure is well known and it has become a standard in many crystallization studies on Earth and in space.

  19. Human serum albumin-coated gold nanoparticles for selective extraction of lysozyme from real-world samples prior to capillary electrophoresis.

    PubMed

    Yeh, Pei-Rong; Tseng, Wei-Lung

    2012-12-14

    This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complex matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limit of detection at a signal-to-noise ratio of 3 for Lyz was down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of Lyz in hen egg white, human milk, and human tear. Also, this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  20. Dimethyl sulfoxide induced structural transformations and non-monotonic concentration dependence of conformational fluctuation around active site of lysozyme.

    PubMed

    Roy, Susmita; Jana, Biman; Bagchi, Biman

    2012-03-21

    Experimental studies have observed significant changes in both structure and function of lysozyme (and other proteins) on addition of a small amount of dimethyl sulfoxide (DMSO) in aqueous solution. Our atomistic molecular dynamic simulations of lysozyme in water-DMSO reveal the following sequence of changes on increasing DMSO concentration. (i) At the initial stage (around 5% DMSO concentration) protein's conformational flexibility gets markedly suppressed. From study of radial distribution functions, we attribute this to the preferential solvation of exposed protein hydrophobic residues by the methyl groups of DMSO. (ii) In the next stage (10-15% DMSO concentration range), lysozome partially unfolds accompanied by an increase both in fluctuation and in exposed protein surface area. (iii) Between 15-20% concentration ranges, both conformational fluctuation and solvent accessible protein surface area suddenly decrease again indicating the formation of an intermediate collapse state. These results are in good agreement with near-UV circular dichroism (CD) and fluorescence studies. We explain this apparently surprising behavior in terms of a structural transformation which involves clustering among the methyl groups of DMSO. (iv) Beyond 20% concentration of DMSO, the protein starts its final sojourn towards the unfolding state with further increase in conformational fluctuation and loss in native contacts. Most importantly, analysis of contact map and fluctuation near the active site reveal that both partial unfolding and conformational fluctuations are centered mostly on the hydrophobic core of active site of lysozyme. Our results could offer a general explanation and universal picture of the anomalous behavior of protein structure-function observed in the presence of cosolvents (DMSO, ethanol, tertiary butyl alcohol, dioxane) at their low concentrations.

  1. Bacteriostatic activity of serum against staphylococci.

    PubMed

    Cybulska, J; Jeljaszewicz, J

    1966-03-01

    Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection

  2. Serum lipolytic activity in Reye's syndrome.

    PubMed

    Kang, E S; Matsuo, N; Nagai, T; Greenhaw, J; Williams, P L

    1989-09-15

    High levels of the serum free fatty acids (FFA) are found in Reye's syndrome (RS). While this is attributed to enhanced adipose tissue lipolysis, the possibility that intravascular lipolysis could augment this process was investigated by measuring lipase activity in sera from RS and other subjects. Ordinarily, lipolytic activity is not detectable in serum from unheparinized subjects. Significant lipolytic activities ranging from 1-3 mumol/ml serum per hour were detected in sera from 5 of the 7 RS patients studied. Similar activities were also found in sera from two other subjects one of whom was a long-term survivor of RS and the other who had recurrent bouts of biliary obstruction and encephalopathy. Lipase activity was negligible in the serum from 2 other RS patients, 4 other long-term survivors of RS, 2 siblings, one RS parent and in 20 disease controls including patients with influenza, diabetic ketoacidosis and cerebral edema, meningitis and febrile infections with diarrhea and vomiting. None of these individuals had received heparin. An inverse relationship was found between LPL and hepatic lipase (HL) activities. Glucose levels tended to correlate directly with LPL and inversely with HL activity. The basis for the presence of LPL activity in RS sera is not known but the presence of serum lipase activity in unheparinized patients supports the notion that the TG in the circulating lipoprotein particles probably also serve as another source of FFA in the sera of RS patients.

  3. Baicalin promotes the bacteriostatic activity of lysozyme on S. aureus in mammary glands and neutrophilic granulocytes in mice.

    PubMed

    Gao, Xuejiao; Guo, Mengyao; Zhang, Zecai; Shen, Peng; Yang, Zhengtao; Zhang, Naisheng

    2017-02-08

    Staphylococcus aureus causes mastitis as a result of community-acquired or nosocomial infections. Lysozyme (LYSO) is an enzyme that is upregulated in many organisms during the innate immune response against infection by bacterial pathogens. Baicalin is a bioactive flavonoid that can bind to enzymes, often to potentiate their effect. Here we tested the effects of baicalin on the activity of LYSO using the S. aureus mastitis mouse model and neutrophilic granulocyte model of S. aureus infection. In our experiments, S. aureus counts decreased with increasing baicalin concentration. Furthermore, qPCR and western blot analyses showed that LYSO expression was unaffected by baicalin, while fluorescence quenching and UV fluorescence spectral analyses showed that baicalin binds to LYSO. To test whether this binding increased LYSO activity, we assessed LYSO-induced bacteriostasis in the presence of baicalin. Our results showed that LYSO-induced S. aureus bacteriostasis increased with increasing concentrations of baicalin, and that baicalin binding to LYSO synergistically increased the antibacterial activity of LYSO. These results demonstrate that baicalin enhances LYSO-induced bacteriostasis during the innate immune response to S. aureus. They suggest baicalin is a potentially useful therapeutic agent for the treatment of bacterial infections.

  4. Shift in aggregation, ROS generation, antioxidative defense, lysozyme and acetylcholinesterase activities in the cells of an Indian freshwater sponge exposed to washing soda (sodium carbonate).

    PubMed

    Mukherjee, Soumalya; Ray, Mitali; Ray, Sajal

    2016-09-01

    Washing soda, chemically identified as anhydrous sodium carbonate, is a popular cleaning agent among the rural and urban populations of India which often contaminates the freshwater ponds and lakes, the natural habitat of sponge Eunapius carteri. Present investigation deals with estimation of cellular aggregation, generation of ROS and activities of antioxidant enzymes, lysozyme and acetylcholinesterase in the cells of E. carteri under the environmentally realistic concentrations of washing soda. Prolonged treatment of washing soda inhibited the degree of cellular aggregation. Experimental exposure of 8 and 16mg/l of sodium carbonate for 48h elevated the physiological level of reactive oxygen species (ROS) generation in the agranulocytes, semigranulocytes and granulocytes of E. carteri, whereas, treatment of 192h inhibited the ROS generation in three cellular morphotypes. Activities of superoxide dismutase, catalase and glutathione-S-transferase were recorded to be inhibited under prolonged exposure of washing soda. Washing soda mediated inhibition of ROS generation and depletion in the activities of antioxidant enzymes were indicative to an undesirable shift in cytotoxic status and antioxidative defense in E. carteri. Inhibition in the activity of lysozyme under the treatment of sodium carbonate was suggestive to a severe impairment of the innate immunological efficiency of E. carteri distributed in the washing soda contaminated habitat. Washing soda mediated inhibition in the activity of acetylcholinesterase indicated its neurotoxicity in E. carteri. Washing soda, a reported environmental contaminant, affected adversely the immunophysiological status of E. carteri with reference to cellular aggregation, oxidative stress, antioxidative defense, lysozyme and acetylcholinesterase activity.

  5. Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins.

    PubMed

    Aslebagh, Roshanak; Pfeffer, Bruce A; Fliesler, Steven J; Darie, Costel C

    2016-10-01

    Modification of proteins by 4-hydroxy-2-nonenal (HNE), a reactive by-product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age-related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff-base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff-base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS "signatures" of HNE-modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE-modified lysozyme into an electrospray quadrupole time-of-flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC-MS/MS, we found that, in addition to N-terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.

  6. Recent advances for the production and recovery methods of lysozyme.

    PubMed

    Ercan, Duygu; Demirci, Ali

    2016-12-01

    Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.

  7. 21 CFR 862.1490 - Lysozyme (muramidase) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... serum, plasma, leukocytes, and urine. Lysozyme measurements are used in the diagnosis and treatment of monocytic leukemia and kidney disease. (b) Classification. Class I (general controls). The device is...

  8. The studies of density, apparent molar volume, and viscosity of bovine serum albumin, egg albumin, and lysozyme in aqueous and RbI, CsI, and DTAB aqueous solutions at 303.15 K.

    PubMed

    Singh, Man; Chand, Hema; Gupta, K C

    2005-06-01

    Density (rho), apparent molar volume (V(phi)), and viscosity (eta) of 0.0010 to 0.0018% (w/v) of bovine serum albumin (BSA), egg albumin, and lysozyme in 0.0002, 0.0004, and 0.0008 M aqueous RbI and CsI, and (dodecyl)(trimethyl)ammonium bromide (DTAB) solutions were obtained. The experimental data were regressed against composition, and constants are used to elucidate the conformational changes in protein molecules. With salt concentration, the density of proteins is found to decrease, and the order of the effect of additives on density is observed as CsI > RbI > DTAB. The trend of apparent molar volume of proteins is found as BSA > egg-albumin > lysozyme for three additives. In general, eta values of BSA remain higher for all compositions of RbI than that of egg-albumin for CsI and DTAB. These orders of the data indicate the strength of intermolecular forces between proteins and salts, and are helpful for understanding the denaturation of proteins.

  9. Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters.

    PubMed

    Noritomi, Hidetaka; Takasugi, Tsubasa; Kato, Satoru

    2008-04-01

    Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25 degrees C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.

  10. New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

    SciTech Connect

    Michalska, Karolina; Brown, Roslyn N.; Li, Hui; Jedrzejczak, Robert; Niemann, George; Heffron, Fred; Cort, John R.; Adkins, Joshua N.; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-03-01

    Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

  11. Covalent attachment of lysozyme to cotton/cellulose materials: protein verses solid support activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Covalent attachment of enzymes to cellulosic materials like cotton is of interest where either release or loss of enzyme activity over time needs to be avoided. The covalent attachment of an enzyme to a cellulosic substrate requires either activation of a protein side chain or an organic functional ...

  12. A complex of equine lysozyme and oleic acid with bactericidal activity against Streptococcus pneumoniae.

    PubMed

    Clementi, Emily A; Wilhelm, Kristina R; Schleucher, Jürgen; Morozova-Roche, Ludmilla A; Hakansson, Anders P

    2013-01-01

    HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET's bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA's bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments - an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes' bactericidal activities, while the proteins are required both to solubilize and/or present the

  13. A novel antifungal protein with lysozyme-like activity from seeds of Clitoria ternatea.

    PubMed

    K, Ajesh; K, Sreejith

    2014-06-01

    An antifungal protein with a molecular mass of 14.3 kDa was isolated from the seeds of butterfly pea (Clitoria ternatea) and designated as Ct protein. The antifungal protein was purified using different methods including ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-50 column. Ct protein formed a single colourless rod-shaped crystal by hanging drop method after 7 days of sample loading. The protein showed lytic activity against Micrococcus luteus and broad-spectrum, fungicidal activity, particularly against the most clinically relevant yeasts, such as Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus laurentii, Candida albicans and Candida parapsilosis. It also exerted an inhibitory activity on mycelial growth in several mould species including Curvularia sp., Alternaria sp., Cladosporium sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp., and Sclerotium sp. The present study adds to the literature on novel seed proteins with antifungal activity.

  14. A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: genome organization, transcriptional profiling and biological activities of recombinant protein.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Kasthuri, Saranya Revathy; Whang, Ilson; Lim, Bong-Soo; Nam, Bo-Hye; Lee, Jehee

    2013-10-01

    Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

  15. Systematic mutation of bacteriophage T4 lysozyme.

    PubMed

    Rennell, D; Bouvier, S E; Hardy, L W; Poteete, A R

    1991-11-05

    Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

  16. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells.

    PubMed

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

    2016-12-01

    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.

  17. Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

    PubMed

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

    2016-06-21

    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.

  18. Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

    PubMed Central

    Myers, Fiona A.; Lefevre, Pascal; Mantouvalou, Evangelia; Bruce, Kimberley; Lacroix, Claire; Bonifer, Constanze; Thorne, Alan W.; Crane-Robinson, Colyn

    2006-01-01

    Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the −2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression. PMID:16914441

  19. Regenerated cellulose fiber and film immobilized with lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present work reports an initial engineering approach for fabricating lysozyme-bound regenerated cellulose fiber and film. Glycine-esterified cotton was dissolved in an ionic liquid solvent 1–Butyl–3–methylimidazolium Chloride (BMIMCl) in which lysozyme was activated and covalently attached to c...

  20. Purification of lysozyme using ultrafiltration.

    PubMed

    Ghosh, R; Cui, Z F

    2000-04-20

    This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.

  1. Digestive function of lysozyme in synanthropic acaridid mites enables utilization of bacteria as a food source.

    PubMed

    Erban, Tomas; Hubert, Jan

    2008-03-01

    The activity of lysozyme, the enzyme that hydrolyzes peptidoglycan in G(+) bacterial cell walls, was detected in whole mite extracts (WME) and in spent growth medium extracts (SGME) of 14 species of synanthropic mites (Acari: Acaridida). The adaptation of lysozyme for digestive activity and bacteriophagy was based on: (i) high lysozyme activity in SGME, and (ii) the correlation of maximum lysozyme activity at acidic pH values, corresponding to pH in the ventriculus and caeca. We show that the digestion of fluorescein-labeled Micrococcus lysodeikticus cells began in ventriculus and continued during the passage of a food bolus through the gut. The fluorescein was absorbed by midgut cells and penetrated to parenchymal tissues. Eight species showed a higher rate of population growth on a M. lysodeikticus diet than on a control diet. The lysozyme activity in SGME was positively correlated to the standardized rate (r (s)) of population growth, although no correlation was found between r (s) and lysozyme activity in WME. The lysozyme activity in WME was negatively correlated to that in SGME. The highest activity of digestive lysozyme was found in Lepidoglyphus destructor, Chortoglyphus arcuatus and Dermatophagoides farinae. All of these findings indicate that lysozyme in acaridid mites possesses both defensive and digestive functions. The enzymatic properties of mite lysozyme are similar to those of the lysozymes present in the ruminant stomach and in the insect midgut.

  2. Adaptive functional diversification of lysozyme in insectivorous bats.

    PubMed

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

    2014-11-01

    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.

  3. Lysozymes in the animal kingdom.

    PubMed

    Callewaert, Lien; Michiels, Chris W

    2010-03-01

    Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. 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 i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.

  4. The boundary molecules in a lysozyme pattern exhibit preferential antibody binding.

    PubMed

    Gao, Pei; Cai, Yuguang

    2008-09-16

    Lysozyme was immobilized on a prefabricated carboxylic acid terminated chemical template, forming a tightly packed, one monolayer thick lysozyme pattern. Polyclonal anti-lysozyme antibodies can bind to the immobilized lysozyme pattern. Atomic force microscope (AFM) observation reveals that the antibodies bind to the lysozyme molecules on the pattern edge before they bind to the lysozyme molecules in the pattern interior. Better spatial accessibility and flexibility of the lysozyme molecules on the pattern edge are used to explain the observed antibody binding preference. The topographies of the lysozyme pattern also affect the antibody binding. The antibodies bind to the edge lysozyme from the top if the lysozyme pattern is half-buried in a 10 A deep channel, whereas the antibodies bind to the edge lysozyme from the side if the lysozyme pattern is immobilized on a protruding terrace. The observed "edge effect" suggests that, for the same protein coverage, reducing the protein pattern feature to the nanoscale will improve the overall binding activity of the immobilized protein toward the antibody.

  5. Design of Synthetic Polymer Nanoparticles That Facilitate Resolubilization and Refolding of Aggregated Positively Charged Lysozyme.

    PubMed

    Nakamoto, Masahiko; Nonaka, Tadashi; Shea, Kenneth J; Miura, Yoshiko; Hoshino, Yu

    2016-04-06

    Designed polymer hydrogel nanoparticles (NPs) capable of facilitating resolubilization and refolding of an aggregated protein, positively charged lysozyme, are prepared. NPs designed to interact strongly with denatured lysozyme and relatively weakly with native lysozyme, facilitated resolubilization and refolding of aggregated lysozyme. Such NPs could be prepared by copolymerizing optimized combinations and populations of functional monomers. The refolded lysozyme showed native conformation and enzymatic activity. Eleven grams of aggregated protein was refolded by 1 g of NPs. However, NPs having low affinity to denatured lysozyme and NPs having high affinity to both denatured and native lysozyme showed relatively low facilitation activity. Our results suggest a potential strategy for the design of artificial chaperones with high facilitating activity.

  6. Dietary fat modulates serum paraoxonase 1 activity in rats.

    PubMed

    Kudchodkar, B J; Lacko, A G; Dory, L; Fungwe, T V

    2000-10-01

    We examined the effects of dietary fats with specific fatty acid compositions, on serum paraoxonase (PON1) activity in rats. Male adult Sprague-Dawley rats were divided randomly into four dietary groups. One group received the control diet [AIN 93M with soybean oil (5 g/100 g diet)], whereas the remaining three groups received the modified control diet supplemented with (15 g/100 g diet) triolein, tripalmitin or fish oil, respectively. After 20 d, blood was obtained after overnight food deprivation and PON1 activity was determined. Serum lipids and lipid components of lipoproteins were also determined. Serum PON1 activity [micromol/(L.min)] was significantly (P: < 0.05) higher in triolein (98 +/- 6) and lower in fish oil (41 +/- 4), compared with tripalmitin-fed rats (63 +/- 11). Serum PON1 activity in tripalmitin-fed rats was comparable to that of controls (67 +/- 9). Serum PON1 activity correlated significantly with serum lecithin:cholesterol acyltransferase (LCAT) activity (r = 0.77, P: < 0.001) and was transported in blood principally in association with the denser subfraction of HDL, very high density lipoprotein (VHDL; d > 1.15 kg/L). Serum PON1 activity correlated strongly with serum lipids as well as lipids of VLDL, HDL and its subfractions. Multiple linear regression analysis, however, showed a significant relationship of serum PON1 activity, principally with the phospholipids of VHDL (r = 0.47, P: < 0.002). These data suggest that the modulation of serum PON1 activity by dietary fat may be mediated via the effect of the specific fatty acids on the synthesis and secretion of VHDL, the subfraction of HDL that transports the majority of PON1 in the blood.

  7. N-ACETYL-β-GLUCOSAMINIDASE ACTIVITY IN SERUM DURING PREGNANCY

    PubMed Central

    Walker, P. G.; Woollen, Mary E.; Pugh, Doreen

    1960-01-01

    A spectrophotometric method for the estimation of N-acetyl-β-glucosaminidase in serum has been devised. Sera from normal adult males and females showed similar levels of activity. The activity in serum rose progressively during pregnancy and fell rapidly after parturition to normal levels. This change resembled closely that which occurs in serum β-glucuronidase. Placenta showed a moderate and chorion a high level of N-acetyl-β-glucosaminidase. High N-acetyl-β-glucosaminidase activity was demonstrated histochemically in decidual cells. The functions of N-acetyl-β-glucosaminidase and β-glucuronidase and factors influencing their activity are discussed. Images PMID:13782743

  8. Improving the Lethal Effect of Cpl-7, a Pneumococcal Phage Lysozyme with Broad Bactericidal Activity, by Inverting the Net Charge of Its Cell Wall-Binding Module

    PubMed Central

    Díez-Martínez, Roberto; de Paz, Héctor; Bustamante, Noemí; García, Ernesto; Menéndez, Margarita

    2013-01-01

    Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from −14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules. PMID:23959317

  9. Lysozyme loading and release from Se doped hydroxyapatite nanoparticles.

    PubMed

    Wang, Yanhua; Hao, Hang; Zhang, Shengmin

    2016-04-01

    Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA-LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA-LSM composites with Se:P=10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of β-sheet and unordered, and the decrease of self-association, α-helix and β-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA-LSM composites. The composites with Se:P=10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity.

  10. [Purification and characterization of a lysozyme from a marine microorganism].

    PubMed

    Zou, Yan-Li; Sun, Mi; Wang, Yue-Jun

    2005-05-01

    A novel lysozyme was purified from a marine microorganism and its major characteristics were studied. Cell-free supernatant was prepared by centrifugation of culture broth, ultrafiltration using a hollow fiber (molecular weight cut off, 50kD) and concentration using a hollow fiber (molecular weight cut off, 10kD). The crude lysozyme was purified 34.7 fold to electrophoretic homogeneity with a recovery of 24.1% by CM-Sepharose FF cationic-exchange and Sephadex G-100 gel chromatography. The relative molecular weight of this lysozyme was determined as about 39 kD. The optimum pH and temperature towards Micrococcus lysodleikticus were pH 8.0 and 35 degrees C respectively, and the enzyme was stable at temperature below 50 degrees C and pH 5.0 - 10.0. The lysozyme activity was slightly enhanced by Zn2+ and Cu2+ and slightly inhibited by Mn2+ and Ag+. The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%) and KH2 PO4 (1.0%). The lysozyme had broad-spectrum against many bacteria, including a number of pathogens, which were resistant to egg-white lysozyme.

  11. The Invertebrate Lysozyme Effector ILYS-3 Is Systemically Activated in Response to Danger Signals and Confers Antimicrobial Protection in C. elegans

    PubMed Central

    Gravato-Nobre, Maria João; Vaz, Filipa; Filipe, Sergio; Chalmers, Ronald; Hodgkin, Jonathan

    2016-01-01

    Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material. PMID:27525822

  12. Mesoscopic coarse-grained simulations of lysozyme adsorption.

    PubMed

    Yu, Gaobo; Liu, Jie; Zhou, Jian

    2014-05-01

    Coarse-grained simulations are adopted to study the adsorption behavior of lysozyme on different (hydrophobic, neutral hydrophilic, zwitterionic, negatively charged, and positively charged) surfaces at the mesoscopic microsecond time scale (1.2 μs). Simulation results indicate the following: (i) the conformation change of lysozyme on the hydrophobic surface is bigger than any other studied surfaces; (ii) the active sites of lysozyme are faced to the hydrophobic surface with a "top end-on" orientation, while they are exposed to the liquid phase on the hydrophilic surface with a "back-on" orientation; (iii) the neutral hydrophilic surface can induce the adsorption of lysozyme, while the nonspecific protein adsorption can be resisted by the zwitterionic surface; (iv) when the solution ionic strength is low, lysozyme can anchor on the negatively charged surface easily but cannot adsorb on the positively charged surface; (v) when the solution ionic strength is high, the positively charged lysozyme can also adsorb on the like-charged surface; (vi) the major positive potential center of lysozyme, especially the residue ARG128, plays a vital role in leading the adsorption of lysozyme on charged surfaces; (vii) when the ionic strength is high, a counterion layer is formed above the positively charged surface, which is the key factor why lysozyme can adsorb on a like-charged surface. The coarse-grained method based on the MARTINI force field for proteins and the BMW water model could provide an efficient way to understand protein interfacial adsorption behavior at a greater length scale and time scale.

  13. Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles.

    PubMed

    Sun, Jun; Su, Yujie; Rao, Shengqi; Yang, Yanjun

    2011-08-01

    Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.

  14. Effect of altitude training on serum creatine kinase activity and serum cortisol concentration in triathletes.

    PubMed

    Wilber, R L; Drake, S D; Hesson, J L; Nelson, J A; Kearney, J T; Dallam, G M; Williams, L L

    2000-01-01

    In this investigation we evaluated the effect of a 5-week training program at 1860 m on serum creatine kinase (CK) activity and serum cortisol concentration in national-caliber triathletes for the purpose of monitoring the response to training in a hypobaric hypoxic environment. Subjects included 16 junior-level female (n = 8) and male (n = 8) triathletes who were training for the International Triathlon Union (ITU) World Championships. After an initial acclimatization period, training intensity and/or volume were increased progressively during the 5-week altitude training camp. Resting venous blood samples were drawn at 0700 hours following a 12-h overnight fast and were analyzed for serum CK activity and serum cortisol concentration. Subjects were evaluated before [7-10 days pre-altitude (SL 1)] and after [7-10 days post-altitude (SL 2)] the 5-week training camp at 1860 m. At altitude, subjects were evaluated within 24-36 h after arrival (ALT 1), 7 days after arrival (ALT 2), 18 days after arrival (ALT 3), and 24-36 h prior to leaving the altitude training camp (ALT 4). A repeated-measures analysis of variance was used to evaluate differences over time from SL 1 to SL 2. Compared to SL 1, serum CK activity increased approximately threefold (P < 0.05) within the initial 24-36 h at altitude (ALT 1), and increased by an additional 70% (P < 0.05) after the 1st week of altitude training (ALT 2). Serum CK activity remained significantly elevated over the duration of the experimental period compared to pre-altitude baseline levels. Serum cortisol concentration was increased (P < 0.05) at the end of the 5-week altitude training period (ALT 4) relative to SL 1, ALT 1 and ALT 3. These data suggest that: (1) the initial increase in serum CK activity observed in the first 24-36 h at altitude was due primarily to acute altitude exposure and was independent of increased training intensity and/or training volume, (2) the subsequent increases in serum CK activity observed over

  15. [Phosphonate esterase activity in human serum (author's transl)].

    PubMed

    Labadie, M; Laplaud, P M; Lachatre, G; Breton, J C

    1980-02-01

    A simple methodology for the spectrophotometric assay of phosphonate esterase activity in human serum samples is described, featuring incubation at 30 degrees C in a medium containing p-nitrophenol and phenyl-phosphonic acid ester. Reproducibility of the method as well a mean values in normal patients vs age and sex are reported. Serum activity appears to be increased almost exclusively during pregnancy or administration of estrogenic drugs (as oral contraceptives or in prostate neoplasms).

  16. Pluronic-lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating.

    PubMed

    Muszanska, Agnieszka K; Busscher, Henk J; Herrmann, Andreas; van der Mei, Henny C; Norde, Willem

    2011-09-01

    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 telechelic groups of the PEO chains. Covalent conjugation of lysozyme proceeded via reductive amination of aldehyde functionalized PEO blocks (CHO-Pluronic) and the amine groups of the lysine residues in the protein. SDS-PAGE gel electrophoresis together with MALDI-TOF mass spectrometry analysis revealed formation of conjugates of one or two lysozyme molecules per Pluronic polymer chain. The conjugated lysozyme showed antibacterial activity towards Bacillus subtilis. Analysis with a quartz crystal microbalance with dissipation revealed that Pluronic-lysozyme conjugates adsorb in a brush conformation on a hydrophobic gold-coated quartz surface. X-ray photoelectron spectroscopy indicated surface coverage of 32% by lysozyme when adsorbed from a mixture of unconjugated Pluronic and Pluronic-lysozyme conjugate (ratio 99:1) and of 47% after adsorption of 100% Pluronic-lysozyme conjugates. Thus, bifunctional brushes were created, possessing both anti-adhesive activity due to the polymer brush, combined with the antibacterial activity of lysozyme. The coating having a lower degree of lysozyme coverage proved to be more bactericidal.

  17. Bioengineered lysozyme in combination therapies for Pseudomonas aeruginosa lung infections

    PubMed Central

    Griswold, Karl E; Bement, Jenna L; Teneback, Charlotte C; Scanlon, Thomas C; Wargo, Matthew J; Leclair, Laurie W

    2014-01-01

    There is increasing urgency in the battle against drug-resistant bacterial pathogens, and this public health crisis has created a desperate need for novel antimicrobial agents. Recombinant human lysozyme represents one interesting candidate for treating pulmonary infections, but the wild type enzyme is subject to electrostatic mediated inhibition by anionic biopolymers that accumulate in the infected lung. We have redesigned lysozyme’s electrostatic potential field, creating a genetically engineered variant that is less susceptible to polyanion inhibition, yet retains potent bactericidal activity. A recent publication demonstrated that the engineered enzyme outperforms wild type lysozyme in a murine model of Pseudomonas aeruginosa lung infection. Here, we expand upon our initial studies and consider dual therapies that combine lysozymes with an antimicrobial peptide. Consistent with our earlier results, the charge modified lysozyme combination outperformed its wild type counterpart, yielding more than an order-of-magnitude reduction in bacterial burden following treatment with a single dose. PMID:24637705

  18. Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; He, Xiao-Min; Lyne, James E.; Stubbs, Gerald; Hash, John H.

    1990-01-01

    The lysozyme from the fungus of the Chalaropsis species has been crystallized. This lysozyme displays no sequence homology with avian, phage, or mammalian lysozymes, however, preliminary studies indicate significant sequence homology with the bacterial lysozyme from Streptomyces. Both enzymes are unusual in possessing beta-1,4-N-acetylmuramidase and beta-1,4-N,6-O-diacetylmuramidase activity. The crystals grow from solutions of ammonium sulfate during growth periods from several months to a year. The space group is P2(1)2(1)2(1) with a = 34.0 A, b = 42.6 A, c = 122.1 A. Preliminary data indicate that there is 1 molecule/asymmetric unit.

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

    PubMed

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

    2012-01-01

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

  20. Studies on antistreptolysin O activity generated in serum by microorganisms.

    PubMed Central

    Watson, K. C.; Kerr, E. J.

    1976-01-01

    An antistreptolysin factor (ASF) was generated in normal human serum by the growth of Staph, aureus and Pseud, aeruginosa. Alpha toxin producing strains of the former were usually positive but activity was not restricted to such strains. Positive strains produce cholesterol esterase which was obtained from DEAE-cellulose column fractions of 18 h broth cultures. Antistreptolysin factor develops slowly in serum, being maximal between the 5th and 10th days and is associated with alterations and disappearance of beta lipoproteins on gel electrophoresis. Activity also appeared in beta lipoproteins precipitated from normal serum with dextran sulphate and redissolved in nutrient broth before inoculation with Staph, aureus. The slow appearance of antistreptolysin activity in serum appears to be due to an esterase inhibitor which is present in high concentrations in some sera. Activity is also modified by the production of a staphylococcal fraction capable of binding to the antistreptolysin factor and reducing its activity. It is suggested that antistreptolysin factor which can be demonstrated in small amounts in normal human serum represents a readily available non-specific defence mechanism capable of binding to certain bacterial products and possible to other foreign protein molecules. Images Fig. 2 PMID:178333

  1. Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum.

    PubMed

    Hata, Keiko; Tochigi, Tatsuo; Sato, Ikuro; Kawamura, Sadafumi; Shiozaki, Kazuhiro; Wada, Tadashi; Takahashi, Kohta; Moriya, Setsuko; Yamaguchi, Kazunori; Hosono, Masahiro; Miyagi, Taeko

    2015-04-01

    Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.

  2. Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Seabass

    PubMed Central

    Fu, Gui Hong; Bai, Zhi Yi; Xia, Jun Hong; Liu, Feng; Liu, Peng; Yue, Gen Hua

    2013-01-01

    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 (Glu50 and Asp67) 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 (Glu71, Asp84, Asp95) 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. PMID:24244553

  3. The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis.

    PubMed

    Tanaka, Tetsuya; Kawano, Suguru; Nakao, Sumihiro; Umemiya-Shirafuji, Rika; Rahman, Md Morshedur; Boldbaatar, Damdinsuren; Battur, Banzragch; Liao, Min; Fujisaki, Kozo

    2010-12-01

    A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41-79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.

  4. Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

    PubMed

    Zdybicka-Barabas, Agnieszka; Mak, Pawel; Klys, Anna; Skrzypiec, Krzysztof; Mendyk, Ewaryst; Fiołka, Marta J; Cytryńska, Małgorzata

    2012-11-01

    Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

  5. Parallel tempering Monte Carlo simulations of lysozyme orientation on charged surfaces

    NASA Astrophysics Data System (ADS)

    Xie, Yun; Zhou, Jian; Jiang, Shaoyi

    2010-02-01

    In this work, the parallel tempering Monte Carlo (PTMC) algorithm is applied to accurately and efficiently identify the global-minimum-energy orientation of a protein adsorbed on a surface in a single simulation. When applying the PTMC method to simulate lysozyme orientation on charged surfaces, it is found that lysozyme could easily be adsorbed on negatively charged surfaces with "side-on" and "back-on" orientations. When driven by dominant electrostatic interactions, lysozyme tends to be adsorbed on negatively charged surfaces with the side-on orientation for which the active site of lysozyme faces sideways. The side-on orientation agrees well with the experimental results where the adsorbed orientation of lysozyme is determined by electrostatic interactions. As the contribution from van der Waals interactions gradually dominates, the back-on orientation becomes the preferred one. For this orientation, the active site of lysozyme faces outward, which conforms to the experimental results where the orientation of adsorbed lysozyme is co-determined by electrostatic interactions and van der Waals interactions. It is also found that despite of its net positive charge, lysozyme could be adsorbed on positively charged surfaces with both "end-on" and back-on orientations owing to the nonuniform charge distribution over lysozyme surface and the screening effect from ions in solution. The PTMC simulation method provides a way to determine the preferred orientation of proteins on surfaces for biosensor and biomaterial applications.

  6. Identification and expression analysis of three c-type lysozymes in Oreochromis aureus.

    PubMed

    Gao, Feng-ying; Qu, Lan; Yu, Shao-guo; Ye, Xing; Tian, Yuan-yuan; Zhang, Li-li; Bai, Jun-jie; Lu, Maixin

    2012-05-01

    Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.

  7. Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease.

    PubMed

    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

    2015-11-01

    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.

  8. Increased serum cortisol binding in chronic active hepatitis

    SciTech Connect

    Orbach, O.; Schussler, G.C.

    1989-01-01

    A high serum cortisol concentration, apparently due to increased cortisol-binding globulin (CBG), was found in a patient (index case) with chronic active hepatitis (CAH). We therefore performed further studies to determine whether increased cortisol binding is generally associated with CAH. Serum samples were obtained from 15 hospitalized patients with long-term liver function test elevations but no evidence of cirrhosis, 15 normal subjects without a history of hepatitis, four healthy pregnant women, and 10 alcoholic patients with stigmata of cirrhosis. Serum cortisol binding was measured by an adaptation of a previously described charcoal uptake method. Thyroxine-binding globulin (TBG) and sex hormone-binding globulin were determined by radioimmunoassays. Charcoal uptake of 125I cortisol from sera of normal subjects and additional patients with CAH revealed that increased serum cortisol binding by a saturable site, presumably CBG, was associated with CAH. Cortisol binding was significantly correlated with immunoassayable TBG, suggesting that in CAH, similar mechanisms may be responsible for increasing the serum concentrations of CBG and TBG.

  9. Low serum alkaline phosphatase activity in Kikuchi-Fujimoto disease

    PubMed Central

    Inamo, Yasuji

    2017-01-01

    Abstract Various laboratory findings are helpful in making a diagnosis of Kikuchi-Fujimoto disease (KFD); however, they are not specific. We found decreased serum alkaline phosphatase (SAP) activity in children with KFD. The levels of SAP fell in the acute phase and recovered during convalescence. We conclude that low SAP activity is a characteristic of KFD and may be an auxiliary diagnostic marker for the disease. PMID:28248884

  10. Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.

    PubMed Central

    Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

    1983-01-01

    The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be

  11. Serum prolidase activity in benign joint hypermobility syndrome

    PubMed Central

    2014-01-01

    Background Moderate joint laxity is widespread in many joints of the body, and this condition is considered to be caused by an abnormality in the collagen structure. This study was carried out to determine the serum prolidase activity in female patients with benign joint hypermobility syndrome (BJHS), and to evaluate its correlation with their clinical features. Methods A total of 45 patients with BJHS and 40 healthy controls were included in the study. All of the patients with BJHS met the Beighton diagnostic criteria. All the patients and the control group underwent a comprehensive examination of the locomotor system and took the New York Posture Rating Test. The examination and test results were recorded. Serum prolidase activity was measured in both the groups. Results Prolidase activity was significantly lower in patients with BJHS (479.52 ± 126.50) compared to the healthy controls (555.97 ± 128.77) (p = 0.007). We found no correlation between serum prolidase activity and Beighton scores or New York rating test scores. On the other hand, mean prolidase activity was significantly lower in patients with pes planus or hyperlordosis compared to those without (p = 0.05, p = 0.03, respectively). We did not find such a correlation with the other clinical features. Conclusions Significantly lower prolidase activity in patients with BJHS suggests that prolidase may affect the collagen metabolism and cause hyperlaxity. PMID:24612543

  12. [Separation and purification of lysozyme from egg white by high performance cation-exchange chromatography].

    PubMed

    Li, Rong; Chen, Guo-liang

    2002-05-01

    A new method used to separate and purify lysozyme from egg white by high performance cation-exchange chromatography has been established. The process conditions for purifying lysozyme were also discussed in detail. The procedure involved that homogenization of the egg white sample, preliminary purification with sodium chloride, and chromatographic separation by the weak cation exchange column (XIDACE-WCX). The experimental results showed that the purified lysozyme and other impurity proteins were completely separated. By using bioactivity assay, the recovery of lysozyme was 107%, and the specific activity was 15,467 U/mg through the column. Its purity was raised 5.6-fold. The collected fraction with activity was detected by size-exclusion chromatography (SEC). The purified lysozyme was homogeneous. Compared with the traditional soft-based low pressure ion-exchange chromatography, the developed method is rapid and effective.

  13. Manipulation of lysozyme phase behavior by additives as function of conformational stability.

    PubMed

    Galm, Lara; Morgenstern, Josefine; Hubbuch, Jürgen

    2015-10-15

    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.

  14. Hydration of thermally denatured lysozyme studied by sorption calorimetry and differential scanning calorimetry.

    PubMed

    Kocherbitov, Vitaly; Arnebrant, Thomas

    2006-05-25

    We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.

  15. Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model.

    PubMed

    Behbehani, G Rezaei; Saboury, A A; Taleshi, E

    2008-01-01

    The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).

  16. The refined structures of goose lysozyme and its complex with a bound trisaccharide show that the "goose-type" lysozymes lack a catalytic aspartate residue.

    PubMed

    Weaver, L H; Grütter, M G; Matthews, B W

    1995-01-06

    The structure of goose egg-white lysozyme (GEWL) has been refined to an R-value of 15.9% at 1.6 A resolution. Details of the structure determination, the refinement and the structure itself are presented. The structure of a complex of the enzyme with the trisaccharide of N-acetyl glucosamine has also been determined and refined at 1.6 A resolution. The trisaccharide occupies sites analogous to the B, C and D subsites of chicken (HEWL) and phage T4 (T4L) lysozymes. All three lysozymes (GEWL, HEWL and T4L) display the same characteristic set of bridging hydrogen bonds between backbone atoms of the protein and the 2-acetamido group of the saccharide in subsite C. Glu73 of GEWL is seen to correspond closely to Glu35 of HEWL (and to Glu11 of T4L) and supports the established view that this group is critically involved in the catalytic mechanism. There is, however, no obvious residue in goose lysozyme that is a counterpart of Asp52 of chicken lysozyme (or of Asp20 in T4L), suggesting that a second acidic residue is not essential for the catalytic activity of goose lysozyme, and may not be required for the activity of other lysozymes.

  17. Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme.

    PubMed

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

    2014-03-01

    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.

  18. Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria

    PubMed Central

    Abergel, Chantal; Monchois, Vincent; Byrne, Deborah; Chenivesse, Sabine; Lembo, Frédérique; Lazzaroni, Jean-Claude; Claverie, Jean-Michel

    2007-01-01

    Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates. PMID:17405861

  19. Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

    PubMed

    Burke, Thomas P; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G; Portnoy, Daniel A

    2014-11-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.

  20. Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

    PubMed Central

    Burke, Thomas P.; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G.

    2014-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood. PMID:25157076

  1. Lysozyme-lysozyme and lysozyme-salt interactions in the aqueous saline solution: a new square-well potential.

    PubMed

    Chang, Bong Ho; Bae, Young Chan

    2003-01-01

    We investigate lysozyme-lysozyme and lysozyme-salt interactions in electrolyte solutions using a molecular-thermodynamic model. An equation of state based on the statistical mechanical perturbation theory is applied to describe the interactions. The perturbation term includes a new square-well potential of mean force, which implies the information about the lysozyme surface and salt type. The attractive energy of the potential of mean force is correlated with experimental cloud-point temperatures of lysozyme in various solution conditions. The same attractive energy is used to predict osmotic pressure of a given system with no additional parameters. The new potential shows a satisfactory improvement in understanding the interactions between lysozymes in aqueous salt solutions.

  2. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

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

  3. Comparative evaluation of multi-purpose solutions in the stabilization of tear lysozyme.

    PubMed

    Barniak, Vicki L; Burke, Susan E; Venkatesh, Srini

    2010-12-01

    The range and extent of tear proteins removed by various multi-purpose solutions has been investigated, but there is little information in the literature about their ability to prevent denaturation of tear proteins, particularly lysozyme. The purpose of this study was to determine the ability of Bausch+Lomb Biotrue™ multi-purpose solution and other care solutions to affect denaturation of lysozyme using a lysozyme activity assay. The test solutions used were: Biotrue multi-purpose solution, Bausch+Lomb renu(®) fresh™, formerly ReNu MultiPlus(®), Alcon OPTI-FREE RepleniSH, Alcon OPTI-FREE EXPRESS, CIBA VISION AQuify, and AMO COMPLETE Multi-Purpose Solution Easy Rub Formula. A phosphate-buffered saline (PBS) solution served as a control. The test and control solutions containing lysozyme were exposed to sodium dodecyl sulfate (SDS), a known denaturant of the enzyme. The assay was based on digestion of the cell wall of Micrococcus luteus in a suspension, a substrate sensitive to active lysozyme. Enzymatic activity against M. luteus was used to assess activity of lysozyme. The decrease in the turbidity of the cell wall suspension, a measure of relative enzyme activity, was determined by following the decrease in absorbance (at 450nm) over time using a spectrophotometer. Statistically significant greater stabilization of lysozyme was observed with Biotrue multi-purpose solution and renu fresh than with OPTI-FREE RepleniSH, OPTI-FREE EXPRESS, AQuify, COMPLETE Multi-Purpose Solution Easy Rub Formula, and a PBS control. The lysozyme activity assay revealed that Biotrue multi-purpose solution and renu fresh have the ability to stabilize lysozyme under conditions that typically denature the protein.

  4. Determination of phosphodiesterase I activity in human blood serum.

    PubMed

    Hynie, I; Meuffels, M; Poznanski, W J

    1975-09-01

    Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis.

  5. Lysozyme separation by hollow-fibre ultrafiltration.

    PubMed

    Ghosh; Silva1; Cui

    2000-08-01

    This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.

  6. Interaction between lysozyme and procyanidin: multilevel structural nature and effect of carbohydrates.

    PubMed

    Liang, Miao; Liu, Rui; Qi, Wei; Su, Rongxin; Yu, Yanjun; Wang, Libing; He, Zhimin

    2013-06-01

    The interaction of procyanidins with proteins has aroused extensive attention due to its important relationship with the bioavailability and astringent property of polyphenols. In the present work, we have investigated the interactions of lysozyme with procyanidin dimer (B3) using various biophysical approaches, which aims to provide insights into the mechanism of protein/polyphenol aggregation. Procyanidin B3 spontaneously binds lysozyme, inducing the multilevel structural changes in lysozyme and the formation of insoluble complexes. The relationship between lysozyme aggregation and the loss of enzymatic activity was monitored using dynamic light scattering and fluorescence quenching. The influences of two carbohydrates (gum arabic and sucrose) on lysozyme/B3 aggregation were also studied. Gum arabic effectively inhibited the formation of insoluble aggregates, but was unable to restore the fluorescence and activity of lysozyme. However, sucrose concomitantly decreased the aggregate size with the recovery of fluorescence and lysozyme activity. These results proposed two probable mechanisms by which these two carbohydrates inhibit protein/polyphenol aggregation.

  7. Serum angiotensin converting enzyme activity in chronic beryllium disease.

    PubMed

    Newman, L S; Orton, R; Kreiss, K

    1992-07-01

    Serum angiotensin converting enzyme (SACE) activity is used as a marker of sarcoidosis activity and severity, but in chronic beryllium disease (CBD) the studies of SACE give conflicting results. We examined SACE activity in 23 CBD patients, five patients with beryllium sensitization, and 25 beryllium-exposed control subjects. CBD patients underwent complete clinical evaluation, including physical examination, pulmonary function testing, exercise physiology testing, chest radiography, and bronchoscopy with bronchoalveolar lavage and biopsy. CBD SACE activity was systematically compared with these clinical markers of disease severity. Of CBD patients, 22% had elevated SACE activity. The test did not discriminate CBD patients from those in the beryllium-sensitized or beryllium-exposed groups. However, SACE activity in CBD correlated with the extent of pulmonary granulomatous inflammation as reflected by the symptom of breathlessness, the number of white cells in bronchoalveolar lavage (r = 0.44), the number of lavage lymphocytes (r = 0.58), the lavage lymphocyte percentage (r = 0.55), and the profusion of small opacities on chest radiograph (r = 0.41). The test-retest reliability of the assay was high (r = 0.84), as was the agreement between fresh and -70 degrees C frozen sera (r = 0.93). We conclude that SACE activity levels may reflect the extent of pulmonary granulomatous inflammation in CBD but that the test does not help discriminate disease from nondisease.

  8. Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity

    PubMed Central

    Soloski, Mark J.; Crowder, Lauren A.; Lahey, Lauren J.; Wagner, Catriona A.

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations. PMID:24740099

  9. Dietary and demographic correlates of serum ß-glucuronidase activity

    PubMed Central

    Maruti, Sonia S.; Li, Lin; Chang, Jyh-Lurn; Prunty, JoAnn; Schwarz, Yvonne; Li, Shuying S.; King, Irena B.; Potter, John D.; Lampe, Johanna W.

    2010-01-01

    Background β-glucuronidase, an acid hydrolase that deconjugates glucuronides, may increase cancer risk; however, little is known about factors associated with human β-glucuronidase. Objective To examine whether dietary and demographic factors were associated with serum β-glucuronidase activity. Design We conducted a cross-sectional study among 279 healthy men and women, aged 20-40 years. Diet, categorized by botanical families and nutrient intakes, was assessed from 3-day food records and a validated semiquantitative food frequency questionnaire. Demographic factors were directly measured or self-reported. Adjusted mean β-glucuronidase activity across categories of exposure variables were calculated by multiple linear regression. Results Higher β-glucuronidase activity was significantly associated with being male, older age (≥30 years), non-Caucasian, overweight (≥ 25 kg/m2), and higher intakes of gamma-tocopherol. Conversely, lower β-glucuronidase activity was significantly associated with higher intakes of calcium, iron, and magnesium. A suggestive decrease in β-glucuronidase activity was observed for the botanical families, Cruciferae, Rutaceae, Compositae, Roseaceae, and Umbelliferae, but tests for trend were not statistically significant. Conclusions Several dietary and nondietary factors were associated with β-glucuronidase activity, however confirmation of these associations are needed. PMID:20099195

  10. Effects of oligomeric lysozyme on structural state of model membranes.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya

    2011-03-01

    The ability of oligomeric lysozyme to modify the molecular organization of the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with phosphatidylglycerol (PG) or cholesterol (Chol) was assessed using fluorescent probes 6-propionyl-2-dimethylaminonaphthalene (Prodan), 4-dimethylaminochalcone (DMC), pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH). The observed changes in the fluorescence characteristics of polarity-sensitive probes Prodan and DMC, located in interfacial bilayer region, were interpreted due to the partial dehydration of the glycerol backbone, which was under the influence of aggregated protein. Cholesterol was found to prevent the perturbations of membrane polar part by lysozyme aggregates. Analysis of the pyrene excimerization data revealed an oligomer-induced reduction in bilayer free volume, presumably caused by an increased packing density of hydrocarbon chains. This effect proved to be virtually independent of membrane composition. It was demonstrated that membranotropic activity of oligomeric lysozyme markedly exceeds that of monomeric protein. The biological significance of the results obtained is twofold, implicating the general membrane-mediated mechanisms of oligomer toxicity and specific pathways of lysozyme fibrillogenesis in vivo associated with familial nonneuropathic systemic amyloidosis.

  11. [Virucidal activity of disinfectants. Influence of the serum protein upon the virucidal activity of disinfectants].

    PubMed

    Noda, M; Matsuda, S; Kobayashi, M

    2000-08-01

    Five disinfectants were tested for virucidal activity on three DNA viruses and three RNA viruses in the presence or absence of serum protein. Disinfectants of the aldehyde and halogen groups had a virucidal activity on human herpes virus, bovine rhabdo virus, human immunodeficiency virus, human adeno virus, porcine parvo virus, and polio virus. Disinfectants of the invert and amphoteric soap groups, and biganide group had a destructive effect on RNA and DNA viruses possessing an envelope. The presence of serum protein exerted great influence upon the virucidal activity of disinfectants of the invert and amphoteric soap groups.

  12. Serum prolidase activity in ankylosing spondylitis and rheumatoid arthritis.

    PubMed

    Uçar, Demet; Em, Serda; Bozkurt, Mehtap; Oktayoglu, Pelin; Yüksel, Hatice Kurt; Caglayan, Mehmet; Gezer, Orhan; Nas, Kemal

    2013-01-01

    The aim of the present study was to emphasize the collagen turnover in 2 of the most common chronic inflammatory rheumatic diseases by evaluating serum prolidase activity (SPA) in ankylosing spondylitis (AS) and rheumatoid arthritis (RA). 30 patients who met the modified New York Criteria for the classification of AS, 29 patients who met the 2010 Rheumatoid Arthritis Classification Criteria for the classification of RA, and 31 healthy controls were enrolled in the study. Serum samples of the patients and the controls were collected and SPA was measured by a spectrophotometric method. The comparison of the SPA in these 3 groups was statistically examined. In both patient groups, the SPA was lower than in the control group. SPA in patients with AS was statistically significantly lower than in the control and RA groups (P < 0.001/P = 0.002). No statistically significant difference was found between the RA and the control groups (P = 0.891). In conclusion, lower SPA is presumably associated with decreased collagen turnover and fibrosis, leading to decreased physical functions in both chronic inflammatory musculoskeletal diseases.

  13. Fluorescence Studies of Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori

    1998-01-01

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

  14. Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

    PubMed

    Muriel-Galet, V; Talbert, J N; Hernandez-Munoz, P; Gavara, R; Goddard, J M

    2013-07-10

    The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm(2) and 5.74 μg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

  15. PROPERTIES OF A LYSOZYME-DISSOCIATED ENDOTOXIC FRACTION FROM ESCHERICHIA COLI.

    PubMed

    MERGENHAGEN, S E; MARTIN, G R

    1964-10-01

    Mergenhagen, Stephan E. (National Institute of Dental Research, Bethesda, Md.), and George R. Martin. Properties of a lysozyme-dissociated endotoxic fraction from Escherichia coli. J. Bacteriol. 88:1169-1174. 1964.-Treatment of a phenol-water preparation of C(14)-labeled Escherichia coli O91-H21 endotoxin of low solubility with lysozyme at pH 5.0 or 8.0 effected a dissociation of the preparation. Such products of dissociation were equally distributed in the chloroform and water phases after extraction. beta-Glucosidase, but not beta-galactosidase, significantly dissociated this endotoxin also. Concomitant with dissociation, recoverable endotoxin after lysozyme treatment had a reduced content of bound lipid, and dissolved easily in aqueous media to yield a clear solution. Examination of lysozyme-treated endotoxin in an analytical ultracentrifuge revealed that it sedimented as a single major boundary with a sedimentation coefficient of 13.3. Lysozyme-treated endotoxin was more potent than was the conventional endotoxin as evidenced by lethal activity in rabbits and pertussis-sensitized mice. Agar-gel diffusion analysis indicated that the higher molecular weight component associated with conventional endotoxin was dissociated by lysozyme treatment. In immunoelectrophoresis, lysozyme-treated endotoxin was observed as a single sharp band of precipitation which migrated toward the cathode.

  16. PROPERTIES OF A LYSOZYME-DISSOCIATED ENDOTOXIC FRACTION FROM ESCHERICHIA COLI

    PubMed Central

    Mergenhagen, Stephan E.; Martin, George R.

    1964-01-01

    Mergenhagen, Stephan E. (National Institute of Dental Research, Bethesda, Md.), and George R. Martin. Properties of a lysozyme-dissociated endotoxic fraction from Escherichia coli. J. Bacteriol. 88:1169–1174. 1964.—Treatment of a phenol-water preparation of C14-labeled Escherichia coli O91–H21 endotoxin of low solubility with lysozyme at pH 5.0 or 8.0 effected a dissociation of the preparation. Such products of dissociation were equally distributed in the chloroform and water phases after extraction. β-Glucosidase, but not β-galactosidase, significantly dissociated this endotoxin also. Concomitant with dissociation, recoverable endotoxin after lysozyme treatment had a reduced content of bound lipid, and dissolved easily in aqueous media to yield a clear solution. Examination of lysozyme-treated endotoxin in an analytical ultracentrifuge revealed that it sedimented as a single major boundary with a sedimentation coefficient of 13.3. Lysozyme-treated endotoxin was more potent than was the conventional endotoxin as evidenced by lethal activity in rabbits and pertussis-sensitized mice. Agar-gel diffusion analysis indicated that the higher molecular weight component associated with conventional endotoxin was dissociated by lysozyme treatment. In immunoelectrophoresis, lysozyme-treated endotoxin was observed as a single sharp band of precipitation which migrated toward the cathode. Images PMID:14219034

  17. The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

    PubMed Central

    Sotelo-Mundo, Rogerio R.; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; Arvizu-Flores, Aldo A.; Velazquez-Contreras, Enrique F.; Valenzuela-Soto, Elisa M.; Rojo-Dominguez, Arturo; Kanost, Michael R.

    2008-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of α-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5−30 °C. These results together with measured thermodynamical activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins. PMID:17979817

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

    SciTech Connect

    Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.

    2007-01-01

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

  19. Establishment of lysozyme gene RNA interference system and its involvement in salinity tolerance in sea cucumber (Apostichopus japonicus).

    PubMed

    Tian, Yi; Jiang, Yanan; Shang, Yanpeng; Zhang, Yu-Peng; Geng, Chen-Fan; Wang, Li-Qiang; Chang, Ya-Qing

    2017-03-27

    The lysozyme gene was silenced using RNA interference (RNAi) to analyze the function of lysozyme in sea cucumber under salt stress. The interfering efficiency of four lysozyme RNAi oligos ranged from 0.55 to 0.70. From the four oligos, p-miR-L245 was used for further interfering experiments because it had the best silencing efficiency. Peristomial film injection of p-miR-L245 (10 μg) was used for further interfering experiments. The lowest gene expression, determined by RT-PCR assay, in muscle, coelomic fluid, and parapodium occurred 48 h after p-miR-L245 injection, while that of body wall and tube foot was 96 h and 24 h, respectively. Lysozyme activity in muscle and body wall was significantly lower than the controls. The lowest lysozyme activity in muscle, body wall and parapodium, was found at 48, 72, and 48 h, respectively, which was consistent with the transcription expression of lysozyme. The lowest point of lysozyme activity was at 96 h in coelomic fluid and tube foot, which was laid behind lysozyme expression in transcription level. The expression profile of the lysozyme transcription level and lysozyme activity in the body wall and tube foot increased at 12 h after p-miR-L245 injection before the interference effect appeared. NKA gene expression was expressed at a high level in the positive control (PC) and negative control (NC) groups at 12, 24, and 48 h, while NKA was expressed at low levels in the lysozyme RNAi injection group at 12 and 24 h. The level of NKA gene expression recovered to the level of the PC and NC group at 48, 72, and 96 h after the lysozyme RNAi injection. NKCC1 gene expression was high in the PC and NC groups at 96 h, while the NKCC1 was expressed at a low level 96 h after lysozyme RNAi injection. The results suggest that lysozyme decay involves NKA and NKCC1 gene expression under salinity 18 psμ. The K(+) and Cl(-) concentration after lysozyme RNAi injection was lower than in the PC and NC group.

  20. Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

    PubMed

    Wellman-Labadie, Olivier; Picman, Jaroslav; Hincke, Maxwell T

    2008-01-01

    Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.

  1. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    SciTech Connect

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.

    2009-06-04

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

  2. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    SciTech Connect

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L.

    2008-07-11

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

  3. Opsonic activity of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes.

    PubMed

    Tamura, Y; Tanaka, M

    1987-05-01

    The role of anti-flagellar serum against Clostridium chauvoei in phagocytosis by mouse polymorphonuclear leucocytes was examined. Anti-flagellar serum markedly increased phagocytic rate against the flagellated strain Okinawa but not against a non-flagellated mutant (NFM) derived from the same strain, while anti-NFM serum increased the phagocytic rate against both strains. These results indicate that anti-flagellar serum exerts its protective effect by opsonic activity.

  4. Serum creatine kinase B subunit activity in diagnosis of acute myocardial infarction.

    PubMed Central

    Ljungdahl, L; Gerhardt, W; Hofvendahl, S

    1980-01-01

    The value of serum creatine kinase B subunit activity (CK B) in the diagnosis of acute myocardial infarction was studied in 238 consecutive cases. All were admitted to a coronary care unit because of suspected acute myocardial infarction. Serum CK B activity was determined by an immunoinhibition procedure, using a CK M subunit inhibiting antibody (anti-M). For the evaluation of serum CK B, patients were classified into acute myocardial infarction and non-acute myocardial infarction groups. This classification was based on electrocardiographic findings, on quantitative determinations of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total serum creatine kinase (CK) activities, and on qualitative electrophoretic determinations of serum CK and serum lactate dehydrogenase (LD) isoenzymes. The prevalence of acute myocardial infarction in the patient material was 0.47. Serum CK B subunit activity was found to be a highly selective indicator of acute myocardial infarction with a predictive value of a positive test result of 0.97 and a predictive value of a negative test result of 0.99. The serum CK B activity increased above the acute myocardial infarction discrimination limit within 12 hours from onset of symptoms. Two non-acute myocardial infarction patients, who were resuscitated after cardiac arrest, had increased serum CK B values caused by the transient presence of CK isoenzyme BB in serum. PMID:7378210

  5. Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

    PubMed Central

    Aoki, Yutaka; Helzlsouer, Kathy J.; Strickland, Paul T.

    2014-01-01

    Context: Cholinesterase (ChE) specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking) with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose. PMID:24473115

  6. Stomach lysozymes of the three-toed sloth (Bradypus variegatus), an arboreal folivore from the Neotropics.

    PubMed

    Pacheco, M Andreína; Concepción, Juan Luís; Rangel, José David Rosales; Ruiz, Marie Christine; Michelangeli, Fabián; Domínguez-Bello, María G

    2007-07-01

    Lysozymes are antimicrobial defences that act as digestive enzymes when expressed in the stomach of herbivores with pre-gastric fermentation. We studied this enzyme in the complex stomach of the three-toed sloth (Bradypus variegatus), a folivore with pre-gastric fermentation. Lysozymes were identified by SDS-PAGE and immunoblotting in all portions: diverticulum, pouch, glandular and muscular prepyloric area with 14.3 kDa of molecular mass. Purified lysozymes from all areas but the diverticulum were characterized by MALDI-TOF, optimal pH, optimal ionic strength, and specific activity. The differences observed suggested at least three isoforms. The optimal pHs were similar to the pH of the stomach portion where the enzymes were isolated. The lysozyme from the pouch (fermentation chamber) exhibited higher specific activity and concentration than the others. The specific activity of the enzyme from the acid muscular prepyloric portion was comparable to that reported in the cow abomasums; however, its concentration was lower than that observed in cow. This distinctive pattern of secretion/specific activity and overall low concentration suggests different roles for the lysozymes in this herbivore compared to Artiodactyla. We postulate that sloth stomach lysozymes may still be antimicrobial defences by protecting the microbial flora of the fermentation chamber against foreign bacteria.

  7. Engineered regulation of lysozyme by the SH3-CB1 binding interaction.

    PubMed

    Pham, Elizabeth; Truong, Kevin

    2012-06-01

    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.

  8. Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

    PubMed Central

    Merchant, Mark; Juneau, Kate; Gemillion, Jared; Falconi, Rodolfo; Doucet, Aaron; Shirley, Matthew H.

    2011-01-01

    Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria. PMID:22110960

  9. Membrane changes induced by exposure of Escherichia coli to human serum.

    PubMed Central

    Kroll, H P; Bhakdi, S; Taylor, P W

    1983-01-01

    The effect of bactericidal concentrations of lysozyme-free human serum on parameters of membrane integrity has been studied in serum-susceptible and serum-resistant Escherichia coli strains. Serum treatment released all of the alkaline phosphatase from the periplasmic space of two rapidly serum-susceptible strains but did so at different rates. In contrast, no periplasmic enzyme was released from two serum-resistant strains or from one moderately susceptible smooth strain. Lysozyme-free serum and heat-inactivated serum released comparable amounts of 86Rb+ from preloaded cells at comparable rates, regardless of serum susceptibility. Serum decreased the rate of phospholipid biosynthesis in both serum-susceptible and serum-resistant strains. In susceptible but not in resistant strains, intracellular ATP pools were depleted after serum exposure. Outer membranes and cytoplasmic membranes were prepared from serum-treated E. coli, and assays for C3 and C5b-9(m) were performed. With rapidly susceptible strains, C3 deposition on the outer membrane without attachment of C5b-9(m) occurred during the short prekilling phase. Subsequent bacterial killing was accompanied by deposition of C5b-9(m), which was recovered with C3 exclusively in outer membrane fractions with increased density and by eventual total loss of recoverable cytoplasmic membranes. Minimal deposition of complement components, without accompanying cytoplasmic membrane loss, occurred with serum-resistant strains. Loss of recoverable cytoplasmic membrane was not due to the action of either serum or bacterial phospholipase A. The results raise the possibilities that C5b-9(m) primarily damages the outer membrane and that the bacteria themselves actively participate in the ensuing, as yet unclarified, metabolic reactions that finally lead to their death. Images PMID:6358036

  10. Acetylated lysozyme as impurity in lysozyme crystals: constant distribution coefficient

    NASA Astrophysics Data System (ADS)

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

    2001-11-01

    Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A and B impurities added were 0.76, 0.38 and 0.1 mg/ml and 0.43, 0.22, 0.1 mg/ml, respectively. The HEWL concentration were 20, 30 and 40 mg/ml. The crystals grown in 18 experiments for each impurity concentration and supersaturation 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±0.13 for A and K=3.42±0.25 for B. According to definition of K by Eq. (1) in the text, the condition K=const is equivalent to a decrease of impurity amount in the crystal as the supersaturation increases. 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 the impurity adsorption and incorporation rates are proportional to the impurity concentration and that the growth rate is proportional to the concentration of crystallizing protein in solution. The frequency at which an impurity molecules irreversibly join the crystal was estimated to be 3 s -1, much higher than such frequency for regular crystal molecules 5×10 -2 s -1 at 30 mg/ml lysozyme concentration. Reasons for this inequality are discussed.

  11. Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2

    PubMed Central

    Roces, Clara; Courtin, Pascal; Kulakauskas, Saulius; Rodríguez, Ana; Chapot-Chartier, Marie-Pierre

    2012-01-01

    Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972, Lactococcus lactis D1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover, L. lactis lacking a functional d,d-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene pip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance. PMID:22504807

  12. Macroporous chitin affinity membranes for lysozyme separation.

    PubMed

    Ruckenstein, E; Zeng, X

    1997-12-20

    Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.

  13. Preparation of a light-sensitive and reversible dissolution copolymer and its application in lysozyme purification.

    PubMed

    Wen, Wen; Wan, Junfen; Cao, Xuejun; Xia, Jinan

    2007-01-01

    A novel light-sensitive and cation-exchange copolymer (PNBCC) has been synthesized by random copolymerization of chlorophyllin sodium copper salt, crylic acid, n-butyl acrylate, and N-isopropylacrylamide. The PNBCC copolymer showed reversible dissolution and could be precipitated by 488 nm laser irradiation with the least light density of 1.70 x 10(5) W/m(2). By optimizing the ratio of monomers, pH, and ion concentration, over 95% copolymer was recovered by laser irradiation. The copolymer was used to purify lysozyme as light-sensitive cation exchanger, and its adsorption matched a Langmuir adsorption isotherm with maximum adsorption capacity of 98,900 U/g and dissociated constant of 852 U/mL. By applying the copolymer to the separation of lysozyme from egg white, the specific activity of lysozyme was improved from 399 to 6346 U/mg and the recovery of lysozyme achieved 81.3%.

  14. A direct calorimetric determination of denaturation enthalpy for lysozyme in sodium dodecyl sulfate.

    PubMed

    Behbehani, G Rezaei; Saboury, A A; Taleshi, E

    2008-02-15

    Thermodynamics of the interaction between sodium dodecyl sulfate (SDS) with lysozyme were investigated at pH 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. A new method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The new solvation model was used to reproduce the enthalpies of lysozyme-SDS interaction over the whole range of SDS concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of SDS, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The enthalpy of denaturation is 160.81+/-0.02 kJ mol(-1) for SDS.

  15. Heat induced aggregation of gold nanorods for rapid visual detection of lysozyme.

    PubMed

    Tohidi Moghadam, Tahereh; Ranjbar, Bijan

    2015-11-01

    Gold nanorods have been nominated as propitious candidates for nanobiodiagnostic applications. Herein, a technique has been introduced for rapid visual detection of lysozyme, as its high level of excretion in biological fluids is a characteristic sign of leukemia and kidney disorders. Gold nanorods were biofunctionalized with lysozyme aptamer and characterized with UV-Visible and FTIR spectroscopy, zeta potential analyzer and transmission electron microscopy. Exposure of the nanoprobe to nano molar levels of lysozyme (20 nmol l(-1)) lead to dictated aggregation of the nanostructures at ambient temperature; which was significantly improved by heat induced morphological perturbations and rapid detection by the naked eye (down to pico molar level). Qualitative analysis of Acute myeloid leukemia, Acute lymphocytic leukemia and Lymphoma blood serums showed sensitivity and specificity of the fabricated aptasensor under both temperature conditions. This report encourages utilization of heat-induced aggregation of gold nanorods as a promising nanodiagnostic technique for the emerging nanotechnologies.

  16. Killing of an encapsulated strain of Escherichia coli by human serum.

    PubMed Central

    Taylor, P W; Kroll, H P

    1983-01-01

    Changes in cell viability and in factors affecting metabolic integrity were examined after exposure of Escherichia coli LP1092 to human serum. Antibody-dependent classical pathway activity accounted for the rapid killing of strain LP1092 by complement. Removal of serum lysozyme by bentonite absorption or by neutralization with anti-human lysozyme immunoglobulin G resulted in a reduction in the rate of killing; optimal activity could be restored by the addition of physiological amounts of egg-white lysozyme. The pattern of 86Rb+ and alkaline phosphatase release obtained after serum treatment did not support the view that complement simultaneously disrupts cytoplasmic and outer membrane integrity. Macromolecular synthesis was affected late in the reaction sequence; complete inhibition of precursor incorporation into RNA, DNA, and protein occurred only after almost total loss of bacterial colony-forming ability. Addition of chloramphenicol, an inhibitor of protein synthesis, to the bactericidal system resulted in a marked reduction in the rate of serum killing. Killing was completely inhibited by an inhibitor (KCN) and an uncoupler (2,4-dinitrophenol) of oxidative phosphorylation. Exposure of LP1092 cells to serum was followed by a rapid and large increase in intracellular ATP levels; ATP synthesis did not occur when bacteria were exposed to dialyzed serum, which killed LP1092 cells at a much reduced rate. Addition of glucose or serum ultrafiltrate to dialyzed serum restored optimal bactericidal activity. We suggest that optimal killing of gram-negative bacteria is an energy-dependent process requiring an input of bacterially generated ATP. PMID:6185430

  17. Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus.

    PubMed

    Takahashi, Hajime; Nakazawa, Moemi; Ohshima, Chihiro; Sato, Miki; Tsuchiya, Tomoki; Takeuchi, Akira; Kunou, Masaaki; Kuda, Takashi; Kimura, Bon

    2015-07-02

    Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40 min at 100 °C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus.

  18. Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies

    NASA Astrophysics Data System (ADS)

    Jing, Mingyang; Song, Wei; Liu, Rutao

    2016-07-01

    Although copper is essential to all living organisms, its potential toxicity to human health have aroused wide concerns. Previous studies have reported copper could alter physical properties of lysozyme. The direct binding of copper with lysozyme might induce the conformational and functional changes of lysozyme and then influence the body's resistance to bacterial attack. To better understand the potential toxicity and toxic mechanisms of copper, the interaction of copper with lysozyme was investigated by biophysical methods including multi-spectroscopic measurements, isothermal titration calorimetry (ITC), molecular docking study and enzyme activity assay. Multi-spectroscopic measurements proved that copper quenched the intrinsic fluorescence of lysozyme in a static process accompanied by complex formation and conformational changes. The ITC results indicated that the binding interaction was a spontaneous process with approximately three thermodynamical binding sites at 298 K and the hydrophobic force is the predominant driven force. The enzyme activity was obviously inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. This study helps to elucidate the molecular mechanism of the interaction between copper and lysozyme and provides reference for toxicological studies of copper.

  19. A comparison of the physical properties of ultrasonically synthesized lysozyme- and BSA-shelled microbubbles.

    PubMed

    Vong, Fiona; Son, Younggyu; Bhuiyan, Sadia; Zhou, Meifang; Cavalieri, Francesca; Ashokkumar, Muthupandian

    2014-01-01

    Ultrasonic technique has been used for synthesising protein microspheres possessing specific physical and functional properties. Various proteins have been used as shell materials under different experimental conditions. In previous studies, thermal or chemical denaturation of the proteins was used to obtain stable bovine-serum albumin (BSA) and lysozyme microbubbles (MBs), respectively. It is ideal to establish a generic procedure to synthesise microspheres irrespective of the nature of the protein. In order to see if a generic procedure can be established, ultrasonic synthesis of lysozyme and BSA MBs was carried out under similar experimental conditions and their properties were evaluated. The size, size distribution and the stability of the MBs were significantly different for the lysozyme and BSA MBs. The size and size distribution of the lysozyme coated MBs were larger than BSA bubbles. The mechanical strength of MBs against the shear forces, generated when irradiated by high frequency ultrasound, was studied using pulsed-sonoluminescence (SL). This study indicated that lysozyme MBs were significantly more stable than BSA MBs. An increase in mechanical strength of the MBs may lead to an increase in their storage lifetime and stability against gas diffusion. Possible reasons for such observations have been discussed.

  20. Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

    PubMed Central

    Peter, A T; Bosu, W T; MacWilliams, P; Gallagher, S

    1987-01-01

    Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum. PMID:3453274

  1. Elastic Properties of Lysozyme Confined in Nanoporous Polymer Films

    NASA Astrophysics Data System (ADS)

    Wang, Haoyu; Akcora, Pinar

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. It is known that confined media provide a protective environment to the encapsulated proteins and prevent diffusion of the denaturant. In this study, different types of proteins (streptavidin, lysozyme and fibrinogen) were chemically attached into the nanopores of poly(methyl methacrylate) thin films. Heterogeneous flat surfaces with varying cylinder pore sizes (10-50 nm) were used to confine proteins of different sizes and shapes. Stiffness of protein functionalized nanopores was measured in nanoindentation experiments. Our results showed that streptavidin behaved more stiffly when pore dimension changed from micron to nanosize. Further, it was found that lysozyme confined within nanopores showed higher specific bioactivity than proteins on flat surfaces. These results on surface elasticity and protein activity may help in understanding protein interactions with surfaces of different topologies and chemistry.

  2. Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines.

    PubMed

    Roldán, Ana; Lasanta, Cristina; Caro, Ildefonso; Palacios, Víctor

    2012-05-01

    Biological aging is a key step in the production of Sherry wine classified as "fine". During this stage, a film of yeast referred to as "flor velum" covers the surface of the wine and substantially alters its characteristics. Other microorganisms may coexist with flor yeasts, such as lactic acid bacteria and non-Saccharomyces yeasts, whose growth may be favored under certain conditions, causing organoleptic deviations and deterioration of the wine. To prevent the development of lactic bacteria, lysozyme usage has been introduced. Lysozyme is a hydrolytic enzyme with muramidase activity that can lyse gram-positive bacteria; its use in winemaking was approved by the OIV in 1997 (resolution OENO 10/97). Thus far, the use of lysozyme during the production of Sherry wines is not widespread despite its effectiveness in controlling lactic acid bacteria. However, there have been no studies on the effect of lysozyme on flor velum. The aim of this study was to determine the influence of lysozyme on yeast growth and the formation, development and metabolism of flor velum during the biological aging process of Sherry wine. The results indicate that lysozyme does not affect the flor yeast during the fermentative stage or biofilm stage. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, low doses of lysozyme (≥12.5 g/hL) affect cell multiplication and the membrane hydrophobicity of the yeast, inhibiting their aggregation and flotation and the subsequent development of flor velum. Thus, the yeast inoculation protocol and the methodology used for the addition of lysozyme influence velum development, its metabolism and the wine characteristics.

  3. Binding of lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association.

    PubMed

    Gorbenko, Galyna P; Ioffe, Valeriya M; Kinnunen, Paavo K J

    2007-07-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  4. Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

    PubMed Central

    Gorbenko, Galyna P.; Ioffe, Valeriya M.; Kinnunen, Paavo K. J.

    2007-01-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5′-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  5. Changes in serum angiotensin I converting enzyme activity due to carbon disulfide exposure.

    PubMed

    Filipović, N; Bilalbegović, Z; Sefić, M; Djurić, D

    1984-01-01

    The activity of serum angiotensin I converting enzyme (ACE) was determined in 50 workers from a viscose factory in Banja Luka, Yugoslavia, and in 50 control subjects. Activity of serum ACE was significantly lower in workers exposed to carbon disulfide than in the control group. No correlation was found between a decrease of serum ACE in exposed workers and duration of exposure. These findings indicate that the serum ACE may be influenced by carbon disulfide, but the mechanism of these changes remains to be elucidated in this case.

  6. Interaction of nonionic detergents with the specific sites of lysozyme amyloidogenic region - inhibition of amyloid fibrillization.

    PubMed

    Siposova, Katarina; Kozar, Tibor; Musatov, Andrey

    2017-02-01

    Two nonionic detergents, Triton X-100 (TX-100) and n-dodecyl-β-d-maltoside (DDM) were tested for their ability to affect lysozyme amyloid aggregation. We have demonstrated that fibrillization of lysozyme is completely inhibited by low sub-micellar concentrations of both of these detergents. The apparent IC50 values were calculated to be 22μM and 26μM for TX-100 and DDM, respectively. The detergent/protein ratio is not the only parameter controlling inhibition. The precise timing of the detergent addition was found to be also crucial. It appears that the primary inhibitory activity of detergents resulted from inhibition of nuclei formation, in addition to inhibition of fibril polymerization at the early stage of protofibrils growth. The docking study revealed that Asn-59, Trp-63 and Ala-107, all present within the lysozyme amyloidogenic region, were involved in the interaction with both detergents. In addition, TX-100 also interacted with Gln-57 and Asp-103 within lysozyme. Moreover, based on our computational results, TX-100 bridges the Gln-57 and Ala-107 amino acids of the amyloidogenic segment of lysozyme and therefore inhibits more effectively the amyloid fibril formation. Along these lines, the knowledge gained from our study indicates that the detergents or their derivatives may be applicable as a promising strategy for the modulation of lysozyme protein aggregation.

  7. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    PubMed Central

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

    2011-01-01

    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

  8. Enhancement of enrofloxacin serum antibacterial activity by calcium primed broilers.

    PubMed

    Aguilera, R; Gutiérrez, O L; Sumano, L H

    2007-02-01

    The aim of this trial was to assess the effect that calcium gluconate priming of 468 broilers has on the antibacterial activity of a standard dose of enrofloxacin. Hence, a series of oral pharmacokinetic studies were carried out in four groups of broilers medicated individually through an oral cannula as follows: group A, medicated only with enrofloxacin 10mg/kg; group B, receiving immediately one after the other, calcium gluconate (200mg/kg) and enrofloxacin 10mg/kg; group C, dosed first with calcium gluconate (200mg/kg) and 1h later enrofloxacin (10mg/kg); and group D, dosed first with calcium gluconate (200mg/kg) and 2h later enrofloxacin (10mg/kg). Broilers were bled at different times after the dose of enrofloxacin and antibacterial activity, measured as concentration of enrofloxacin, was measured by an agar diffusion assay. Results revealed that group D the greatest values of maximum serum concentration (Cs(max)), area under the concentration vs. time curve (AUC) and area under the moment curve (AUMC). These values were statistically higher than the corresponding ones derived from groups A, B and C (P<0.05). Taking Cs(max) and AUC values of group A as reference baseline, an increase of 24% and 50%, respectively, was obtained in group D. Group B had the lowest Cs(max), AUC, AUMC and elimination half life (T(1/2)beta) and these values were statistically different from groups A, C and D (P<0.05). The T(1/2)beta was statistically longer in groups C and D as compared with A and B, and the former groups were also different between each other (P<0.05). These results show that if calcium gluconate is first dosed to broilers and 2h later enrofloxacin is administered (as in group D), a more pronounced antibacterial activity of enrofloxacin can be obtained. A challenge of this sequential dosing scheme in a field trial may reveal its clinical value.

  9. [Two cases of Lambert-Eaton syndrome with an increase of serum cholinesterase activity].

    PubMed

    Ciechanowski, K; Cebula, D

    1997-02-01

    Paraneoplastic Lambert-Eaton myasthenia syndrome is presented in two cases with small cell lung cancer. An increase of serum cholinesterase activity was explained by induced release of biologically active proteins by neoplastic tissue.

  10. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  11. A first survey on the biochemical composition of egg yolk and lysozyme-like activity of egg envelopment in the cuttlefish Sepia officinalis from the Northern Adriatic Sea (Italy).

    PubMed

    Matozzo, Valerio; Conenna, Irene; Riedl, Verena Maria; Marin, Maria Gabriella; Marčeta, Tihana; Mazzoldi, Carlotta

    2015-08-01

    The cuttlefish Sepia officinalis is an important fishery resource in the Northern Adriatic Sea (Italy). During reproduction, fertilised eggs are released by adult females in coastal waters and embryo development can take over two months. During this period, embryos rely on nutrients and other substances, such as immune factors, provided by the female in egg yolk. In cephalopods in general, and specifically in the common cuttlefish, little information is available on yolk biochemical composition and substances included in egg envelopment. In the present study, the main biochemical components of egg yolk and the presence of antimicrobial substances in egg envelopment of S. officinalis were determined for the first time. Statistically significant differences in total egg weight and egg yolk weight were observed among batches from different females. Egg and yolk weights were positively correlated, with yolk representing the 13% (±5%) of the total egg weight. Total proteins were the main biochemical component (46%) of egg yolk, followed by total carbohydrates plus glycogen (39%) and lipids (15%). Statistically significant differences among batches were recorded in egg yolk total protein amounts, lipids, carbohydrates and glycogen, but no correlations were found between egg yolk weight and the biochemical components. The Petri dish and the quantitative spectrophotometric assays revealed the presence of lysozyme-like activity in egg gelatinous envelopment.

  12. Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis)

    PubMed Central

    Kong, Hailong; Lv, Min; Mao, Nian; Wang, Cheng; Cheng, Yunxia; Zhang, Lei; Jiang, Xingfu; Luo, Lizhi

    2016-01-01

    There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions. PMID:27575006

  13. IgG, IgA, and lysozyme in Martina Franca donkey jennies and their foals.

    PubMed

    Veronesi, Maria C; Dall'Ara, Paola; Gloria, Alessia; Servida, Francesco; Sala, Elisabetta; Robbe, Domenico

    2014-04-01

    Because immune transfer from jenny to donkey foal is mostly unknown, the aim of the present study was to evaluate, from 5 days before to 10 days after foaling, immunoglobulin (Ig)G, IgA, and lysozyme peripartal concentrations in serum and mammary secretions of 10 healthy, spontaneously foaling Martina Franca jennies and in serum of their mature, viable, healthy foals, in the first 10 days after birth. The results showed that, in jennies, mammary secretion of IgG levels (ranging between 16 and 75 mg/mL) and IgA (0.9-2 mg/mL), and IgG (6.8-13.5 mg/mL) and IgA (0.5-2.4 mg/mL) serum concentrations were not different along the time of study. Also, IgG concentrations in serum of foals did not show significant differences although a high level was observed at 12 hours after birth (8 mg/mL), and IgA concentrations in serum of foals did not show any significant difference, although a high level was observed at 12 hours after birth (1.2 mg/mL). Lysozyme increased significantly at Day 2 after parturition in mammary secretions of jennies (551.9 μg/mL) and at 12 hours in serum of foals (25.9 μg/mL). The study demonstrated that the pattern of passive immune transfer in donkey foals seems to be similar to that reported for the horse foal, with IgG predominating IgA in serum and mammary secretions of the jenny and also in serum of foals. The most significant early increase in foals' serum concerns lysozyme, which probably plays an important role in the innate immunity of the donkey foal in the first challenging hours after birth.

  14. Design and structural analysis of an engineered thermostable chicken lysozyme.

    PubMed Central

    Shih, P.; Kirsch, J. F.

    1995-01-01

    A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive. PMID:8535242

  15. Locations of Halide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    surrounding the protein was accurately determined. This was achieved by solving the linearized version of the Poisson-Boltzmann equation for the protein in solution. The solution was computed employing the commercial code Delphi which uses a finite difference technique. This has recently become available as a module in the general protein visualization code Insight II. Partial charges were assigned to the polar groups of lysozyme for the calculations done here. The calculations showed the complexity of the electrostatic field surrounding the protein. Although most of the region near the protein surface had a positive field strength, the active site cleft was negatively charged and this was projected a considerable distance. This might explain the occurrence of "head-to-side" interactions in the formation of lysozyme aggregates in solution. Pockets of high positive field strength were also found in the vicinity of the anion locations obtained from the crystallographic part of this study, confirming the validity of these calculations. This study clearly shows not only the importance of determining the counterion locations in protein crystals and the electrostatic fields surrounding the protein, but also the advantage of performing them together.

  16. In vitro evaluation of synergistic activity between ciprofloxacin and broad snouted caiman serum against Escherichia coli.

    PubMed

    Siroski, P A; Russi, N B; Ortega, H H; Formentini, E A

    2015-02-01

    The in vitro synergistic activity between ciprofloxacin and serum of broad snouted caiman on Escherichia coli was studied. The estimated MIC value of ciprofloxacin was 0.0188 µg/ml, and two assays of kill curve during 5 hours were performed: the first one in a standard culture medium and the second one in the presence of caiman serum. Different concentrations of ciprofloxacin were tested. Ciprofloxacin showed higher values of bacterial elimination rate in the presence of caiman serum in all concentrations tested. The combined activity of sub-inhibitory concentrations of ciprofloxacin and the humoral immune factors present in caiman serum determined an increase in the bacterial elimination observed in this assay. We suggest that the antibacterial activity of complement and natural antibodies present in caiman serum, which can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway, can inhibit bacterial growth of Escherichia coli by lysis.

  17. Identification of serum component involved in generation of neo-lectin with agglutinating and phenoloxidase activities in human serum.

    PubMed

    Manikandan, Beulaja; Ramar, Manikandan; Munusamy, Arumugam

    2014-01-01

    Human serum albumin (HSA) was identified as the component involved in generation of neo-lectin molecules with both lectin and phenoloxidase activities. Pronase treated HSA was able to agglutinate hen RBC and oxidize hydroquinone. Sodium dodecyl sulphate (SDS) treated HSA agglutinated both hen and sheep RBC as well as oxidized dopamine. The hemagglutinating activities of pronase/SDS treated HSA observed against hen RBC were dosimetric. The oxidation of pronase/SDS treated HSA with hydroquinone/dopamine, respectively, was inhibitable by inhibitors of phenoloxidase, namely, phenylthiourea and tropolone. Very low concentrations of HSA could generate these humoral neo-lectin molecules.

  18. Serum-induced platelet procoagulant activity: an assay for the characterization of prothrombotic disorders.

    PubMed

    Warner, M N; Pavord, S; Moore, J C; Warkentin, T E; Hayward, C P; Kelton, J G

    1999-02-01

    Platelets contribute to hemostasis by forming a platelet plug and by providing a procoagulant surface for the assembly and activation of the coagulation factors. The contribution of platelets to prothrombotic disorders has been difficult to analyze. Recently an assay was reported that measured the procoagulant activity of test platelets by making the platelet lipid surface the limiting factor in the production of thrombin. In this report we describe a novel technique, based on this assay, that we used to study patient serum factors that activate control platelets and in turn initiate measurable procoagulant activity. Using this assay we investigated a group of patients with prothrombotic disorders. The patient test serum was incubated with normal platelets in the presence of activated factor Xa. The resultant thrombin was measured in a chromogenic assay. The rate-limiting step was the presence of any potential platelet-activating factors, such as antibodies in the heat-treated test serum, that would allow the Xa to bind to the platelet phospholipid surface. Serum samples from patients with heparin-induced thrombocytopenia (HIT) and the anti-phospholipid antibody syndrome enhanced platelet procoagulant activity, while samples from patients with idiopathic thrombocytopenic purpura and disseminated intravascular coagulation (DIC) did not. HIT serum samples also induced platelet activation, as measured by platelet microparticle shedding, carbon 14-labeled serotonin release, and platelet aggregation. The measurement of serum-induced platelet procoagulant activity provides a method for the investigation of circulating platelet agonists in prothrombotic disorders.

  19. Bunching effect in single-molecule T4 lysozyme nonequilibrium conformational dynamics under enzymatic reactions.

    PubMed

    Wang, Yuanmin; Lu, H Peter

    2010-05-20

    The bunching effect, implying that conformational motion times tend to bunch in a finite and narrow time window, is observed and identified to be associated with substrate-enzyme complex formation in T4 lysozyme conformational dynamics under enzymatic reactions. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. On the basis of the single-molecule spectroscopic results, molecular dynamics simulation, and a random walk model analysis, multiple intermediate states have been estimated in the evolution of T4 lysozyme enzymatic reaction active complex formation (Chen, Y.; Hu, D.; Vorpagel, E. R.; Lu, H. P. Probing single-molecule T4 lysozyme conformational dynamics by intramolecular fluorescence energy transfer. J. Phys. Chem. B 2003, 107, 7947-7956). In this Article, we report progress on the analysis of the reported experimental results, and we have identified the bunching effect of the substrate-enzyme active complex formation time in T4 lysozyme enzymatic reactions. We show that the bunching effect, a dynamic behavior observed for the catalytic hinge-bending conformational motions of T4 lysozyme, is a convoluted outcome of multiple consecutive Poisson rate processes that are defined by protein functional motions under substrate-enzyme interactions; i.e., convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. We suggest that the bunching effect is likely common in protein conformational dynamics involved in conformation-gated protein functions.

  20. Serum metabolites related to cardiorespiratory fitness, physical activity energy expenditure, sedentary time and vigorous activity.

    PubMed

    Wientzek, Angelika; Floegel, Anna; Knüppel, Sven; Vigl, Matthaeus; Drogan, Dagmar; Adamski, Jerzy; Pischon, Tobias; Boeing, Heiner

    2014-04-01

    The aim of our study was to investigate the relationship between objectively measured physical activity (PA) and cardiorespiratory fitness (CRF) and serum metabolites measured by targeted metabolomics in a population- based study. A total of 100 subjects provided 2 fasting blood samples and engaged in a CRF and PA measurement at 2 visits 4 months apart. CRF was estimated from a step test, whereas physical activity energy expenditure (PAEE), time spent sedentary and time spend in vigorous activity were measured by a combined heart rate and movement sensor for a total of 8 days. Serum metabolite concentrations were determined by flow injection analysis tandem mass spectrometry (FIA-MS/MS). Linear mixed models were applied with multivariable adjustment and p-values were corrected for multiple testing. Furthermore, we explored the associations between CRF, PA and two metabolite factors that have previously been linked to risk of Type 2 diabetes. CRF was associated with two phosphatidylcholine clusters independently of all other exposures. Lysophosphatidylcholine C14:0 and methionine were significantly negatively associated with PAEE and sedentary time. CRF was positively associated with the Type 2 diabetes protective factor. Vigorous activity was positively associated with the Type 2 diabetes risk factor in the mutually adjusted model. Our results suggest that CRF and PA are associated with serum metabolites, especially CRF with phosphatidylcholines and with the Type 2 diabetes protective factor. PAEE and sedentary time were associated with methionine. The identified metabolites could be potential mediators of the protective effects of CRF and PA on chronic disease risk.

  1. CMKLR1 activation ex vivo does not increase proportionally to serum total chemerin in obese humans

    PubMed Central

    Toulany, Jay; Parlee, Sebastian D; Sinal, Christopher J; Slayter, Kathryn; McNeil, Shelly

    2016-01-01

    Prochemerin is the inactive precursor of the adipokine chemerin. Proteolytic processing is obligatory for the conversion of prochemerin into active chemerin and subsequent regulation of cellular processes via the chemokine-like receptor 1 (CMKLR1). Elevated plasma or serum chemerin concentrations and differential processing of prochemerin have been reported in obese humans. The impact of these changes on CMKLR1 signalling in humans is unknown. The objective of this pilot study was to develop a cellular bioassay to measure CMKLR1 activation by chemerin present in human serum and to characterise how obesity modifies serum activation of CMKLR1 under fasted and fed conditions. Blood samples were collected from control (N = 4, BMI 20–25) and obese (N = 4, BMI >30) female subjects after an overnight fast (n = 2) and at regular intervals (n = 7) following consumption of breakfast over a period of 6 h. A cellular CMKLR1-luminescent reporter assay and a pan-chemerin ELISA were used to determine CMKLR1 activation and total chemerin concentrations, respectively. Serum total chemerin concentration (averaged across all samples) was higher in obese vs control subjects (17.9 ± 1.8 vs 10.9 ± 0.5 nM, P < 0.05), but serum activation of CMKLR1 was similar in both groups. The CMKLR1 activation/total chemerin ratio was lower in obese vs control subjects (0.33 ± 0.04 vs 0.58 ± 0.05, P < 0.05). After breakfast, serum total chemerin or CMKLR1 activation did not differ from baseline values. In conclusion, the unexpected observation that obese serum activation of CMKLR1 did not match increased total chemerin concentrations suggests impaired processing to and/or enhanced degradation of active chemerin in serum of obese humans. PMID:27881447

  2. [Lysozyme-antilysozyme interactions in protozoa-bacteria communities (a model Tetrahymena-Escherichia community)].

    PubMed

    Bukharin, O V; Nemtseva, N V

    2001-01-01

    Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme-antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme-antilysozyme interactions in a model Tetrahymena--Escherichia community showed that the antilysozyme activity of Escherichia coli leads to incomplete phagocytosis, thus enhancing bacterial survival in a mixed culture with infusoria. The selection and reproduction of bacterial cells resistant to grazing by infusoria determine the character of host-parasite interactions and allow bacteria to survive. It was demonstrated that the antilysozyme activity of microorganisms, which is responsible for bacterial persistency in natural biocenoses, is involved in the maintenance of protozoa-bacteria communities in bodies of water.

  3. Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification.

    PubMed

    Arica, M Yakup; Yilmaz, Meltem; Yalçin, Emine; Bayramoğlu, Gülay

    2004-06-15

    Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.

  4. Influence of some formulation and process parameters on the stability of lysozyme incorporated in corn flour- or corn starch-based extruded materials prepared by melt blending processing.

    PubMed

    Jbilou, Fouzia; Galland, Sophie; Telliez, Camille; Akkari, Zied; Roux, Roselyne; Oulahal, Nadia; Dole, Patrice; Joly, Catherine; Degraeve, Pascal

    2014-12-01

    In order to obtain an antimicrobial biodegradable material, corn flour was extruded with 1% of lysozyme. Since the limited stability of natural preservatives such as lysozyme is a common bottleneck to the elaboration of active biomaterials by melt blending processes, the influence of formulation and of extrusion processing temperature on its residual enzymatic activity was investigated. To assess the contribution of process parameters such as temperature, shear stress and of related formulation parameters such as glycerol and moisture contents, the stability of lysozyme following its extrusion or its thermoforming with plasticized corn starch or thermal treatments in aqueous glycerol solutions was also studied. Increasing glycerol content from 25% to 30% significantly limited inactivation of lysozyme during extrusion, while increasing initial moisture content of the mixture from 14.5% to 28.5% had the opposite effect. These observations open the possibility to prepare active materials retaining more than 60±7% of initial lysozyme activity.

  5. Single-Molecule Measurements of T4 Lysozyme using Carbon Nanotube Electronic Circuits

    NASA Astrophysics Data System (ADS)

    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.

  6. Thermophysical properties of lysozyme (protein) solutions

    NASA Technical Reports Server (NTRS)

    Liu, Jiaching; Yang, Wen-Jei

    1992-01-01

    Thermophysical properties of protein solutions composed of the lysozyme crystals with a 0.1 M sodium acetate and 5 percent NaCl solution as the buffer (pH = 4.0) are determined. The properties being measured include specific heat, thermal conductivity, dynamic viscosity, and surface tension. The protein concentrations are varied. Thermal diffusivity is calculated using the measured results. The purpose of the research is to measure thermophysical properties of lysozyme solutions which would serve as the data bank for controlling and modeling the crystal growth process on earth as well as in space.

  7. A serum factor that activates the phosphatidylinositol phosphate signaling system in Xenopus oocytes.

    PubMed Central

    Tigyi, G; Dyer, D; Matute, C; Miledi, R

    1990-01-01

    Blood sera from many vertebrate species elicit large oscillatory chloride currents in oocytes from the frog Xenopus laevis. Rabbit serum was active at dilutions as great as one part in 10 million. Intracellularly applied serum was ineffective, and externally applied serum failed to trigger oscillatory currents when the intracellular level of ionized calcium was prevented from rising by loading the oocyte with EGTA. The serum also caused an increase of inositol 1,4,5-trisphosphate in the oocyte. We conclude that serum contains a factor which activates a membrane receptor that is coupled to the phosphatidylinositol second messenger system. The active factor is a protein with an apparent molecular mass of 60-70 kDa in gel permeation chromatography. Although the normal function of the serum factor is still unknown, it may have far-reaching implications, because it acts on the multifunctional phosphatidylinositol phosphate signaling system. Also, because of its great potency the serum factor and Xenopus oocytes are very useful for probing the operation of the phosphatidylinositol system. PMID:1689488

  8. Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme.

    PubMed

    Hardy, L W; Poteete, A R

    1991-10-01

    Replacement of Asp20 in T4 lysozyme by Cys produces a variant with (1) nearly wild-type specific activity, (2) a newly acquired sensitivity to thiol-modifying reagents, and (3) a pH-activity profile that is very similar to that of the wild-type enzyme. These results indicate that the residue at position 20 has a significant nucleophilic function rather than merely an electrostatic role. The intermediate in catalysis by lysozyme is probably a covalent glycosyl-enzyme instead of the ion pair originally proposed.

  9. Lysozyme immobilization onto PVC catheters grafted with NVCL and HEMA for reduction of bacterial adhesion

    NASA Astrophysics Data System (ADS)

    Guadarrama-Zempoalteca, Yesica; Díaz-Gómez, Luis; Meléndez-Ortiz, H. Iván; Concheiro, Angel; Alvarez-Lorenzo, Carmen; Bucio, Emilio

    2016-09-01

    The aim of the present work was to functionalize poly(vinyl chloride) (PVC) urinary catheters with grafted copolymers that can improve the biocompatibility and serve as binding points of lysozyme. PVC catheters were modified by grafting a mixture of N-vinylcaprolactam (NVCL) and 2-hydroxyethylmethacrylate (HEMA) applying a gamma-ray pre-irradiation method. The effect of absorbed dose, monomer concentration, temperature, and reaction time on the grafting percentage was evaluated. The grafted catheters were characterized regarding surface composition (FTIR-ATR spectroscopy), thermal properties (DSC and TGA) and swelling in aqueous medium. Lysozyme was directly coupled onto PVC-g-(NVCL/HEMA) previously activated using carbonyldiimidazole. Antimicrobial lytic activity of the modified catheters over time was tested against Micrococcus lysodeikticus. Lysozyme diminished the adhesion of Staphylococcus aureus onto the functionalized catheters, which may be suitable to prevent biofilm formation.

  10. The role of the confined water in the dynamic crossover of hydrated lysozyme powders.

    PubMed

    Kurzweil-Segev, Y; Greenbaum Gutina, A; Popov, I; Golodnitsky, D; Feldman, Yu

    2016-04-28

    Water is of fundamental importance for life since it plays a critical role in biological systems. An organism can only function if its macromolecules and other bioactive molecules are hydrated. However, currently there is a gap in the understanding of how protein interfaces affect water's structure and properties. This work presents combined dielectric and calorimetric measurements of hydrated lysozyme powders with different levels of hydration in a broad temperature interval. We chose lysozyme as a test sample since this globular protein has a well-defined pore with an active hydrophilic center inside. Based on the dielectric and calorimetric tests it was shown that a water quasi-solution, which contains the protein residues, has a glass transition temperature at around 155 ± 3 K. The water confined in the pore of the active center of the lysozyme has its melting temperature at around 186 ± 3 K. Melting of confined water is believed to liberate the internal motions of protein macromolecules.

  11. Expression, Purification, and Characteristic of Tibetan Sheep Breast Lysozyme Using Pichia pastoris Expression System

    PubMed Central

    Li, Jianbo; Jiang, Mingfeng; Wang, Yong

    2014-01-01

    A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and 45°C. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification. PMID:25049990

  12. Serum calreticulin as a novel biomarker of juvenile idiopathic arthritis disease activity

    PubMed Central

    Hashaad, Nashwa Ismail; Fawzy, Rasha Mohamed; Elazem, Abeer Ahmed Abo; Youssef, Mohamed Ibrahim

    2017-01-01

    Objective This study aimed to investigate the relations between calreticulin (CRT) serum level and both disease activity and severity parameters in juvenile idiopathic arthritis (JIA). Material and Methods In this study, 60 children with JIA and 50 age-and-sex-matched healthy subjects were enrolled. The assessment of the disease activity was done using juvenile arthritis disease activity score 27 (JADAS-27). The assessment of disease severity was done via gray-scale ultrasonography (US) and power Doppler US (PDUS). Enzyme-linked immunosorbent assay (ELISA) was used to assay the serum level of human CRT. Results The mean serum CRT levels in JIA patients was 8.6±1.2 ng/mL and showed a highly significant increase (p=0.001) as compared to the mean serum levels in the controls (5.02±0.77 ng/mL). There were statistically significant positive correlations between the serum CRT levels and disease duration, tender joint count, swollen joint count, visual analog scale, erythrocyte sedimentation rate, JADAS-27, C-reactive protein, rheumatoid factor titer, and ultrasonographic grading for synovitis and neovascularization. Conclusion Elevated serum CRT levels in JIA patients and its correlations with JIA disease activity and severity parameters signified that CRT might be used as a novel biomarker for disease activity and severity in JIA. PMID:28293448

  13. Serum dopamine-beta-hydroxylase activity in rat during post-natal development.

    PubMed

    Lamprecht, F; Wooten, G F

    1976-01-01

    Rat serum dopamine-beta-hydroxylase (DBH) activity is quite high in the immediate post-natal period reaching peak activity (75 units) at 16 days of age. Activity then decreases rapidly over the following weeks to approach adult levels (10 units) by seven weeks of age. Also, specific activity of DBH in heart increased rapidly during the first 2 1/2 weeks of life attaining adult levels by 18 days of age. In contrast, heart weight and total DBH activity in whole heart increased in a coordinate fashion at a relatively constant rate throughout the first seven weeks of life. Serum levels of non-copper sensitive endogenous inhibitor (s) of DBH increased throughout the first seven weeks of life while no change in copper sensitive inhibition was observed. Also, the rapid phase of decrease in serum DBH activity corresponded to the period of the most rapid increase in body weight.

  14. [Forensic-biochemical indices of serum amilase and cholinesterase activity in lethal poisoning with heroin].

    PubMed

    Gabadadze, G D; Kinle, A F

    2006-01-01

    The examination of the activity of serum amilase and cholinesterase in the blood from subjects who had died of heroin poisoning showed significant elevation of such activity. These findings allow experts to use the activity of the above enzymes as effective markers in detection of narcotic drugs in cadaveric blood, opiate narcotics, in particular.

  15. A zinc complex of heparan sulfate destabilises lysozyme and alters its conformation

    SciTech Connect

    Hughes, Ashley J.; Hussain, Rohanah; Cosentino, Cesare; Guerrini, Marco; Siligardi, Giuliano; Yates, Edwin A.; Rudd, Timothy R.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Zinc-heparan sulfate complex destabilises lysozyme, a model amyloid protein. Black-Right-Pointing-Pointer Addition of zinc, without heparan sulfate, stabilises lysozyme. Black-Right-Pointing-Pointer Heparan sulfate cation complexes provide alternative protein folding routes. -- Abstract: The naturally occurring anionic cell surface polysaccharide heparan sulfate is involved in key biological activities and is implicated in amyloid formation. Following addition of Zn-heparan sulfate, hen lysozyme, a model amyloid forming protein, resembled {beta}-rich amyloid by far UV circular dichroism (increased {beta}-sheet: +25%), with a significantly reduced melting temperature (from 68 to 58 Degree-Sign C) by fluorescence shift assay. Secondary structure stability of the Zn-heparan sulfate complex with lysozyme was also distinct from that with heparan sulfate, under stronger denaturation conditions using synchrotron radiation circular dichroism. Changing the cation associated with heparan sulfate is sufficient to alter the conformation and stability of complexes formed between heparan sulfate and lysozyme, substantially reducing the stability of the protein. Complexes of heparan sulfate and cations, such as Zn, which are abundant in the brain, may provide alternative folding routes for proteins.

  16. Serum thiols as a biomarker of disease activity in lupus nephritis.

    PubMed

    Lalwani, Pritesh; de Souza, Giselle Katiane Bonfim Bacelar; de Lima, Domingos Savio Nunes; Passos, Luiz Fernando Souza; Boechat, Antonio Luiz; Lima, Emerson Silva

    2015-01-01

    Lupus Nephritis (LN) develops in more than half of the Systemic Lupus Erythematous (SLE) patients. However, lack of reliable, specific biomarkers for LN hampers clinical management of patients and impedes development of new therapeutics. The goal of this study was to investigate whether oxidative stress biomarkers in patients with SLE is predictive of renal pathology. Serum biochemical and oxidative stress markers were measured in patients with inactive lupus, active lupus with and without nephritis and compared to healthy control group. To assess the predictive performance of biomarkers, Receiver Operating Characteristic (ROC) curves were constructed and cut-offs were used to identify SLE patients with nephritis. We observed an increased oxidative stress response in all SLE patients compared to healthy controls. Among the several biomarkers tested, serum thiols had a significant inverse association with SLE Disease Activity Index (SLEDAI). Interestingly, thiols were able too aptly differentiate between SLE patients with and without renal pathology, and serum thiol levels were not affected by immunosuppressive drug therapy. The decreased thiols in SLE correlated significantly with serum creatinine and serum C3 levels. Further retrospective evaluation using serum creatinine or C3 levels in combination with thiol's cutoff values from ROC analysis, we could positively predict chronicity of renal pathology in SLE patients. In summary, serum thiols emerge as an inexpensive and reliable indicator of LN, which may not only help in early identification of renal pathology but also aid in the therapeutic management of the disease, in developing countries with resource poor settings.

  17. Judging disease activity in rheumatoid arthritis by serum free kappa and lambda light chain levels.

    PubMed

    Ye, Yun; Li, Su-Liang; Xie, Ming; Jiang, Ping; Liu, Kai-Ge; Li, Ya-Jun

    2013-10-01

    The study aimed to evaluate the levels of serum free kappa (κ) and lambda (λ) light chains in patients with rheumatoid arthritis (RA) as well as exploring the association between serum free κ and λ light chains and activity of RA. For this purpose, healthy individuals and patients with active RA and RA in remission were enrolled, and their serum levels of free κ and λ light chains were measured using rate nephelometry. The diagnostic accuracy of serum free κ and λ light chains was evaluated by receiver operating characteristic curves and 95% confidence intervals for areas under the curve (AUC). The results obtained indicated that the levels of serum free κ and λ light chains in patients with active RA were significantly higher than those of patients in remission and of healthy controls (p < 0.05). Further, the AUC values in patients with active RA were 0.871 for free κ light chain and 0.781 for free λ light chain. When the optimal cut-off point for serum κ light chain was 8.02 g/L, the maximum sensitivity and specificity were 82.5% and 82.5%, respectively, and when the optimal cut-off point for serum λ light chain was 3.57 g/L, the maximum sensitivity and specificity were 80% and 82.5%, respectively. It was thus found that serum levels of free κ and λ light chains were positively correlated with disease activity in RA, the Disease Activity Score 28 (DAS28), and values for C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count (PLT), rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) (p < 0.05). In conclusion, high serum levels of free κ and λ light chains in patients with active RA are closely correlated with disease activity parameters including DAS28, CRP, ESR, PLT, RF, and ACPA. Thus, the above-mentioned levels of serum free κ and λ light chains may be used as important indicators of activity of RA.

  18. Monolithic molecularly imprinted cryogel for lysozyme recognition.

    PubMed

    Rabieizadeh, Mohammadmahdi; Kashefimofrad, Seyed Mohammadreza; Naeimpoor, Fereshteh

    2014-10-01

    The application of molecularly imprinted polymers in the selective adsorption of macromolecules such as proteins by monolithic protein-imprinted columns requires a macroporous structure, which can be provided by cryogelation at low temperature in which the formation of ice crystals gives a porous structure to the molecularly imprinted polymer. In this study, we applied this technique to synthesize lysozyme-imprinted polyacrylamide cryogels containing 8% w/v of total monomers and 0.3% w/v of lysozyme. The synthesized cryogel was sponge-like and elastic with very fast swelling and reshaping properties, showing a swelling ratio of 24.5 ± 3 and gel fraction yield of about 72%. It showed an imprinting effect of 1.58 and a separation factor of 1.37 for cytochrome c as the competing protein. Adsorption studies on the cryogel revealed that it follows the Langmuir isotherm, with a maximum theoretical adsorption capacity of 36.3 mg lysozyme per gram of cryogel. Additionally, it was shown that a salt-free rebinding solution at low flow rate and pH = 7.0 is favorable for lysozyme rebinding. This kind of monolithic column promises a wide range of application in separation of various biomolecules due to its preparation simplicity, good rebinding characteristics, and macroporosity.

  19. Crystal structures of pheasant and guinea fowl egg-white lysozymes.

    PubMed Central

    Lescar, J.; Souchon, H.; Alzari, P. M.

    1994-01-01

    The crystal structures of pheasant and guinea fowl lysozymes have been determined by X-ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6(1)22 with cell dimensions a = 89.2 A and c = 61.7 A. The structure was refined to a final crystallographic R-factor of 17.0% for 8,854 observed reflections in the resolution range 6-1.9 A. Crystals of pheasant lysozyme are tetragonal, space group P4(3)2(1)2, with a = 98.9 A, c = 69.3 A and 2 molecules in the asymmetric unit. The final R-factor is 17.8% to 2.1 A resolution. The RMS deviation from ideality is 0.010 A for bond lengths and 2.5 degrees for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N-terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3-dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the C alpha atom of Gly 41 and absent in lysozymes from other species (which have a large side-chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other c-type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody-bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen-antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation in c-type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes. PMID:8061608

  20. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

    PubMed Central

    Jońca, Joanna; Żuk, Monika; Wasąg, Bartosz; Janaszak-Jasiecka, Anna; Lewandowski, Krzysztof; Wielgomas, Bartosz; Waleron, Krzysztof; Jasiecki, Jacek

    2015-01-01

    Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. PMID:26444431

  1. Production and Ultrastructure of Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli1

    PubMed Central

    Birdsell, D. C.; Cota-Robles, E. H.

    1967-01-01

    Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 m sucrose in 0.01 m tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 m also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment. Images PMID:4960155

  2. The protective effect of salicylic acid on lysozyme against riboflavin-mediated photooxidation

    NASA Astrophysics Data System (ADS)

    Li, Kun; Wang, Hongbao; Cheng, Lingli; Zhu, Hui; Wang, Mei; Wang, Shi-Long

    2011-06-01

    As a metabolite of aspirin in vivo, salicylic acid was proved to protect lysozyme from riboflavin-mediated photooxidation in this study. The antioxidative properties of salicylic acid were further studied by using time-resolved laser flash photolysis of 355 nm. It can quench the triplet state of riboflavin via electron transfer from salicylic acid to the triplet state of riboflavin with a reaction constant of 2.25 × 10 9 M -1 s -1. Mechanism of antioxidant activities of salicylic acid on lysozyme oxidation was discussed. Salicylic acid can serve as a potential antioxidant to quench the triplet state of riboflavin and reduce oxidative pressure.

  3. Structural study of lysozyme in two ionic liquids at cryogenic temperature

    NASA Astrophysics Data System (ADS)

    Yoshimura, Yukihiro; Takekiyo, Takahiro; Mori, Takahiro

    2016-11-01

    We have investigated the structure and activity of chicken egg-white lysozyme in aqueous solutions of two typical ionic liquids, 1-butyl-3-methylimidazolium chloride and ethylammonium nitrate, at cryogenic temperature. An increase in structural disorder due to the unfolding and a decrease in the α-helical structure of lysozyme were noticeable upon glass formation. However, a decrease in the structural stability after cooling was less than that before cooling. The secondary and tertiary structures showed good reversibility upon cooling to 77 K and then reverting back to ambient temperature. We discussed an influence of a cooling upon the structure in aqueous ionic liquid solutions.

  4. Activation peptide of carboxypeptidase B in serum and urine in acute pancreatitis

    PubMed Central

    Appelros, S; Thim, L; Borgstrom, A

    1998-01-01

    Background—The pathophysiology of acute pancreatitis involves activation of the pancreatic proenzymes. Levels of the trypsinogen activation peptide in urine in acute pancreatitis has been shown to correlate with the severity of disease. However, this peptide is unstable in urine and, because of its low molecular mass, difficult to measure. Procarboxypeptidase B has a larger activation peptide which could be more suitable for analysis in serum and urine. 
Aims—To study the presence of the activation peptide from procarboxypeptidase B (CAPAP) in serum and urine in acute pancreatitis. 
Patients—Urine and serum samples were obtained within 48 hours of admittance from 40 patients with acute pancreatitis. Severity was classified retrospectively according to levels of C-reactive protein and clinical course. Thirty four patients with abdominal pain from other causes were studied as controls. 
Methods—CAPAP was purified from human pancreatic juice. Specific antibodies were obtained and a radioimmunoassay was developed. 
Results—Levels of CAPAP in serum and urine in acute pancreatitis correlate with the severity of the attack. CAPAP is very stable, and urine contains only CAPAP whereas, in serum, cross reacting procarboxypeptidase B is found together with CAPAP. 
Conclusions—CAPAP could be a valuable tool in the diagnosis and early determination of severity in acute pancreatitis. 

 Keywords: carboxypeptidase B; activation peptide; acute pancreatitis PMID:9505893

  5. Serum paraoxonase 1 activity in patients with iron deficiency anemia

    PubMed Central

    Gedikbasi, Asuman; Akalin, Nilgul; Gunaldi, Meral; Yilmaz, Deniz; Mert, Meral; Harmankaya, Ozlem; Soylu, Aliye; Karakaya, Pinar; Kumbasar, Abdulbaki

    2016-01-01

    Introduction In this study we aimed to detect paraoxonase 1 (PON-1) activity in iron deficiency anemia (IDA) and to compare it with healthy controls by observing the change after iron therapy. Material and methods In this study, 50 adult patients with IDA and 40 healthy subjects were enrolled. All patients were analyzed at the beginning and after treatment according to laboratory assessments. Results Mean paraoxonase and arylesterase activities in the iron deficiency anemia group were significantly lower than mean activities of the control group (102.4 ±19.2 U/l and 163.3 ±13.68 U/l, respectively and 157.3 ±26.4 U/l and 256.1 ±24.6 U/l, respectively; p = 0.0001 for both). Paraoxonase and arylesterase activities significantly increased after treatment for IDA (143.2 ±13.9 and 197.6 ±27.9 U/l, respectively, p = 0.0001). Mean activities after treatment with iron were significantly lower than mean activities in the control group (p = 0.002; p = 0.0001 respectively). Conclusions Paraoxonase and arylesterase activities in patients with IDA significantly increased after treatment with iron therapy. In adults IDA may also be one of the factors associated with increased risk of atherosclerosis. PMID:27478448

  6. Photoactive terthiophenes: the influence of serum on anti-HIV (human immunodeficiency virus) activities.

    PubMed

    Hudson, J B; Marles, R J; Soucy-Breau, C; Harris, L; Arnason, J T

    1994-12-01

    A number of carboxylic acid derivatives of the photoactive terthiophene, alpha-terthienyl, were found to possess impressive UVA-dependent activity against the human immunodeficiency virus, HIV-1; but only when assayed in the absence of serum, indicating that the latter contained interfering components. Good antiviral activity required a high rate of singlet oxygen production, in accordance with previous observations on thiophenes.

  7. Characterization of phospholipase A(2) activity in serum of the American alligator (Alligator mississippiensis).

    PubMed

    Merchant, Mark; Heard, Raphiel; Monroe, Caleb

    2009-11-01

    PLA(2) is a diverse class of enzymes with a broad spectrum of physiological functions. Secretory PLA(2) isoforms have been reported to exhibit important innate immune function in higher vertebrates. This study was conducted to characterize PLA(2) activity in the serum of the American alligator (Alligator mississippiensis). We used a glycerophospholipid with a fatty acid in the sn-2 position labeled with a fluorescent probe (BODIPY) to detect and quantify alligator serum PLA(2) activity. Incubation of BODIPY-labeled bacteria with different concentrations of alligator serum resulted in a concentration-dependent detection of PLA(2) activity. Kinetic studies showed that product formation was rapid, with substantial activity within 5 min, and maximal activity at approximately 20 min. The alligator PLA(2) activity was temperature-dependent, with activity at lower temperatures (5-10 degrees C) approximately half of that observed at temperatures of 30-40 degrees C. In addition, the generation of fluorescent product was reduced by a specific inhibitor (p-bromophenacyl bromide) of secretory PLA(2) in a concentration-dependent manner, enforcing the idea that the observed activities are due to a secretory PLA(2) enzyme in alligator serum.

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

    PubMed

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

    2015-01-01

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

  9. Proteolytic activity of IgGs from blood serum of Wistar rats at experimental rheumatoid arthritis.

    PubMed

    Kit, Yu Ya; Myronovsky, S L; Kril, I I; Havrylyuk, A M; Chop'yak, V V; Stoika, R S

    2014-01-01

    The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  10. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  11. Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity.

    PubMed Central

    Tomás, J M; Benedí, V J; Ciurana, B; Jofre, J

    1986-01-01

    The ability of Klebsiella pneumoniae strains to resist the bactericidal activity of serum was quantitated. The K. pneumoniae strains tested included mutants lacking the capsular polysaccharide and mutants having a modified lipopolysaccharide structure. The last mutants were obtained as phage-resistant mutants, and their lipopolysaccharide was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chemical analysis. Serum-resistant mutants derived from phage-resistant mutants (lipopolysaccharide mutants) were also characterized. Resistance to the bactericidal activity of complement was mediated by the lipopolysaccharide, especially by the O-antigen polysaccharide chains. The capsular polysaccharide seemed not to play any important role in resistance to serum bactericidal activity in this bacterium. Images PMID:3531020

  12. Activation of muscarinic K+ current in guinea-pig atrial myocytes by a serum factor.

    PubMed Central

    Banach, K; Hüser, J; Lipp, P; Wellner, M C; Pott, L

    1993-01-01

    1. Atrial myocytes obtained by enzymatic perfusion of hearts from adult guinea-pigs and cultured for 0-14 days were studied using the whole-cell voltage-clamp technique. 2. Superfusion of the myocytes with diluted sera (1:100 to 1:10,000) from different species (human, horse, guinea-pig) evoked an inward rectifying K+ current. The voltage-dependent properties of this current were identical to those of the K+ current activated by acetylcholine (IK(ACh)). Current density in the presence of horse serum (1:100) approximately corresponded to the non-desensitizing fraction of IK(ACh) during superfusion with 1-2 x 10(-6) M ACh. 3. During a maximal serum-evoked current, application of ACh (10(-6) M) failed to evoke additional K+ current. After switching superfusion from serum-containing to serum-free solution, the K+ current decayed 1-2 orders of magnitude slower than ACh-activated IK(ACh). During the decay of the serum-evoked current, a proportional increase in responsiveness to ACh was recorded. During submaximal activation of K+ current by serum, a saturating concentration of ACh resulted in a total current that was identical to the current evoked by ACh alone minus the desensitizing component. Thus, activation of K+ current by serum caused desensitization of IK(ACh). From these results it is concluded that sera contain a factor that activates the same population of K+ channels as ACh. 4. Irreversible activation of IK(ACh) by ACh in myocytes dialysed with the GTP-analogue GTP-gamma-S abolished sensitivity to serum and vice versa. 5. The effect of serum was not modified by atropine (10(-6) M) which completely blocked the response to 2 x 10(-6) M ACh. Furthermore, theophylline (1 mM), which completely inhibited IK(ACh) activation by adenosine (100 microM), failed to inhibit the effect of serum. Thus, neither muscarinic nor purinergic (A1) receptors are involved. 6. The peptide somatostatin (10(-6) M) and the alpha 1-agonist phenylephrine (1 microM) which previously have

  13. Silver metallation of hen egg white lysozyme: X-ray crystal structure and NMR studies.

    PubMed

    Panzner, Matthew J; Bilinovich, Stephanie M; Youngs, Wiley J; Leeper, Thomas C

    2011-12-14

    The X-ray crystal structure, NMR binding studies, and enzyme activity of silver(I) metallated hen egg white lysozyme are presented. Primary bonding of silver is observed through His15 with secondary bonding interactions coming from nearby Arg14 and Asp87. A covalently bound nitrate completes a four coordinate binding pocket.

  14. Frequent hard physical activity lowered serum beta-carotene level in a population study of a rural city of Japan.

    PubMed

    Takatsuka, N; Kawakami, N; Ohwaki, A; Ito, Y; Matsushita, Y; Ido, M; Shimizu, H

    1995-07-01

    To determine the effect of physical activity on serum beta-carotene, we analyzed data about life styles including 3-day food records and blood samples collected from 57 men and 74 women in a rural city of Japan. Physical activity was asked as mean frequency of hard physical activities per week last year. A declining trend in serum beta-carotene was observed with increasing frequency of hard physical activities in men. In multiple regression analyses, the frequency of hard physical activities showed a negative partial correlation coefficient (r = -0.38, p = 0.007) with serum beta-carotene in men when controlled by age, BMI (body mass index), dietary factors (carotene intake, alcohol consumption and vitamin supplements use), smoking status, serum total cholesterol and serum triglycerides. These results suggest that frequent hard physical activity decreases serum beta-carotene especially in men.

  15. [Modifying effect of the blood serum from patients with soft-tissue sarcomas on interleukin-2 production and activity].

    PubMed

    Goretskiĭ, B A; Berezhnaia, N M; Palivets, A Iu; Konovalenko, V F

    1989-01-01

    Interleukin-2 (IL-2) production by lymphocytes of a peripheral blood (LPB) in patients with soft tissue sarcomas is considerably lower in comparison with that in donors. Autological serum had either suppressive or stimulative effect on LPB IL-2 production in the observed patients. The investigated serum affected LPB of donors in the same way. Serum of patients with soft tissue sarcomas had the same dual effect on the ability of the active drug IL-2 to proliferate human T-lymphoblasts. Moreover serum-inhibitors of production acted as IL-2 activity stimulators, while serum-stimulators of IL-2 production inhibited the mediator effects.

  16. Serum levels of lipids, lipoproteins and paraoxonase activity in pre-eclampsia.

    PubMed

    Demir, B; Demir, S; Atamer, Y; Guven, S; Atamer, A; Kocyigit, Y; Hekimoglu, A; Toprak, G

    2011-01-01

    Serum paraoxonase 1 (PON1) activity and the oxidation of lipoproteins were investigated in 35 women with pre-eclampsia and in 35 healthy control women with normal pregnancies. Blood pressure, body mass index (BMI), serum levels of total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and lipoprotein (a) (Lp[a]), and PON1 activity were assessed. There were no significant between-group differences in subject age, gestational age at diagnosis of pre-eclampsia, BMI, serum total cholesterol, triglycerides, LDL and ApoB levels. Mean systolic and diastolic blood pressures and serum Lp(a) were significantly higher in subjects with pre-eclampsia than in controls. Mean serum HDL, ApoA1 and PON1 activity were significantly lower in subjects with pre-eclampsia compared with controls. In conclusion, lipids and oxidized lipoproteins may play important roles in the pathogenesis of pre-eclampsia.

  17. Preparation of anionic polyelectrolyte modified magnetic nanoparticles for rapid and efficient separation of lysozyme from egg white.

    PubMed

    Chen, Jia; Lin, Yuexin; Jia, Li

    2015-04-03

    Poly(sodium 4-styrenesulfonate) modified magnetic nanoparticles (PSS-MNPs) were successfully synthesized and characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, vibrating sample magnetometry, and Fourier-transform infrared spectrometry. The PSS-MNPs were found to enable effective separation of lysozyme from egg white. The impacts of solution pH, ionic strength, and contact time on the adsorption process were investigated. The adsorption kinetic data were well fitted using a pseudo-second-order kinetic model and the adsorption equilibrium can be reached in 3 min. The adsorption isotherm data could be well described by the Langmuir equation. The maximum adsorption capacity of PSS-MNPs for lysozyme was calculated to be 476.2 mg g(-1) according to the Langmuir adsorption isotherm. The fast and efficient adsorption of lysozyme by PSS-MNPs was mainly based on electrostatic interactions between them. The adsorbed lysozyme can be eluted using 20mM phosphate buffer (pH 7.0) containing 1.0M NaCl with a recovery of 96%. The extracted lysozyme from egg white demonstrated high purity, retaining about 90.7% of total lysozyme activity.

  18. 25(OH) vitamin D serum values and rheumatoid arthritis disease activity (DA S28 ESR)

    PubMed Central

    Sahebari, Maryam; Mirfeizi, Zahra; Rezaieyazdi, Zahra; Rafatpanah, Houshang; Goshyeshi, Ladan

    2014-01-01

    Background: The role of vitamin D in the pathogenesis of rheumatoid arthritis is under investigation. This study was designed to evaluate the correlation between serum values of 25(OH) vitamin D [25(OH)D] and disease activity in rheumatoid arthritis (RA) patients according to Disease Activity Score 28 joints and ESR (DA S28 ESR). Methods: Ninety-nine patients according to ACR classification criteria for RA and 68 healthy controls were included in this study. The participants with known confounding risk factors affecting serum values of 25(OH)D were excluded. All patients were under treatment with supplementary calcium carbonate (1500mg), 25(OH)D (800U), and Hydroxychloroquine (6mg/kg). The control group was mostly recruited from patients’ relatives who lived with them to minimize the impact of diverse lifestyles on 25(OH)D status. Disease activity was assessed by DA S28 ESR. Serum concentrations of 25(OH)D were measured. Serum values of 25(OH)D less than 50 nmol/L were considered 25(OH)D deficiency. Results: The mean 25(OH)D serum values were 83.74±46.45 nmol/L in patients and 46.53±34.07 nmol/L in controls. After adjustment for age, sex and BMI, multivariate analysis showed no correlation between 25(OH)D serum levels and DAS in RA (P=0.29, rp=0.11). However, 25(OH)D serum values were significantly lower in patients with early diagnosed RA compared with the other patients (p=0.012). In the early diagnosed patients, 25(OH)D and anti-CCP serum values were negatively correlated (P=0.04, rs=-0.5). Conclusion: This study showed that there was no correlation between 25(OH)D serum values and DAS over a short duration of disease course. However, in early RA, 25(OH) D serum values were lower than the established RA. PMID:25202442

  19. Preparation of lysozyme molecularly imprinted polymers and purification of lysozyme from egg white.

    PubMed

    Wang, Xuejiao; Dong, Shaohua; Bai, Quan

    2014-06-01

    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.

  20. Elasticity and Strength of Biomacromolecular Crystals: Lysozyme

    NASA Technical Reports Server (NTRS)

    Holmes, A. M.; Witherow, W. K.; Chen, L. Q.; Chernov, A. A.

    2003-01-01

    The static Young modulus, E = 0.1 to 0.5 GPa, the crystal critical strength (sigma(sub c)) and its ratio to E,sigma(sub c)/E is approximately 10(exp 3), were measured for the first time for non cross-linked lysozyme crystals in solution. By using a triple point bending apparatus, we also demonstrated that the crystals were purely elastic. Softness of protein crystals built of hard macromolecules (26 GPa for lysozyme) is explained by the large size of the macromolecules as compared to the range of intermolecular forces and by the weakness of intermolecular bonds as compared to the peptide bond strength. The relatively large reported dynamic elastic moduli (approximately 8 GPa) from resonance light scattering should come from averaging over the moduli of intracrystalline water and intra- and intermolecular bonding.

  1. Dynamics of Lysozyme in Trehalose solutions

    NASA Astrophysics Data System (ADS)

    Ghatty, Pavan; Uberbacher, Edward C.

    2008-03-01

    Anhydrobiosis in Tardigrades and Nematodes has been a topic of constant interest and intrigue in the scientific community. An increase in the concentration of Trehalose has been attributed to the ability of some organisms to survive extreme conditions of temperature, pressure and pH. Although there exist many experimental studies attributing this effect to Trehalose, the molecular details governing the interaction between Trehalose and proteins remains unclear. We have conducted a 20ns study of Lysozyme in varying concentrations of Trehalose in water. Strong and weak hydrogen bonds and hydrophobic interactions between water, Trehalose and protein seem to dictate the interactions in the system. We have observed a hydrogen bonded network of Trehalose around the protein entrapping a layer of water between itself and protein. Lysozyme remains in a near-native conformation throughout the simulation giving hints on the ability of Trehalose in preserving the structure of protiens.

  2. Membrane effects of lysozyme amyloid fibrils.

    PubMed

    Kastorna, Anna; Trusova, Valeriya; Gorbenko, Galyna; Kinnunen, Paavo

    2012-04-01

    The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.

  3. [Method of determining tissue renin activity using heterologous serum].

    PubMed

    Orbetsova, V Ts; Kiprov, D

    1979-01-01

    The authors described a method for determination of tissue renin activity with heterologous substrate. The preparation of the substrate was performed at several stages: salting with amonium sulfate; dialisis of the precipitate till complete separation of amonium sulfate molecules; distruction of angiotensinases by interchangeble souring and alcalization of the medium; lyophylization of the pure substrate. The obtained renin-substrate was preserved in ampules and its usage had a series of advantages--duration, economic, a possibility for standartization of the determination, etc., which were described in details in the article. The described in details also the quantitative determination of the renin activity in the tissues (renal and cerebral) with the help of the obtained substrate as the moments, modiied by the authors, were indicated.

  4. Growth and dissolution kinetics of tetragonal lysozyme

    NASA Technical Reports Server (NTRS)

    Monaco, L. A.; Rosenberger, F.

    1993-01-01

    The growth and dissolution kinetics of lysozyme in a 25 ml solution bridge inside a closed growth cell was investigated. It was found that, under all growth conditions, the growth habit forming (110) and (101) faces grew through layer spreading with different growth rate dependence on supersaturation/temperature. On the other hand, (100) faces which formed only at low temperatures underwent a thermal roughening transition around 12 C.

  5. Evaluation of serum antioxidant activity in patients with liver pathology by the chemiluminescent method.

    PubMed

    Titov, V N; Sazhina, N N

    2014-12-01

    Total antioxidant activity of the serum in patients with liver pathology was assessed by two chemiluminescent methods based on different models of free radical oxidation: Hb-H2O2-luminol and ABAP-luminol. Comparative analysis showed a significant, but not high correlation of the results (r=0.798), which can be explained by different mechanisms of induction of free radicals and effects of various serum components (proteins and bilirubin) on the initiation process. The influence of aphysiological concentration of analyzed values manifested in the Hb-H2O2 model. Disagreement between the results of measurements was more pronounced in patients with abnormally high serum bilirubin content. The results suggested that ABAP-luminol chemiluminescent model is more preferable for evaluation of antioxidant activity in clinical practice.

  6. THz characterization of lysozyme at different conformations

    NASA Astrophysics Data System (ADS)

    Globus, Tatiana; Khromova, Tatyana; Lobo, Rebecca; Woolard, Dwight; Swami, Nathan; Fernandez, Erik

    2005-05-01

    This work demonstrates application of Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm-1 to discriminate between different protein conformations and evaluate possible application of THz spectroscopy for monitoring of protein folding-unfolding process. A specific procedure developed earlier for unfolding lysozyme by salt (KSCN) precipitation and refolding the lysozyme molecules by removing of KSCN and dissolving in sodium acetate was used to prepare three different forms of lysozyme. In addition, two standard procedures were used to prepare samples in unfolded conformation: denaturation at high temperature ~95° C followed by fast freezing, and dissolution in 6 M guanidine. Thin, air dried protein films were characterized as well as material in the form of gel. Spectra reveal resonance features in transmission which represent vibrational modes in the protein samples. A great variability of spectral features for the different conformational states showed the sensitivity of vibrational frequencies to the three dimensional structure of proteins. The results obtained on liquid (gel) samples indicate that THz transmission spectroscopy can be used for monitoring folding-unfolding process in a realistic, aqueous environment.

  7. Lysozyme binds onto functionalized carbon nanotubes.

    PubMed

    Bomboi, Francesca; Tardani, Franco; Gazzoli, Delia; Bonincontro, Adalberto; La Mesa, Camillo

    2013-08-01

    Single walled carbon nanotubes have singular physicochemical properties making them attractive in a wide range of applications. Studies on carbon nanotubes and biological macromolecules exist in literature. However, ad hoc investigations are helpful to better understand the interaction mechanisms. We report on a system consisting of single walled carbon nanotubes and lysozyme. The phenomenology of nanotube-protein interactions and its effects on protein conformation were determined. We investigated the formation of oxidized nanotube-lysozyme conjugates, by studying the effect of both protein concentration and pH. Electrophoretic mobility, dielectric spectroscopy and dynamic light scattering were used to determine the interaction pathways, monitoring the surface charge density and the size of the complexes. The results allowed identifying the conditions of surface saturation at different pH values. The secondary structure of nanotube-adsorbed protein was controlled by circular dichroism; it was observed that it substantially retains its native conformation. Interestingly, we found that the interactions among oxidized nanotubes and lysozyme molecules are mainly of electrostatic nature and easily tunable by varying the pH of the solutions.

  8. Lysozyme binding onto cat-anionic vesicles.

    PubMed

    Bonincontro, A; Spigone, E; Ruiz Peña, M; Letizia, C; La Mesa, C

    2006-12-15

    Mixing aqueous sodium dodecylsulfate with cetyltrimethylammonium bromide solutions in mole ratios close to (1.7/1.0) allows the formation of cat-anionic vesicles with an excess of negative charges on the outer surface. The vesicular dispersions are mixed with lysozyme, and interact electrostatically with the positive charges on the protein, forming lipo-plexes. Dielectric relaxation, zeta-potential, and light scattering indicate the occurrence of interactions between vesicles and the protein. According to CD, the vesicle-adsorbed protein retains its native conformation. Binding and surface saturation, inferred by dielectric relaxation and zeta-potential, fulfil a charge neutralisation stoichiometry. Adsorbed lysozyme promotes the vesicle clustering and is concomitant with the lipo-plexes flocculation. Above the charge neutralisation threshold, lysozyme in excess remains dispersed in molecular form. Attempts were made to determine in what conditions protein release from the vesicles occurs. Accordingly, the full neutralisation of sodium dodecylsulfate in excess by cetyltrimethylammonium bromide ensures the lipo-plexes break-up, the precipitation of the mixed surfactants and the protein release in native form.

  9. Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein

    PubMed Central

    Dostal, Sarah M.; Fang, Yongliang; Guerrette, Jonathan C.; Scanlon, Thomas C.; Griswold, Karl E.

    2015-01-01

    The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed towards chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial co-culture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors. PMID:25607237

  10. Activation of the human neutrophil respiratory burst with zymosan-activated serum.

    PubMed

    Smith, R J; Iden, S S; Bowman, B J

    1984-06-15

    Zymosan-activated serum ( ZAS ) stimulated a time- and concentration-dependent generation of superoxide anion (O-2) by human neutrophils. O-2 production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS . O-2 generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS . The amount of O-2 produced by ZAS stimulated neutrophils was enhanced in the presence of extra-cellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS -elicited O-2 production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O-2 in response to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC),N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), demonstrates the stimulus specific nature of ZAS -induced desensitization. Thus, ZAS , which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represents a relevant probe for investigating neutrophil function.

  11. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    SciTech Connect

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  12. Low-frequency spectroscopic analysis of monomeric and fibrillar lysozyme.

    PubMed

    Zakaria, Hidayatul A; Fischer, Bernd M; Bradley, Andrew P; Jones, Inke; Abbott, Derek; Middelberg, Anton P J; Falconer, Robert J

    2011-03-01

    Terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared (FT-IR) spectroscopy were used to generate far-infrared and low-frequency spectral measurements of monomeric lysozyme and lysozyme fibrils. The formation of lysozyme fibrils was verified by the Thioflavin T assay and transmission electron microscopy (TEM). It was evident in the FT-IR spectra that between 150 and 350 cm(-1) the two spectra diverge, with the lysozyme fibrils showing higher absorbance intensity than the monomeric form. The broad absorption phenomenon is likely due to light scattered from the fibrillar architecture of lysozyme fibrils as supported by simulation of Rayleigh light scattering. The lack of discrete phonon-like peaks suggest that far-infrared spectroscopy cannot detect vibrational modes between the highly ordered hydrogen-bonded beta-pleated sheets of the lysozyme subunit.

  13. Direct evidence for interaction between human erythroid progenitor cells and a hemoglobin switching activity present in fetal sheep serum.

    PubMed Central

    Stamatoyannopoulos, G; Nakamoto, B; Kurachi, S; Papayannopoulou, T

    1983-01-01

    An activity that induces Hb F to Hb A switching in human cells is present in fetal sheep serum. To test directly the role of cell-to-environment interactions in hemoglobin switching and to define the level of erythroid cell differentiation at which this activity operates, colony transfer experiments were done. Clones grown in the presence of switching activity-containing medium (fetal sheep serum) or control medium (fetal calf serum) were transferred, at the 16- to 30-cell stage, to either fetal sheep serum or fetal calf serum plates and Hb F synthesis was determined in the fully mature erythroid bursts. Fetal calf serum-to-fetal calf serum transfers produced colonies with the high Hb F levels characteristic of undisturbed fetal calf serum-grown clones. Fetal sheep serum-to-fetal calf serum transfers resulted in significant decrease in Hb F synthesis, revealing an interaction between hemoglobin switching activity and cells at an early stage of progenitor cell development. The reduction of Hb F synthesis in fetal calf serum-to-fetal sheep serum transfers indicated that hemoglobin switching activity interacts with cells at later stages of progenitor cell development. Maximal decrease in Hb F synthesis was observed in fetal sheep serum-to-fetal sheep serum transfers, indicating that optimal effects on Hb switching are obtained when the environment that induces Hb switching is present throughout the development of progenitor cells. By splitting single early clones into two parts and transferring them to either a fetal sheep serum or a fetal calf serum environment, these interactions were further demonstrated in the progeny of a single erythroid burst-forming unit. Since all clone transfers were done on cell-free plates, the results of fetal calf serum-to-fetal sheep serum and of fetal sheep serum-to-fetal sheep serum transfers indicated that the switching activity does not require helper cells for its action. These studies show directly that (i) Hb F synthesis is

  14. Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity.

    PubMed

    Tymecki, Łukasz; Strzelak, Kamil; Koncki, Robert

    2013-10-03

    A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.

  15. Dose-dependent effect of lysozyme upon Candida albicans biofilm

    PubMed Central

    Sebaa, Sarra; Hizette, Nicolas; Boucherit-Otmani, Zahia; Courtois, Philippe

    2017-01-01

    The present study investigated the in vitro effect of lysozyme (0–1,000 µg/ml) on Candida albicans (C. albicans) biofilm development. Investigations were conducted on C. albicans ATCC 10231 and on 10 clinical isolates from dentures. Strains were cultured aerobically at 37°C in Sabouraud broth. Yeast growth was evaluated by turbidimetry. Biofilm biomass was quantified on a polystyrene support by crystal violet staining and on acrylic surfaces by counts of colony forming units. Lysozyme affected biofilm formation to a greater extent than it affected growth. For the ATCC 10231 reference strain, lysozyme acted as a biofilm promotor on polystyrene at the highest concentration tested (1,000 µg/ml, non-physiological). When the reference strain was investigated on acrylic resin support, lysozyme acted as a significant biofilm promotor on rough resin, but less on smooth resin. The attached biomass in the presence of physiological concentrations of lysozyme (10–30 µg/ml) was significantly decreased compared with the hypothetical value of 100% using a one-sample t-test, but a comparison between the different lysozyme conditions using analysis of variance and post hoc tests did not reveal significant differences. In 10 wild strains, different patterns of biofilm formation on polystyrene were observed in the presence of lysozyme. Some strains, characterized by large amounts of biofilm formation in the presence of 1,000 µg/ml lysozyme, were poor biofilm producers at low concentrations of lysozyme. In contrast, some strains that were poor biofilm producers with a high lysozyme concentration were more inhibited by low concentrations of lysozyme. The present study emphasizes the need to develop strategies for biofilm control based on in vitro experiments, and to implement these in clinical trials prior to approval of hygiene products enriched with exocrine proteins, such as lysozyme. Further studies will extend these investigations to other Candida species, and to fungi

  16. The activity of lysosomal exoglycosidases in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E.

    PubMed

    Zaniewska, Agnieszka; Borzym-Kluczyk, Malgorzata; Szajda, Slawomir D; Romatowski, Jacek; Gil, Andrzej; Knas, Malgorzata; Dobryniewski, Jacek; Zwierz, Krzysztof

    2009-08-01

    The aim of this study was to determine the activity of the lysosomal exoglycosidases: alpha-mannosidase (MAN), alpha-fucosidase (FUC), and beta-glucuronidase (GLUCUR) in serum of alcohol-dependent men supplemented and not supplemented with borage oil enriched with vitamin E. Serum was collected from eight social drinkers and 16 alcohol-dependent men after a drinking period. The activity of exoglycosidases and the concentration of protein in serum were determined. The increase in specific activity of MAN and GLUCUR was significant in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E, in comparison with the specific activity in serum of social drinkers. In serum of alcohol-dependent men treated with borage oil enriched with vitamin E, specific activity of MAN and GLUCUR fluctuated in comparison with alcohol-dependent men not supplemented. Specific activity of FUC in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E showed a tendency to increase, in comparison with social drinkers. Specific activity of FUC had a tendency to decrease in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E, in comparison with alcohol-dependent men not supplemented. Thus, supplementation of alcohol-dependent men after a long-lasting drinking period with borage oil and vitamin E did not change the rate of catabolism of the oligosaccharide chains of glycoconjugates, as evaluated by serum activity of exoglycosidases.

  17. Lysozyme pattern formation in evaporating droplets

    NASA Astrophysics Data System (ADS)

    Gorr, Heather Meloy

    Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ring-like structures due to the redistribution of solute during evaporation (the "coffee ring effect"). The forms of the deposited patterns depend on complex interactions between solute(s), solvent, and substrate in a rapidly changing, far from equilibrium system. Solute self-organization during evaporation of colloidal sessile droplets has attracted the attention of researchers over the past few decades due to a variety of technological applications. Recently, pattern formation during evaporation of various biofluids has been studied due to potential applications in medical screening and diagnosis. Due to the complexity of 'real' biological fluids and other multicomponent systems, a comprehensive understanding of pattern formation during droplet evaporation of these fluids is lacking. In this PhD dissertation, the morphology of the patterns remaining after evaporation of droplets of a simplified model biological fluid (aqueous lysozyme solutions + NaCl) are examined by atomic force microscopy (AFM) and optical microscopy. Lysozyme is a globular protein found in high concentration, for example, in human tears and saliva. The drop diameters, D, studied range from the micro- to the macro- scale (1 microm -- 2 mm). In this work, the effect of evaporation conditions, solution chemistry, and heat transfer within the droplet on pattern formation is examined. In micro-scale deposits of aqueous lysozyme solutions (1 microm < D < 50 microm), the protein motion and the resulting dried residue morphology are highly influenced by the decreased evaporation time of the drop. The effect of electrolytes on pattern formation is also investigated by adding varying concentrations NaCl to the lysozyme solutions. Finally, a novel pattern recognition program is described and implemented which classifies deposit images by their solution chemistries. The results presented in this Ph

  18. Serum 5'nucleotidase activity in rats: a method for automated analysis and criteria for interpretation.

    PubMed

    Carakostas, Michael C.; Power, Richard J.; Banerjee, Asit K.

    1990-01-01

    A manual kit for determining serum 5'nucleotidase (5'NT, EC 3.1.3.5) activity was adapted for use with rat samples on a large discrete clinical chemistry analyzer. The precision of the method was good (within-run C.V. = 2.14%; between-run C.V. = 5.5%). A comparison of the new automated method with a manual and semi-automated method gave regression statistics of y = 1.18X -3.66 (Sy. x = 4.54), and y = 0.733X + 1.97 (Sy. x = 1.69), respectively. Temperature conversion factors provided by the kit manufacturer for human samples were determined to be inaccurate for converting results from rat samples. Analysis of components contributing to normal variation in rat serum 5'NT activity showed age and sex to be major factors. Increased serum 5'NT activity was observed in female rats when compared to male rats beginning at about 5 to 6 weeks of age. An analysis of variance of serum 5'NT, alkaline phosphatase, and GGT activities observed over a 9-week period in normal rats suggests several advantages for 5'NT as a predictor of biliary lesions in rats.

  19. Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles.

    PubMed

    Merchant, Mark; Britton, Adam

    2006-04-01

    We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentration-dependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25-30 degrees C and reduced activities below 25 degrees C and above 35 degrees C.

  20. A Single-Step Purification of Cauliflower Lysozyme and Its Dual Role Against Bacterial and Fungal Plant Pathogens.

    PubMed

    Manikandan, Muthu; Balasubramaniam, R; Chun, Se-Chul

    2015-09-01

    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 %).

  1. Stabilization of lysozyme by benzyl alcohol: surface tension and thermodynamic parameters.

    PubMed

    Goyal, Monu Kumari; Roy, Ipsita; Amin, Aeshna; Banerjee, Uttam Chand; Bansal, Arvind Kumar

    2010-10-01

    The aim of the study was to understand the effect of benzyl alcohol on biological activity, aggregation behavior, denaturant and heat-induced unfolding of lysozyme. Compatibility studies of lysozyme carried out with a number of anti-microbial preservatives, indicated benzyl alcohol to be the best suppressor of protein aggregation against heat stress. The effect of this preservative was checked at various pH values ranging from 4.0 to 9.0. In spite of reducing the thermal denaturation temperature (T(m)) at all pH values, benzyl alcohol had a stabilizing effect on lysozyme in terms of retaining the biological activity when the enzyme was incubated at 75 degrees C. The reduction in T(m) with increasing benzyl alcohol concentration was correlated with decreasing surface tension of surrounding medium. A detailed thermodynamic study of lysozyme in the presence of benzyl alcohol was carried out at pH 6.2. Change in Gibb's free energy of thermal unfolding at 25 degrees C was found to remain constant in the presence of benzyl alcohol, indicating no interaction of benzyl alcohol with the native protein at room temperature. Both the enthalpy and entropy change at mid point of thermal unfolding were found to increase in the presence of benzyl alcohol indicating the stabilization of partially unfolded state.

  2. Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma.

    PubMed

    Bilyy, Rostyslav; Tomin, Andriy; Mahorivska, Iryna; Shalay, Olga; Lohinskyy, Volodymyr; Stoika, Rostyslav; Kit, Yuriy

    2011-01-01

    Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m)  = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.

  3. Serum Cytokine Profile in Asian Indian Patients with Takayasu Arteritis and its Association with Disease Activity

    PubMed Central

    Goel, Ruchika; Kabeerdoss, Jayakanthan; Ram, Babu; Prakash, John Antony Jude; Babji, Sudhir; Nair, Aswin; Jeyaseelan, Lakshmanan; Jeyaseelan, Visalakshi; Mathew, John; Balaji, Veeraraghavan; Joseph, George; Danda, Debashish

    2017-01-01

    Background: Arterial inflammation Takayasu arteritis (TA) is an outcome of balance between pro- and anti-inflammatory cytokines. Comprehensive assessment of these cytokines is important for understanding pathogenesis and assessing disease activity. Objective: To study pro- and anti-inflammatory cytokines representing different T-helper cell pathway in serum samples of Asian Indian patients with TA and to assess their association with disease activity. Methods: Consecutive Indian patients with TA were assayed for serum interferon-γ, interleukin-6, interleukin-23, interleukin-17, interleukin-10 and transforming growth factor- β levels at baseline and follow up visit. Patients were grouped into active and stable disease based on Indian Takyasu Arteritis clinical Activity Score-2010. Serum levels of these cytokines between active and stable disease and between baseline and follow up visits were compared by non-parametric tests. Results: Among 32 patients enrolled, 15 were classified as active while 17 as stable disease at baseline. IFN-γ levels were significantly higher in active disease than stable disease (p=0.0129) while other cytokines did not differ significantly between 2 groups. Serum levels of none of the cytokines changed significantly over 2 visits in both responders and non-responders. IL23 levels positively correlate with disease duration ((r=0.999; p<0.005). Modest correlation was observed between IFN-γ and IL23 levels at both baseline and follow up and between IFN-γ and IL-6 and CRP at follow up. Conclusion: IFN-γ levels are raised in active disease in TA and correlates well with other biomarkers of disease activity and proinflammatory cytokines. There is also a direct correlation between Il-23 levels and disease duration.

  4. Brain Injury Alters Ectonucleotidase Activities and Adenine Nucleotide Levels in Rat Serum

    PubMed Central

    Laketa, Danijela; Savić, Jasmina; Bjelobaba, Ivana; Lavrnja, Irena; Vasić, Vesna; Stojiljković, Mirjana; Nedeljković, Nadežda

    2015-01-01

    Summary Background Cortical stab injury (CSI) induces changes in the activity, expression and cellular distribution of specific ectonucleotidases at the injury site. Also, several experimentally induced neuropathologies are associated with changes in soluble ectonucleotidase activities in the plasma and serum, whilst various insults to the brain alter purine compounds levels in cerebrospinal fluid, but also in serum, indicating that insults to the brain may induce alterations in nucleotides release and rate of their hydrolysis in the vascular system. Since adenine nucleotides and adenosine regulate diverse cellular functions in the vascular system, including vascular tone, platelet aggregation and inflammatory responses of lymphocytes and macrophages, alterations of ectonucleotidase activities in the vascular system may be relevant for the clinical outcome of the primary insult. Methods We explored ectonucleotidase activities using specific enzyme assays and determined adenine nucleotides concentrations by the UPLC method in the rat serum after cortical stab injury. Results At 4-h post-injury, ATP and AMP hydrolysis increased by about 60% and 40%, respectively, while phosphodiesterase activity remained unchanged. Also, at 4-h post-injury a marked decrease in ATP concentration and more than 2-fold increase in AMP concentration were recorded. Conclusions CSI induces rapid up-regulation of nucleotide catabolizing soluble ectonucleotidases in rat serum, which leads to the observed shift in serum nucleotide levels. The results obtained imply that ectonucleotidases and adenine nucleotides participate in the communication between the brain and the vascular system in physiological and pathological conditions and thereby may be involved in the development of various human neuropathologies.

  5. Chronic Cadmium Exposure Lead to Inhibition of Serum and Hepatic Alkaline Phosphatase Activity in Wistar Rats.

    PubMed

    Treviño, Samuel; Andrade-García, Alejandra; Herrera Camacho, Irma; León-Chavez, Bertha Alicia; Aguilar-Alonso, Patricia; Flores, Gonzalo; Brambila, Eduardo

    2015-12-01

    Alkaline phosphatase (ALP) activity in the serum and liver from rats administered with cadmium (Cd) in drinking water was studied. After metal administration, Cd showed a time-dependent accumulation in the liver, meanwhile metallothionein had a maximum increase at 1 month, remaining in this level until the end of the study. On the other hand, serum and liver ALP activity was decreased after 3 months exposure. To determine if Cd produced an inhibition on enzyme, apo-ALP prepared from both nonexposed and exposed rats was reactivated with Zn, showing 60% more activity as compared with the enzyme isolated from nonexposed rats. In vitro assays showed that Cd-ALP was partially reactivated with Zn; however, in the presence of cadmium, Zn-ALP was completely inhibited. Kinetic studies indicate a noncompetitive inhibition by Cd; these results suggest that Cd can substitute Zn, and/or Cd can interact with nucleophilic ligands essential for the enzymatic activity.

  6. Functional analysis of two invertebrate-type lysozymes from red swamp crayfish, Procambarus clarkii.

    PubMed

    Zhang, Hong-Wei; Sun, Chen; Sun, Shan-Shan; Zhao, Xiao-Fan; Wang, Jin-Xing

    2010-12-01

    Lysozyme is an important immune effector and is widely distributed in many organisms. In the present study, two novel invertebrate-type lysozymes (Pclysi1 and Pclysi2) were cloned from red swamp crayfish, Procambarus clarkii. Alignment analysis showed that these two genes were different in catalytic residues. Results of RT-PCR showed that these two genes share similar tissue distribution patterns, and both were upregulated after bacteria challenge. The mature recombinant proteins were expressed in Escherichia coli system and purified. Activity analysis revealed that rPclysi1 had no muramidase activity and isopeptidase activity, but had antimicrobial activity. Meanwhile, rPclysi2 was muramidase-deficient and had no antimicrobial activity, but possessed isopeptidase activity. Above data suggest that Pclysi1 and 2 may offer different functions in the crayfish immunity.

  7. Serum Malondialdehyde Concentration and Glutathione Peroxidase Activity in a Longitudinal Study of Gestational Diabetes

    PubMed Central

    Miranda, María; Muriach, María; Romero, Francisco J.; Villar, Vincent M.

    2016-01-01

    Aims The main goal of this study was to evaluate the presence of oxidative damage and to quantify its level in gestational diabetes. Methods Thirty-six healthy women and thirty-six women with gestational diabetes were studied in the three trimesters of pregnancy regarding their levels of oxidative stress markers. These women were diagnosed with diabetes in the second trimester of pregnancy. Blood glucose levels after 100g glucose tolerance test were higher than 190, 165 or 145 mg/dl, 1, 2 or 3 hours after glucose intake. Results The group of women with gestational diabetes had higher serum malondialdehyde levels, with significant differences between groups in the first and second trimester. The mean values of serum glutathione peroxidase activity in the diabetic women were significantly lower in the first trimester. In the group of women with gestational diabetes there was a negative linear correlation between serum malondialdehyde concentration and glutathione peroxidase activity in the second and third trimester. Conclusions In this observational and longitudinal study in pregnant women, the alterations attributable to oxidative stress were present before the biochemical detection of the HbA1c increase. Usual recommendations once GD is detected (adequate metabolic control, as well as any other normally proposed to these patients) lowered the concentration of malondialdehyde at the end of pregnancy to the same levels of the healthy controls. Serum glutathione peroxidase activity in women with gestational diabetes increased during the gestational period. PMID:27228087

  8. Comparative serum bactericidal activities of three doses of ciprofloxacin administered intravenously.

    PubMed Central

    Dan, M; Poch, F; Quassem, C; Kitzes, R

    1994-01-01

    The pharmacokinetics and serum bactericidal activities of three intravenous doses of ciprofloxacin were studied comparatively in 30 patients. Single 200-, 300-, and 400-mg intravenous doses of ciprofloxacin were given over 30 min to 10 patients each, and serum samples were obtained at 0.5, 1, 2, 3, 4, 8, and 12 h after the start of the infusion. Serum drug concentrations were determined by high-pressure liquid chromatography. Pharmacokinetic parameters were estimated by using noncompartmental analysis methods. Serum bactericidal activity against clinical isolates of Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, and Staphylococcus aureus was determined for samples obtained at 0.5, 4, 8, and 12 h. Excellent activity was demonstrated up to 12 h by all doses against E. coli and E. cloacae. Much poorer titers were observed for the remaining organisms, although the 400-mg dose prompted improved results against P. aeruginosa with a mean bactericidal titer of 1:2.9 at 8 h. In conclusion, while the 200-mg dose appears to be largely adequate for infections caused by members of the family Enterobacteriaceae, it seems that when P. aeruginosa is involved, 400 mg twice a day or even three times a day is more appropriate. Intravenous ciprofloxacin performs poorly against A. calcoaceticus and S. aureus, even at a higher dose. PMID:8031055

  9. A rapid microtiter plate method for the detection of lysozyme release from human neutrophils.

    PubMed

    Moreira-Ludewig, R; Healy, C T

    1992-04-01

    An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Participation of decreased serum cholesteryl ester transfer activity, independent of increased serum lipoprotein(a), in angina pectoris in normolipemic elderly subjects.

    PubMed

    Miyashita, Y; Morimoto, S; Fukuo, K; Imanaka, S; Koh, E; Tamatani, M; Ogihara, T

    1992-01-01

    The cholesteryl ester transfer activity (CETA) is a measurement of the transfer of cholesteryl ester from HDL to VLDL, LDL or peripheral cells. Its role in the development of early coronary heart disease is not clear. In the present study, serum levels of CETA, lipoprotein(a) [Lp(a)] and other lipid-related factors were compared in 10 normal young subjects, 28 healthy elderly subjects and 14 normolipemic elderly patients with angina pectoris. Compared to the young normals and healthy elderly subjects, the elderly patients with angina pectoris showed significantly decreased mean serum CETA levels, and significantly increased mean serum levels of Lp(a) and apoprotein B. These results may indicate that decreased serum values of CETA participate in the development of angina pectoris in normolipemic elderly patients.

  11. Identification of haptoglobin as a natural inhibitor of trypanocidal activity in human serum.

    PubMed Central

    Smith, A B; Hajduk, S L

    1995-01-01

    Trypanosomes are protozoan parasites of medical and veterinary importance. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense infect humans, causing African sleeping sickness. However, Trypanosoma brucei brucei can only infect animals, causing the disease Nagana in cattle. Man is protected from this subspecies of trypanosomes by a toxic subtype of high density lipoproteins (HDLs) called the trypanosome lytic factor (TLF). The toxic molecule in TLF is believed to be the haptoglobin-related protein that when bound to hemoglobin kills the trypanosome via oxidative damage initiated by its peroxidase activity. The amount of lytic activity in serum varies widely between different individuals with up to a 60-fold difference in activity. In addition, an increase in the total amount of lytic activity occurs during the purification of TLF, suggesting that an inhibitor of TLF (ITLF) exists in human serum. We now show that the individual variation in trypanosome lytic activity in serum correlates to variations in the amount of ITLF. Immunoblots of ITLF probed with antiserum against haptoglobin recognize a 120-kDa protein, indicating that haptoglobin is present in partially purified ITLF. Haptoglobin involvement is further shown in that it inhibits TLF in a manner similar to ITLF. Using an anti-haptoglobin column to remove haptoglobin from ITLF, we show that the loss of haptoglobin coincides with the loss of inhibitor activity. Addition of purified haptoglobin restores inhibitor activity. This indicates that haptoglobin is the molecule responsible for inhibition and therefore causing the individual variation in serum lytic activity. Images Fig. 2 Fig. 4 PMID:7479764

  12. Alteration of serum inflammatory cytokines in active pulmonary tuberculosis following anti-tuberculosis drug therapy.

    PubMed

    Chowdhury, Imran Hussain; Ahmed, Albin Mostaque; Choudhuri, Subhadip; Sen, Aditi; Hazra, Avijit; Pal, Nishith Kumar; Bhattacharya, Basudev; Bahar, Bojlul

    2014-11-01

    Active pulmonary tuberculosis (APTB) is associated with a failure of the host immune system to control the invading Mycobacterium tuberculosis (Mtb). The objective of this study was to quantify and assess the role of serum inflammatory cytokines in active pulmonary tuberculosis patients following anti-tuberculosis drug (ATD) therapy. Blood samples were collected from APTB patients and normal healthy subjects (NHS) (total n=204) at baseline and 2, 4 and 6 months post-therapy and the abundance of serum inflammatory cytokines were measured by cytokine specific ELISA. Compared to NHS, APTB patients at baseline had higher levels of serum pro-inflammatory cytokines IL-12p40 (P<0.001), IFN-γ (P<0.001), TNF-α (P<0.01), IL-1β (P<0.001) and IL-6 (P<0.001) and anti-inflammatory cytokines IL-10 (P<0.001) and TGF-β1 (P<0.001) while there was no change in the level of IL-4. In APTB patients, the serum levels of IFN-γ, TNF-α, IL-6 and TGF-β1 directly relate to the bacterial load while the TNF-α, IL-1β, IL-6 and TGF-β1 relate to radiological severity. At baseline, the IL-6 level in NHS and APTB patients differed most and following ATD therapy, this level rapidly decreased and stabilized by 4-month in APTB patients. It is concluded that a subtle reduction in the serum level of IL-6 of the APTB patients following ATD therapy might play a vital role in immune-protection of the host against Mtb infection and hence the serum IL-6 level can be a useful marker to diagnose the effectiveness of therapy in the patients.

  13. Enhancing effect of serum ultrafiltrate on the activity of cephalosporins against gram-negative bacilli.

    PubMed Central

    Leggett, J E; Craig, W A

    1989-01-01

    A few studies have suggested that the inhibitory effect of serum on activity of broad-spectrum cephalosporins is less than that predicted by the degree of protein binding. Microdilution MICs of ceftriaxone, cefoperazone, moxalactam, and ceftizoxime were therefore determined against ATCC and clinical strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus in Mueller-Hinton broth containing either human albumin (as 0, 2.5, or 5% solution) or heat-inactivated human serum (as 0, 25, 50, or 95% solution). Arithmetic linear dilutions were used to improve accuracy. For standard bacterial strains, MICs in the presence of 5% albumin were higher than in broth alone by multiples of 10.9 to 21 for ceftriaxone, 5.5 to 16.4 for cefoperazone, 1.9 to 3.7 for moxalactam, and 1.1 to 1.4 for ceftizoxime, as expected by their protein binding. MICs in the presence of 95% serum were similar to those in 5% albumin for all four drugs against S. aureus and P. aeruginosa but were 2.2- to 4.8-fold lower (P less than 0.001) against E. coli and K. pneumoniae. Similar findings were observed at lower protein concentrations and with clinical isolates, except that for some strains of P. aeruginosa MICs were lower in serum than in albumin. Individual sera from five subjects gave comparable results. The addition of serum ultrafiltrate to albumin-containing solutions reduced MICs of ceftriaxone and cefoperazone 1.6- to 7.4-fold against E. coli and K. pneumoniae (P less than 0.01) but did not alter the MICs for S. aureus. Serum may contain an ultrafiltrable component(s) that enhances the activity of third-generation cephalosporins against many gram-negative bacilli. PMID:2496656

  14. Serum Paraoxonase 1 Activity Is Associated with Fatty Acid Composition of High Density Lipoprotein

    PubMed Central

    Boshtam, Maryam; Pourfarzam, Morteza; Ani, Mohsen; Naderi, Gholam Ali; Basati, Gholam; Mansourian, Marjan; Dinani, Narges Jafari; Asgary, Seddigheh; Abdi, Soheila

    2013-01-01

    Introduction. Cardioprotective effect of high density lipoprotein (HDL) is, in part, dependent on its related enzyme, paraoxonase 1 (PON1). Fatty acid composition of HDL could affect its size and structure. On the other hand, PON1 activity is directly related to the structure of HDL. This study was designed to investigate the association between serum PON1 activity and fatty acid composition of HDL in healthy men. Methods. One hundred and forty healthy men participated in this research. HDL was separated by sequential ultracentrifugation, and its fatty acid composition was analyzed by gas chromatography. PON1 activity was measured spectrophotometrically using paraxon as substrate. Results. Serum PON1 activity was directly correlated with the amount of stearic acid and dihomo-gamma-linolenic acid (DGLA). PON1/HDL-C was directly correlated with the amount of miristic acid, stearic acid, and DGLA and was inversely correlated with total amount of ω6 fatty acids of HDL. Conclusion. The fatty acid composition of HDL could affect the activity of its associated enzyme, PON1. As dietary fats are the major determinants of serum lipids and lipoprotein composition, consuming some special dietary fatty acids may improve the activity of PON1 and thereby have beneficial effects on health. PMID:24167374

  15. Relationship between serum leptin level and disease activity in patients with systemic sclerosis.

    PubMed

    Budulgan, Mahmut; Dilek, Banu; Dağ, Şevin Buluttekin; Batmaz, Ibrahim; Yıldız, İsmail; Sarıyıldız, Mustafa Akif; Çevik, Remzi; Nas, Kemal

    2014-03-01

    To determine the relationship between serum leptin levels and disease activity in systemic sclerosis (SSc). A total of 60 subjects (30 controls and 30 patients) were included. The inflammatory markers and leptin levels were evaluated and body mass index (BMI) was measured for both groups. The assessment of the skin involvement was performed based on the modified Rodnan skin score (mRSS). Disease activity was evaluated according to the Valentini scleroderma disease activity index. There was a significant difference between the patient and control groups in terms of BMI (p < 0.05); however there was no difference with regards to age and gender (p > 0.05). Valentini scores and mRSS were determined to be significantly higher in active patients (n = 14) than in inactive patients (n = 16) (p < 0.05). No significant difference was determined between groups in terms of leptin levels (p > 0.05). However, leptin levels were significantly lower in active patients than in inactive patients (p < 0.05). We found a significant positive correlation between serum leptin and BMI (p < 0.05), and leptin and serum C3 levels (p < 0.05); no relationship was detected between leptin and other parameters. Leptin can be used as an activity marker in SSc. Further studies, including larger series, should be carried out to clarify this relationship.

  16. Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

    PubMed

    Akiyama, Y; Griffith, R; Miller, P; Stevenson, G W; Lund, S; Kanapa, D J; Stevenson, H C

    1988-03-01

    Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate. We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process. Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5'-N activity and decreasing peroxidase activity of suspension cultured monocytes. Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity. Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab')2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule. Fibrinogen and fibronectin also had maturation promoting activity. Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte

  17. Bacterial Endotoxin Activity in Human Serum Is Associated With Dyslipidemia, Insulin Resistance, Obesity, and Chronic Inflammation

    PubMed Central

    Lassenius, Mariann I.; Pietiläinen, Kirsi H.; Kaartinen, Kati; Pussinen, Pirkko J.; Syrjänen, Jaana; Forsblom, Carol; Pörsti, Ilkka; Rissanen, Aila; Kaprio, Jaakko; Mustonen, Jukka; Groop, Per-Henrik; Lehto, Markku

    2011-01-01

    OBJECTIVE To investigate whether bacterial lipopolysaccharide (LPS) activity in human serum is associated with the components of the metabolic syndrome (MetS) in type 1 diabetic patients with various degrees of kidney disease and patients with IgA glomerulonephritis (IgAGN). RESEARCH DESIGN AND METHODS Serum LPS activity was determined with the Limulus Amoebocyte Lysate chromogenic end point assay in type 1 diabetic patients with a normal albumin excretion rate (n = 587), microalbuminuria (n = 144), macroalbuminuria (n = 173); patients with IgAGN (n = 98); and in nondiabetic control subjects (n = 345). The relationships of the LPS/HDL ratio and MetS-associated variables were evaluated with Pearson correlation. RESULTS The MetS was more prevalent in type 1 diabetic patients (48%) than in patients with IgAGN (15%). Diabetic patients with macroalbuminuria had a significantly higher serum LPS/HDL ratio than patients with IgAGN. In the normoalbuminuric type 1 diabetic group, patients in the highest LPS/HDL quartile were diagnosed as having the MetS three times more frequently than patients in the lowest quartile (69 vs. 22%; P < 0.001). High LPS activity was associated with higher serum triglyceride concentration, earlier onset of diabetes, increased diastolic blood pressure, and elevated urinary excretion of monocyte chemoattractant protein-1. CONCLUSIONS High serum LPS activity is strongly associated with the components of the MetS. Diabetic patients with kidney disease seem to be more susceptible to metabolic endotoxemia than patients with IgAGN. Bacterial endotoxins may thus play an important role in the development of the metabolic and vascular abnormalities commonly seen in obesity and diabetes-related diseases. PMID:21636801

  18. Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

    PubMed

    Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

    2016-03-10

    Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.

  19. Capacity of a hydroxyapatite–lysozyme combination against Streptococcus mutans for the treatment of dentinal caries

    PubMed Central

    Pinheiro, Sérgio Luiz; da Rocha, Nathany Nunes; Peres, Mariane de Lourdes Hernandes Martins

    2016-01-01

    Background: One current strategy for the treatment of carious lesions is the use of biomaterials with antimicrobial activity. Aims: The aim of this study was to evaluate a combination of hydroxyapatite and lysozyme for the treatment of dentinal caries by measuring Streptococcus mutans counts before carious tissue sealing, and 24 h, 1 month, and 6 months after treatment. Materials and Methods: Forty permanent third molars were selected, and flat dentin surfaces were prepared. The teeth were exposed to a cariogenic challenge with S. mutans. After challenge, the dentinal caries were collected from five specimens. The remaining specimens were treated with a mixture of hydroxyapatite and lysozyme in sodium laureth sulfate and sealed with composite resin. S. mutans counts were obtained 24 h, 1 month, and 6 months after sealing. Statistical Analysis: The results were evaluated by descriptive statistics and Wilcoxon signed-rank test. Results: a significant reduction in S. mutans (CFU/mL) was observed in dentinal lesions 1 month after treatment with hydroxyapatite/lysozyme in sodium laureth sulfate (P = 0.0254). Comparison of S. mutans counts obtained 24 h, 1 month, and 6 months after treatment revealed reductions only at the 1-month time point (P = 0.0318). Conclusions: the combination of hydroxyapatite and lysozyme may be an alternative for reducing the S. mutans burden in dentinal caries. PMID:27656068

  20. Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity

    PubMed Central

    Detree, Camille; Chabenat, Apolline; Lallier, François H.; Satoh, Nori; Shoguchi, Eiichi; Tanguy, Arnaud; Mary, Jean

    2016-01-01

    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 -1700m, 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. PMID:26882089

  1. Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity.

    PubMed

    Detree, Camille; Chabenat, Apolline; Lallier, François H; Satoh, Nori; Shoguchi, Eiichi; Tanguy, Arnaud; Mary, Jean

    2016-01-01

    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.

  2. Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone-ion exchange chromatography.

    PubMed

    Wang, Chaozhan; Zhang, Qinming; Cheng, Yan; Wang, Lili

    2010-01-01

    Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.

  3. Superior Catalytic Performance of Gold Nanoparticles Within Small Cross-Linked Lysozyme Crystals.

    PubMed

    Liu, Mingyue; Wang, Libing; Huang, Renliang; Yu, Yanjun; Su, Rongxin; Qi, Wei; He, Zhimin

    2016-10-08

    Bionanomaterials synthesized by bio-inspired templating methods have emerged as a novel class of composite materials with varied applications in catalysis, detection, drug delivery, and biomedicine. In this study, two kinds of cross-linked lysozyme crystals (CLLCs) of different sizes were applied for the in situ growth of Au nanoparticles (AuNPs). The resulting composite materials were characterized by light microscopy, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The catalytic properties of the prepared materials were examined in the catalytic reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). It was found that the size of the AuNPs increased with an increase in Au loading for both small and large crystals. In addition, small crystals favored homogeneous adsorption and distribution of the metal precursors. And the size of the AuNPs within small crystals could be maintained below 2.5 nm by managing the HAuCl4/lysozyme molar ratio. Furthermore, the lysozyme functional groups blocked the AuNP activity sites, therefore reducing their catalytic activity. This effect was more pronounced for small AuNPs. Moreover, the mass transfer of reactants (4-NP) from solution to AuNPs within the crystals restricted their catalytic reduction, leading to superior catalytic performance of the AuNPs within small cross-linked lysozyme crystals (Au@S-CLLCs) compared to those within large cross-linked lysozyme crystals (Au@L-CLLCs) at similar Au loadings. Finally, an increase in Au loading clogged the crystal channels with increased quantities of larger aggregated AuNPs, thus impeding the catalytic performance of Au@S-CLLCs.

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

    PubMed Central

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

    2015-01-01

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

  5. Heme Oxygenase Activity Correlates with Serum Indices of Iron Homeostasis in Healthy Nonsmokers

    PubMed Central

    Ghio, Andrew J.; Schreinemachers, Dina M.

    2016-01-01

    Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirmed such participation of HO in iron homeostasis of humans. Carbon monoxide produced through HO activity will bind to hemoglobin in circulating erythrocytes, and therefore, blood carboxyhemoglobin (COHb) can be used as an index of HO activity. Using the second National Health and Nutrition Examination Survey, we tested the postulate that HO activity correlates with serum indices of iron homeostasis in healthy nonsmokers. The investigation included 844 lifetime nonsmokers (586 females) 18 years of age and older in the study population. Significant correlations were demonstrated between COHb and several indices of iron homeostasis including serum levels of both ferritin and iron and percentage iron saturation of transferrin. There was no significant association between COHb and hemoglobin, the largest repository of heme in the human body, which functions as the substrate for HO. We conclude that HO activity contributes to human iron homeostasis with significant correlations between COHb and serum ferritin and iron levels and percentage iron saturation of transferrin. PMID:27199547

  6. Tetragonal Lysozyme, From Monomer to Crystal

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    The data now leads us to a comprehensive model for the process by which tetragonal lysozyme crystals are nucleated and subsequently grow. Lysozyme is typically desolubilized by addition of ionic salts. The salt anions bind to basic and other sites on the protein and promote protein-protein interactions, i.e., initiate the nucleation self assembly process. Formation of protein-protein interactions occurs at the expense of the protein-anion interactions, with the anions being released to the solution. The association follows a defined pattern, forming the "head to side" interactions of the crystal 4(3) helix. The presence of the high salt also promotes hydrophobic interactions between the protein molecules, further tightening their interaction. The solute assembly process persists after crystal nucleation, and the 4(3) helical structures form the subsequent growth units. AFM measurements show that the growth units follow the dimensions of these helices, and that those on the surface are more compact about the c-axis than in the bulk crystal, with adjacent helices riot being in contact. This further supports the role of hydrophobic interactions, as the surface is still in contact with the bulk solution. Once buried within the crystal the protein:salt ratio radically changes and the hydrophobic interactions relax to those measured crystallographically. Thus the crystal growth process recapitulates the initial stages of the nucleation process, and the two seamlessly merge. Experimental evidence, based upon face growth rate, AFM, and fluorescence energy transfer data, for a postulated model of the nucleation of tetragonal lysozyme crystals and how it transitions into crystal growth will be presented.

  7. Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions.

    PubMed

    Schinkel, J E; Downer, N W; Rupley, J A

    1985-01-15

    The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison

  8. Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo

    PubMed Central

    Gnanadhas, Divya Prakash; Ben Thomas, Midhun; Thomas, Rony; Raichur, Ashok M.

    2013-01-01

    The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes. PMID:23877702

  9. High-throughput asparaginase activity assay in serum of children with leukemia

    PubMed Central

    Fernandez, Christian A; Cai, Xiangjun; Elozory, Allie; Liu, Chengcheng; Panetta, J Carl; Jeha, Sima; Molinelli, Alejandro R; Relling, Mary V

    2013-01-01

    Asparaginase is an antineoplastic agent used in combination therapy for acute lymphoblastic leukemia (ALL). The asparaginase activity measured in serum reflects the effectiveness of the drug. However, the wide inter-individual variability in the pharmacokinetics of asparaginase suggests that the serum activity should be closely monitored in patients during therapy. In order to identify patients with low asparaginase exposure during treatment, a fast, sensitive, and high-throughput assay is required for measuring asparaginase activity in patient sera. In this study, asparaginase activity was determined by monitoring the enzymatically-coupled oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD+ in a 96-well format. The rate of disappearance of NADH (ΔmOD/minute) was directly proportional to the activity of asparaginase, and the linear range of the assay was established from 0.025 to 2.2 IU/mL (R2 = 0.998) with a reportable range that was extended to 4.0 IU/mL by dilution with serum albumin. Inter-assay precision was established (low control CV% = 8.8, high control CV% = 9.0), as was intra-assay precision (low control CV% = 3.3, high control CV% = 2.7). The method is high-throughput and provides a broader linear range of detection compared to previously described assays. The speed, ease, and accuracy of the assay make it suitable for assessing serum asparaginase activity after standard doses of native E. coli, Erwinia, and PEGylated E. coli asparaginase given to children during the treatment of leukemia. PMID:23936585

  10. Purification of Lysozyme by Intrinsically Shielded Hydrogel Beads

    NASA Astrophysics Data System (ADS)

    Li, Cong; Zhang, R.; Wang, L.; Bowyer, A.; Eisenthal, R.; Shen, Yehua; Hubble, J.

    2013-07-01

    Macro-sized intrinsically shielded hydrogel beads have been prepared from BSA and CM-dextran grafted with CB using a technique based on freeze-thawing gelation method. The size of the beads lies in around 500 μm. Isothemal titration calorimetry (ITC) showed that the relative binding affinities of the lysozyme for CB, compared with BSA, at pH 3.0 was stronger than that at pH 7.4. They were employed for the affinity separation of lysozyme using chromatography column. Their adsorption capacity for lysozyme at pH 3.0 is higher than that at pH 9. In a binary mixture of lysozyme and ovalbumin, the beads showed very high selectivity toward lysozyme. Lysozyme of very high purity (> 93%) was obtained from a mixture of lysozyme and ovalbumin, and 85% from egg white solution. The results indicate that the macro-sized bead can be used for the separation, purification, and recovery of lysozyme in a chromatograph column.

  11. Co-option of bacteriophage lysozyme genes by bivalve genomes

    PubMed Central

    Wang, Chunyang; Jin, Min; Lan, Jiangfeng; Ye, Ting; Hui, Kaimin; Tan, Jingmin; Wang, Zheng; Wang, Wen; Han, Guan-Zhu

    2017-01-01

    Eukaryotes have occasionally acquired genetic material through horizontal gene transfer (HGT). However, little is known about the evolutionary and functional significance of such acquisitions. Lysozymes are ubiquitous enzymes that degrade bacterial cell walls. Here, we provide evidence that two subclasses of bivalves (Heterodonta and Palaeoheterodonta) acquired a lysozyme gene via HGT, building on earlier findings. Phylogenetic analyses place the bivalve lysozyme genes within the clade of bacteriophage lysozyme genes, indicating that the bivalves acquired the phage-type lysozyme genes from bacteriophages, either directly or through intermediate hosts. These bivalve lysozyme genes underwent dramatic structural changes after their co-option, including intron gain and fusion with other genes. Moreover, evidence suggests that recurrent gene duplication occurred in the bivalve lysozyme genes. Finally, we show the co-opted lysozymes exhibit a capacity for antibacterial action, potentially augmenting the immune function of related bivalves. This represents an intriguing evolutionary strategy in the eukaryote–microbe arms race, in which the genetic materials of bacteriophages are co-opted by eukaryotes, and then used by eukaryotes to combat bacteria, using a shared weapon against a common enemy. PMID:28100665

  12. High Levels of Serum DPP-4 Activity Are Associated with Low Bone Mineral Density in Obese Postmenopausal Women

    PubMed Central

    2016-01-01

    Background Dipeptidyl peptidase 4/CD26 (DPP-4) is a widely expressed cell surface serine protease. DPP-4 inhibitors, one of common anti-diabetic agents play a protective role in bone metabolism in recent studies. A soluble form of DPP-4 is found in serum, and exhibits DPP-4 enzymatic activity. However, the physiological role of serum or soluble DPP-4 and its relationship with DPP-4 enzymatic function remain poorly understood. The aims of current study were to determine the association between serum DPP-4 activity and bone mineral density (BMD) in postmenopausal women. Methods We recruited data and serum samples from 124 consecutive healthy postmenopausal women aged >50 years. We divided study subjects into obese (body mass index [BMI] ≥25 kg/m2) and non-obese (BMI <25 kg/m2) postmenopausal women and examined the correlation between serum DPP-4 activity and clinical variables in each groups. Results A total of 124 postmenopausal women was enrolled, with a mean age of 59.9±7.1 years. The mean BMI of the study patients was 24.4±2.8 kg/m2. Regarding bone turnover markers, serum DPP-4 activity was positively correlated with serum calcium concentrations, intact parathyroid hormone, and serum C-telopeptide levels in all of the study subjects. However, there was no association between serum DPP-4 activity and BMD in the spine or femoral neck in all of the study subjects. Serum DPP-4 activity was negatively correlated (R=−0.288, P=0.038) with BMD of the spine in obese postmenopausal women. Conclusion This study demonstrated for the first time that serum soluble DPP-4 activity was negatively correlated with BMD in obese postmenopausal women. PMID:26676330

  13. Structural and Mechanistic Studies of Pesticin, a Bacterial Homolog of Phage Lysozymes*

    PubMed Central

    Patzer, Silke I.; Albrecht, Reinhard; Braun, Volkmar; Zeth, Kornelius

    2012-01-01

    Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme. PMID:22593569

  14. Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.

    PubMed

    Del Olmo, E; García-Álvarez, O; Maroto-Morales, A; Ramón, M; Jiménez-Rabadán, P; Iniesta-Cuerda, M; Anel-Lopez, L; Martinez-Pastor, F; Soler, A J; Garde, J J; Fernández-Santos, M R

    2016-01-15

    Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

  15. Comparative investigation of antioxidant activity of human serum blood by amperometric, voltammetric and chemiluminescent methods

    PubMed Central

    Korotkova, Elena; Voronova, Olesya; Sazhina, Natalia; Petrova, Ekatherina; Artamonov, Anton; Chernyavskaya, Ludmila; Dorozhko, Elena

    2015-01-01

    Introduction A blood test can provide important information about the functional state of the antioxidant system. Malfunction of this system increases the concentration of free radicals and can cause oxidative stress. A difficulty in assessing oxidative stress is the lack of a universal method for determining the antioxidant activity (AOA) of blood components, because of their different nature. Material and methods The objects of investigation were sera of 30 male patients with a diagnosis of alcohol dependence syndrome and healthy donors. Comparative investigation of total antioxidant activity (TAA) of human serum blood was carried out by voltammetric (VA), amperometric (AM) and chemiluminescent (HL) methods. Results All applied methods revealed that serum TAA of the patients with alcoholism is lower than TAA of healthy donors (control group); according to amperometric method the average value of serum TAA was 850 ±210 nA × s, and 660 ±150 nA × s for healthy donors and alcoholics respectively (p < 0.05). Similar trend was revealed by chemiluminescence and voltammetry methods. The results confirm that thiol compounds make a significant contribution to the antioxidant activity of serum. The average thiol concentrations were 0.94 ±0.34 mmol/l and 1.21 ±0.36 mmol/l (p < 0.05) for alcoholics and healthy donors respectively. Decreasing thiol concentration in blood of alcoholics leads to depletion of antioxidant systems of blood. However, the differences between the results of AM, VA and HL methods were significant, because they reflected different aspects of antioxidant activity. Conclusions For objective assessment of antioxidant activity of biological objects, we suggest using methods based on different model systems. PMID:27695499

  16. NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

    SciTech Connect

    Pecorelli, Alessandra; Bocci, Velio; Acquaviva, Alessandra; Belmonte, Giuseppe; Gardi, Concetta; Virgili, Fabio; Ciccoli, Lucia; Valacchi, Giuseppe

    2013-02-15

    During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O{sub 3} per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H{sub 2}O{sub 2}) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H{sub 2}O{sub 2} are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment.

  17. A correlation of pregnancy term, disease activity, serum female hormones, and cytokines in uveitis

    PubMed Central

    Chan, C-C; Reed, G F; Kim, Y; Agrón, E; Buggage, R R

    2004-01-01

    Background/aims: Pregnancy and the postpartum period are associated with the activity of autoimmune diseases including uveitis. Although the exact mechanism is unknown, hormones are reported to alter inflammatory cytokines and influence disease activity. The authors studied ocular inflammation, female hormones, and serum cytokine levels during and after pregnancy. Methods: A prospective, observational case study was conducted. Four pregnant women in their first trimester with chronic non-infectious uveitis were followed monthly until 6 months after delivery. Serum female hormones (oestrogen, progesterone, prolactin) and various cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ, and TGF-β) were measured by ELISA. Results: The four patients had five full term pregnancies. Uveitis activity decreased after the first trimester but flared in the early postpartum period. Serum female hormones, highly elevated during pregnancy, drastically dropped post partum. Cytokine levels except TGF-β were mostly undetectable. Conclusion: Female hormones and TGF-β may contribute to the activity of uveitis during pregnancy and the postpartum period. PMID:15548800

  18. Hydroxyproline substitutions stabilize non-glycosylated drosocin against serum proteases without challenging its antibacterial activity.

    PubMed

    Knappe, Daniel; Cassone, Marco; Nollmann, Friederike Inga; Otvos, Laszlo; Hoffmann, Ralf

    2014-04-01

    The increasing incidence of multi- and pan-resistant pathogens demands novel compounds to fight Grampositive and especially Gram-negative bacteria. Among the currently investigated compound classes, antimicrobial peptides (AMPs) inhibiting specific bacterial targets appear especially promising for systemic therapy of infections, although unmodified linear peptides are typically rapidly degraded by serum proteases. Proline-rich AMPs have been heavily investigated in recent years due to their low toxicity and proven in vivo efficacy. Here, we report novel unglycosylated drosocin analogs with extended half-life in mouse serum and improved activity against Gram-negative pathogens Escherichia coli and Klebsiella pneumoniae. Substituting proline (Pro) residues in positions 3, 5, 10, and 14 with trans-4-hydroxy-Lproline ((t)Hyp) improved the antibacterial activity, whereas substitution of Pro-16 reduced the activity. Drosocin analogs with (t)Hyp in positions 3 and 5 were also four to eight times more stable in mouse serum than the unmodified analog. The new compounds were not toxic against human HeLa, HEK293, and HepG2 cell lines and showed no hemolytic activity against human erythrocytes at peptide concentrations of at least 600 µg/mL.

  19. Associations between PON1 enzyme activities in human ovarian follicular fluid and serum specimens

    PubMed Central

    Kim, Keewan; Fujimoto, Victor Y.; Browne, Richard W.

    2017-01-01

    The importance of high-density lipoprotein (HDL) particle components to reproduction is increasingly recognized, including the constituent paraoxonase 1 (PON1). However, the reliability characteristics of PON1 enzymes in ovarian follicular fluid (FF) as biomarkers for clinical and epidemiologic studies have not been described. Therefore, we characterized PON1 enzymes in FF and serum and assessed the impact of the PON1 Q192R polymorphism on associations between enzyme activities in two compartments. We also evaluated associations between HDL particle size and enzyme activities. We collected FF and serum from 171 women undergoing in vitro fertilization. PON1 activities were measured as paraoxonase and arylesterase activities, and HDL particle size was determined by 1H NMR spectrometry. Reliability indices for PON1 activities were characterized and we evaluated HDL particle sizes as predictors of PON1 enzyme activities. We found that PON1 enzyme activities were correlated between compartments, but higher in serum than in FF. For FF, the index of individuality (II) was low and the coefficient of variation (CV%) was high for paraoxonase activity overall (0.12 and 11.51%, respectively). However, IIs increased (0.33–1.30) and CV%s decreased (5.58%-8.52%) when stratified by PON1 Q192R phenotype. The intraclass correlation coefficient (ICC) for FF paraoxonase activity was high overall (0.89) but decreased when stratified by PON1 Q192R phenotype (0.43–0.75). We found similar, although more modest, patterns for FF arylesterase activity. For enzyme activities in serum, ICCs were close to 1.00 across all phenotypes. Additionally, different HDL particle sizes predicted PON1 enzyme activities according to PON1 Q192R phenotype. Overall, stratification by PON1 Q192R phenotype improved the reliability characteristics of FF PON1 enzymes as biomarkers for use in clinical investigations but diminished usefulness for epidemiologic studies. Thus, we recommend stratification by PON1 Q

  20. Effects of single-walled carbon nanotubes on lysozyme gelation.

    PubMed

    Tardani, Franco; La Mesa, Camillo

    2014-09-01

    The possibility to disperse carbon nanotubes in biocompatible matrices has got substantial interest from the scientific community. Along this research line, the inclusion of single walled carbon nanotubes in lysozyme-based hydrogels was investigated. Experiments were performed at different nanotube/lysozyme weight ratios. Carbon nanotubes were dispersed in protein solutions, in conditions suitable for thermal gelation. The state of the dispersions was determined before and after thermal treatment. Rheology, dynamic light scattering and different microscopies investigated the effect that carbon nanotubes exert on gelation. The gelation kinetics and changes in gelation temperature were determined. The effect of carbon and lysozyme content on the gel properties was, therefore, determined. At fixed lysozyme content, moderate amounts of carbon nanotubes do not disturb the properties of hydrogel composites. At moderately high volume fractions in carbon nanotubes, the gels become continuous in both lysozyme and nanotubes. This is because percolating networks are presumably formed. Support to the above statements comes by rheology.

  1. Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

    PubMed

    Tonin, Alexandre A; Pimentel, Victor C; da Silva, Aleksandro S; de Azevedo, Maria Isabel; Souza, Viviane C G; Wolkmer, Patrícia; Rezer, João F P; Badke, Manoel R T; Leal, Daniela B R; Schetinger, Maria Rosa C; Monteiro, Silvia G; Lopes, Sonia T A

    2012-04-01

    Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 2×10(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (P<0.05). In conclusion, our results showed that the ADA activity is altered in serum, lymphocytes and erythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters.

  2. [The antioxidant activity of components of blood serum of donors and recipients before liver transplantation].

    PubMed

    Sazhina, N N; Titov, V N; Popov, I N; Solonin, S A; Godkov, M A

    2014-08-01

    The antioxidant activity is implemented in human blood serum by ascorbic acid, uric acid, amino acids, glucose, mono unsaturated fatty acids (in the first instance Ω-9 oleic acid), essential polyenoic fatty acids, thiol groups of albumins and proteins, tripeptide and pigment of bilirubin. The antioxidant activity of blood serum of donors and recipients before liver transplantation was determined The input of particular biochemical analytes into liver transplantation was determined too. The antioxidant parameters were detected using technique of termo-induced chemiluminescence under application of set of corresponding reagents. The analysis of antioxidant activity of blood serum in donors and recipients with hepatic pathology revealed in vivo a significant disorder in the syndrome of compensatory anti-inflammatory defense. Under hepatic pathology, absence of endogenous ascorbic acid, deficiency of exogenous ascorbic acid and disorder of antioxidant activity the uric acid and bilirubin become the major hydrophilic acceptors of active forms of oxygen and inhibitors of oxidative processes in vivo. In patients with physiological level of bilirubin the uric acid provides 40%-80% of antioxidant activity. In case of high hyperbilirubinemia in recipients only 9.6%. It is possible to consider hyperiricosuria under aphysiological processes as a nonspecific test of activation of biological reaction of inflammation, syndrome of compensatory anti-inflammatory defense and test of disorder of biological function of endoecology. To activate the syndrome of compensatory anti-inflammatory defense it is very important to decrease both hyperiricosuria and compensatory function of uric acid as an acceptor of active forms of oxygen by force of prolonged intake of optimal amount of ascorbic acid.

  3. Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.

    PubMed

    Akhterov, Maxim V; Choi, Yongki; Olsen, Tivoli J; Sims, Patrick C; Iftikhar, Mariam; Gul, O Tolga; Corso, Brad L; Weiss, Gregory A; Collins, Philip G

    2015-06-19

    Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.

  4. Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white.

    PubMed

    Abeyrathne, E D N S; Lee, H Y; Ahn, D U

    2014-04-01

    Ovalbumin, ovotransferrin, ovomucin, and lysozyme are a few of the egg white proteins that can be used as functional components. The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using FPC3500 cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin first using 5.0% (wt/vol) (NH4)2SO4 and 2.5% (wt/vol) citric acid combination. After centrifugation, the supernatant (S1) was used for ovalbumin separation and the precipitant was dissolved in water, and reprecipitated using 2.0% ammonium sulfate (wt/vol) and 1.5% citric acid (wt/vol) combination. The precipitant was used as ovotransferrin fraction, and the supernatant (S2) was pooled with the first supernatant (S1), desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was >98% and that of ovotransferrin and lysozyme was >82% for both laboratory and scale-up preparations. The SDS-PAGE and western blotting of the separated proteins, except for ovomucin, showed >90% purity. The ELISA results indicated that the activities of separated ovalbumin, ovotransferrin, and lysozyme were >96%. The protocol separated 4 major proteins in sequence, and the method was simple and easily scaled up.

  5. Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

    PubMed

    Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

    2017-03-01

    Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m(2) or <45 mL/min/1.73 m(2), respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly

  6. Preparation and in vitro photodynamic activity of novel silicon(IV) phthalocyanines conjugated to serum albumins.

    PubMed

    Huang, Jian-Dong; Lo, Pui-Chi; Chen, Yan-Mei; Lai, Janice C; Fong, Wing-Ping; Ng, Dennis K P

    2006-05-01

    The interactions of four novel silicon(IV) phthalocyanines (SiPc), namely SiPc[OC(3)H(5)(NMe(2))(2)](2) (1), SiPc[OC(3)H(5)(NMe(2))(2)](OMe) (2), {SiPc[OC(3)H(5)(NMe(3))(2)](2)}I(4) (3), and {SiPc[OC(3)H(5)(NMe(3))(2)](OMe)}I(2) (4) with human serum albumin (HSA), bovine serum albumin (BSA), and maleylated bovine serum albumin (mBSA) were studied by fluorescence spectroscopy. The fluorescence emission of the serum albumins was effectively quenched by these phthalocyanines mainly through a static quenching mechanism. The higher Stern-Volmer quenching constants for the unsymmetrically substituted phthalocyanines 2 and 4 suggested that they have a stronger interaction with these proteins than the symmetrically substituted analogues 1 and 3. A series of non-covalent BSA or mBSA conjugates of these phthalocyanines were also prepared and evaluated for their in vitro photodynamic activity against HepG2 human hepatocarcinoma cells. The bioconjugation could enhance the photocytotoxicity of 1 and 4 by up to eight folds, but the effects on 2 and 3 were negligible. The results could be partly explained by two counter-balancing effects, namely the enhanced uptake and increased aggregation tendency of phthalocyanine due to BSA conjugation. As shown by absorption spectroscopy, the tetracationic phthalocyanine 3 was significantly aggregated in the protein cavity and its photocytotoxicity remained the lowest among the four photosensitizers.

  7. Lack of sexual activity from erectile dysfunction is associated with a reversible reduction in serum testosterone.

    PubMed

    Jannini, E A; Screponi, E; Carosa, E; Pepe, M; Lo Giudice, F; Trimarchi, F; Benvenga, S

    1999-12-01

    The role of androgenic hormones in human sexuality, in the mechanism of erection and in the pathogenesis of impotence is under debate. While the use of testosterone is common in the clinical therapy of male erectile dysfunction, hypogonadism is a rare cause of impotence. We evaluated serum testosterone levels in men with erectile dysfunction resulting either from organic or non-organic causes before and after non-hormonal impotence therapy. Eighty-three consecutive cases of impotence (70% organic, 30% non-organic, vascular aetiology being the most frequent) were subjected to hormonal screening before and after various psychological, medical (prostaglandin E1, yohimbine) or mechanical therapies (vascular surgery, penile prostheses, vacuum devices). Thirty age-matched healthy men served as a control group. Compared to controls, patients with impotence resulting from both organic and non-organic causes showed reduced serum levels of both total testosterone (11.1 +/- 2.4 vs. 17.7 +/- 5.5 nmol/L) and free testosterone (56.2 +/- 22.9 vs. 79.4 +/- 27.0 pmol/L) (both p < 0.001). Irrespective of the different aetiologies and of the various impotence therapies, a dramatic increase in serum total and free testosterone levels (15.6 +/- 4.2 nmol/L and 73.8 +/- 22.5 pmol/L, respectively) was observed in patients who achieved normal sexual activity 3 months after commencing therapy (p < 0.001). On the contrary, serum testosterone levels did not change in patients in whom therapies were ineffective. Since the pre-therapy low testosterone levels were independent of the aetiology of impotence, we hypothesize that this hormonal pattern is related to the loss of sexual activity, as demonstrated by its normalization with the resumption of coital activity after different therapies. The corollary is that sexual activity may feed itself throughout the increase in testosterone levels.

  8. Impact of Polychlorinated Biphenyls Contamination on Estrogenic Activity in Human Male Serum

    PubMed Central

    Plíšková, Martina; Vondráček, Jan; Canton, Rocio Fernandez; Nera, Jiřií; Kočan, Anton; Petrík, Ján; Trnovec, Tomáš; Sanderson, Thomas; van den Berg, Martin; Machala, Miroslav

    2005-01-01

    Polychlorinated biphenyls (PCBs) are thought to cause numerous adverse health effects, but their impact on estrogen signaling is still not fully understood. In the present study, we used the ER-CALUX bioassay to determine estrogenic/antiestrogenic activities of the prevalent PCB congeners and PCB mixtures isolated from human male serum. The samples were collected from residents of an area with an extensive environmental contamination from a former PCB production site as well as from a neighboring background region in eastern Slovakia. We found that the lower-chlorinated PCBs were estrogenic, whereas the prevalent higher-chlorinated PCB congeners 138, 153, 170, 180, 187, 194, 199, and 203, as well as major PCB metabolites, behaved as anti-estrogens. Coplanar PCBs had no direct effect on estrogen receptor (ER) activation in this in vitro model. In human male serum samples, high levels of PCBs were associated with a decreased ER-mediated activity and an increased dioxin-like activity, as determined by the DR-CALUX assay. 17β-Estradiol (E2) was responsible for a major part of estrogenic activity identified in total serum extracts. Significant negative correlations were found between dioxin-like activity, as well as mRNA levels of cytochromes P450 1A1 and 1B1 in lymphocytes, and total estrogenic activity. For sample fractions containing only persistent organic pollutants (POPs), the increased frequency of anti-estrogenic samples was associated with a higher sum of PCBs. This suggests that the prevalent non-dioxin-like PCBs were responsible for the weak antiestrogenic activity of some POPs fractions. Our data also suggest that it might be important to pay attention to direct effects of PCBs on steroid hormone levels in heavily exposed subjects. PMID:16203234

  9. Impact of polychlorinated biphenyls contamination on estrogenic activity in human male serum.

    PubMed

    Plísková, Martina; Vondrácek, Jan; Canton, Rocio Fernandez; Nera, Jirí; Kocan, Anton; Petrík, Ján; Trnovec, Tomás; Sanderson, Thomas; van den Berg, Martin; Machala, Miroslav

    2005-10-01

    Polychlorinated biphenyls (PCBs) are thought to cause numerous adverse health effects, but their impact on estrogen signaling is still not fully understood. In the present study, we used the ER-CALUX bioassay to determine estrogenic/antiestrogenic activities of the prevalent PCB congeners and PCB mixtures isolated from human male serum. The samples were collected from residents of an area with an extensive environmental contamination from a former PCB production site as well as from a neighboring background region in eastern Slovakia. We found that the lower-chlorinated PCBs were estrogenic, whereas the prevalent higher-chlorinated PCB congeners 138, 153, 170, 180, 187, 194, 199, and 203, as well as major PCB metabolites, behaved as antiestrogens. Coplanar PCBs had no direct effect on estrogen receptor (ER) activation in this in vitro model. In human male serum samples, high levels of PCBs were associated with a decreased ER-mediated activity and an increased dioxin-like activity, as determined by the DR-CALUX assay. 17beta-Estradiol (E2) was responsible for a major part of estrogenic activity identified in total serum extracts. Significant negative correlations were found between dioxin-like activity, as well as mRNA levels of cytochromes P450 1A1 and 1B1 in lymphocytes, and total estrogenic activity. For sample fractions containing only persistent organic pollutants (POPs), the increased frequency of antiestrogenic samples was associated with a higher sum of PCBs. This suggests that the prevalent non-dioxin-like PCBs were responsible for the weak antiestrogenic activity of some POPs fractions. Our data also suggest that it might be important to pay attention to direct effects of PCBs on steroid hormone levels in heavily exposed subjects.

  10. Effects of perfluorooctane sulfonate on the conformation and activity of bovine serum albumin.

    PubMed

    Wang, Yanqing; Zhang, Hongmei; Kang, Yijun; Cao, Jian

    2016-06-01

    Perfluorooctane sulfonate (PFOS) is among the most prominent contaminates in human serum and has been reported to possess potential toxicity to the human body. In this study, the effects of PFOS on the conformation and activity of bovine serum albumin (BSA) were investigated in vitro. The results indicated that the binding interaction of PFOS with BSA destroyed the tertiary and secondary structures of protein with the loss of α-helix structure and the increasing of hydrophobic microenvironment of the Trp or Tyr residues. During the thermal denaturation protein, PFOS increases the protein stability of BSA. The proportion of α-helix decreased on increasing the PFOS concentration and the microenvironment of the Trp or Tyr residues becomes more hydrophobic. The results from molecular modeling indicated that BSA had not only one possible binding site to bind with PFOS by the polar interaction, hydrogen bonds and hydrophobic forces. In addition, the BSA relative activities were decreased with the increase of PFOS concentration. Such loss of BSA activity in the presence of PFOS indicated that one of the binding sites in BSA is located in subdomain IIIA, which is in good agreement with the fluorescence spectroscopic experiments and molecular modeling results. This study offers a comprehensive picture of the interactions of PFOS with serum albumin and provides insights into the toxicological effect of perfluoroalkylated substances.

  11. Serum enzyme activities following long-distance running: comparison between Ethiopian and white athletes.

    PubMed

    Eliakim, A; Nemet, D; Shenkman, L

    1995-11-01

    Ethiopian runners are famous for their achievements in long-distance running. The recent immigration of Ethiopians to Israel provided an opportunity to compare some physiological variables between elite Ethiopian and white Israeli runners. Six Ethiopian and five white Israeli runners, aged 20 to 40 years, were studied before and after an 11 km race. Venous blood was sampled from each runner prior to the race, and 1 h, 48 h, and 5 days following the race. The activities of creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured, and levels of serum electrolytes, urea, creatinine, phosphorus, albumin, cholesterol, and alkaline phosphatase were also determined. CK activity rose 2.6-fold with a peak 5 days after the race. LDH and AST levels rose as well (1.4-1.3-fold, respectively). Significant elevations also occurred in serum phosphorus, uric acid, and creatinine concentrations 1 h after the race. In contrast to previous studies in which higher enzyme activities were reported in blacks, we did not detect any difference in serum enzyme values between black and white runners.

  12. Secretory phospholipase A2 activity in blood serum: the challenge to sense.

    PubMed

    Alekseeva, A S; Korotaeva, A A; Samoilova, E V; Volynsky, P E; Vodovozova, E L; Boldyrev, I A

    2014-11-07

    Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.

  13. Serum pyrrolidone carboxypeptidase activity in N-methyl-nitrosourea induced rat breast cancer.

    PubMed

    Carrera, M P; Ramírez-Expósito, M J; Valenzuela, M T; García, M J; Mayas, M D; Martínez-Martos, J M

    2003-08-01

    Pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3) is an omega peptidase widely distributed in animal fluids and tissues and hydrolyses N-terminal pyroglutamic residues from biologically active peptides such as gonadotropin releasing hormone (GnRH). Previous results obtained by us showed a decrease in human breast cancer Pcp activity, suggesting that this enzyme activity or its putative substrates may play a major role in breast cancer pathogenesis. The aim of the present work is to analyse serum Pcp activity in N-methyl-nitrosourea (NMU) induced rat mammary tumours using pyroglutamyl-beta-naphthylamide as substrate. Serum Pcp activity was significantly lower in NMU-treated rats than in controls. Moreover, multiple regression analysis showed a significant correlation between Pcp activity and the number and size of tumours and the body weight of the animals. Since NMU-induced carcinomas are mainly oestrogen-dependent, the decrease observed in Pcp activity may reflect an increase in circulating levels of GnRH that lead to an increase in gonadal steroid hormones production responsible, at least in part, for the initiation and promotion of the disease.

  14. Activation of the serum response factor by p65/NF-kappaB.

    PubMed Central

    Franzoso, G; Carlson, L; Brown, K; Daucher, M B; Bressler, P; Siebenlist, U

    1996-01-01

    This study demonstrates that the NF-kappaB subunit p65 can act like an accessory protein for the serum response factor (SRF) in transfection assays. p65 functionally synergizes with SRF to activate the transcription of a reporter construct dependent only on the serum response element (SRE). The synergy of the two factors requires neither a kappaB motif nor direct contact of p65 with DNA. Consistent with these results, a physical complex containing p65 and SRF is observed in vitro. Synergy of the factors is independent of the previously described activation domains present on p65, ruling out indirect effects of p65, but synergy is dependent on the activation domain of SRF. The complexing of p65 and SRF is mediated by a segment of the SRF DNA binding domain, a region of the protein which has also been reported to inhibit its own activation domain. We speculate that p65, upon direct or facilitated interaction with SRF, may relieve the inhibitory activity of this segment, thus enabling the activation domain of SRF to become fully functional. In contrast to p65, the p50 subunit of NF-kappaB does not interact significantly with SRF, either functionally or physically. The data suggest the intriguing possibility that NF-kappaB may participate in the regulation of SRE-dependent promoters, expanding the range of activities of this rapidly activatable transcription factor. Images PMID:8670842

  15. Effects of penicillinase on bactericidal and complement activities in normal human serum.

    PubMed Central

    Biggs, W H; Wunderlich, A C; Corbeil, L C; Davis, C E; Curd, J G

    1983-01-01

    During routine addition of penicillinase (beta-lactamase) to patients sera, we found that the capacity of some of these sera to kill serum-sensitive gram-negative organisms was significantly decreased. Further controlled studies showed that penicillinase decreased both the bactericidal activity of normal human sera and the total hemolytic activity (CH50) of complement in these sera. The decreased bactericidal activity correlated significantly (r = 0.57, P less than 0.05) with the reduction of CH50 in eight normal sera. These effects of penicillinase were time and temperature dependent. Measurement of individual complement component activities showed that penicillinase decreased the activity of C2, C4, and C3-C9, suggesting that the penicillinase preparation activated the classical pathway. These results cast doubts on the validity of bactericidal determinations when sera are pretreated with penicillinase. PMID:6603195

  16. The Serum Complement System: A Simplified Laboratory Exercise to Measure the Activity of an Important Component of the Immune System

    ERIC Educational Resources Information Center

    Inglis, Jordan E.; Radziwon, Kimberly A.; Maniero, Gregory D.

    2008-01-01

    The immune system is a vital physiological component that affords animals protection from disease and is composed of innate and adaptive mechanisms that rely on cellular and dissolved components. The serum complement system is a series of dissolved proteins that protect against a variety of pathogens. The activity of complement in serum can be…

  17. Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis

    PubMed Central

    Bianchini, A.A.C.; Petroni, T.F.; Fedatto, P.F.; Bianchini, R.R.; Venancio, E.J.; Itano, E.N.; Ono, M.A.

    2009-01-01

    The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis. PMID:24031350

  18. Two-step chromatography purification of IgGs possessing sialidase activity from human blood serum.

    PubMed

    Kit, Yury; Bilyy, Rostyslav; Korniy, Nataliya; Tomin, Andriy; Chop'yak, Valentyna; Tolstyak, Yaroslav; Antonyuk, Volodymyr; Stoika, Rostyslav

    2015-03-01

    Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G-Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin-Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum.

  19. Evaluation of a new substrate for measurement of serum PON arylesterase activity.

    PubMed

    Mu, Xiaojing; Yu, Nana; Wang, Chengshan; Zou, Xiaobing; Abulimite, Zulipiyan; Xia, Zhining

    2012-01-15

    It was found that the hydrolysis of 9-(4-chlorophenyloxycarbonyl)-10-methylacridinium triflate (CPOCMA) could be catalyzed by recombinant human PON1. Based on this property, the CPOCMA was evaluated as a substrate for serum PON activity assay. The apparent K(m) value of a serum sample for the substrate was determined as 85nmol/L, close to the K(m) value (83nmol/L) of rHuPON1. The interferences by other esterase such as acetylcholinerase and lipases were investigated. The NaCl and CaCl(2) as PON activity enhancers were able to improve the specific signal, respectively. The rHuPON1 in presence of CaCl(2) showed at least 7.8 times selectivity over acetylcholinerase and lipases. By comparing with the UV methods based on phenyl acetate and diazinon, respectively, the proposed chemiluminescent method was validated with 30 serum samples. The method based on CPOCMA allows reliable, cost-saving, and specific determination in a buffer of physiological pH.

  20. Observed activities of serum creatine kinase: total and B subunit activity and other enzymes in young persons abusing solvents.

    PubMed Central

    Spooner, R J; Birrell, R C; McLelland, A S; Baird, J A; Sourindhrin, I

    1984-01-01

    Serum enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase (ALP), gamma-glutamyltransferase, and creatine kinase (CK] were measured in 296 young persons who admitted to recent inhalation of solvents, usually toluene based glues. In general, results fell within expected adult reference ranges except for ALP and CK. About 60% of subjects had CK activities above the upper reference limit and these activities were investigated in terms of their isoenzyme composition. CK B subunit activity was measured in 90 subjects with raised total CK activities. In five instances the CK B subunit activity was judged abnormal and in two subjects the presence of CK BB was confirmed. These two subjects were thought to have a circulating macro CK, type 1. It is concluded that the increased total CK activity found in this group of solvent abusers was due to physical activity, but a contribution from specific muscle toxicity by solvents cannot be excluded. PMID:6142904

  1. The Effects of Acetate Buffer Concentration on Lysozyme Solubility

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth L.; Pusey, Marc L.

    1996-01-01

    The micro-solubility column technique was employed to systematically investigate the effects of buffer concentration on tetragonal lysozyme solubility. While keeping the NaCl concentrations constant at 2%, 3%, 4%, 5% and 7%, and the pH at 4.0, we have studied the solubility of tetragonal lysozyme over an acetate buffer concentration range of 0.01M to 0.5M as a function of temperature. The lysozyme solubility decreased with increasing acetate concentration from 0.01M to 0.1M. This decrease may simply be due to the net increase in solvent ionic strength. Increasing the acetate concentration beyond 0.1M resulted in an increase in the lysozyme solubility, which reached a peak at - 0.3M acetate concentration. This increase was believed to be due to the increased binding of acetate to the anionic binding sites of lysozyme, preventing their occupation by chloride. In keeping with the previously observed reversal of the Hoffmeister series for effectiveness of anions in crystallizing lysozyme, acetate would be a less effective precipitant than chloride. Further increasing the acetate concentration beyond 0.3M resulted in a subsequent gradual decrease in the lysozyme solubility at all NaCl concentrations.

  2. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

    PubMed Central

    Yamamoto, N; Kumashiro, R; Yamamoto, M; Willett, N P; Lindsay, D D

    1993-01-01

    A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited. PMID:8225612

  3. Extra-Articular Symptoms in Constellation with Selected Serum Cytokines and Disease Activity in Spondyloarthritis

    PubMed Central

    Fischer, Katarzyna; Brzosko, Marek

    2016-01-01

    Objectives. In this study, we assessed the extra-articular symptoms in constellation with selected serum cytokines and disease activity in spondyloarthritis (SpA). Patients and Methods. We studied 287 SpA patients: 131 had AS, 110 had PsA, and 46 had SAPHO. We assessed extra-articular symptoms in all cases. In 191 SpA patients, we measured serum interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-23 (IL-23), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). Results. Patients with acute anterior uveitis (AAU) had higher VAS (P = 0.0008), BADSDAI (P = 0.0001), ASDAS-ESR (P = 0.04), CRP (P = 0.006), IL-6 (P = 0.02), and IL-18 (P = 0.03) levels. Patients with inflammatory bowel disease (IBD) had higher VAS (P = 0.03), CRP (P = 0.0009), and IL-6 (P = 0.0003) levels. Patients with skin psoriasis had lower VAS (P = 0.001) and BASDAI (P = 0.00007) levels. Patients with psoriatic onycholysis had lower VAS (P = 0.006), BASDAI (P = 0.00001), and CRP (P = 0.02) and higher IL-23 (P = 0.04) levels. Patients with PPP had lower BASDAI (P = 0.04) and higher ET-1 (P = 0.001) levels. Conclusions. SpA patients with increased serum IL-18 and decreased serum ET-1 had an increased risk of extra-articular symptoms. In SpA patients, increased disease activity was associated with an increased risk of AAU and IBD and a decreased risk of skin psoriasis, psoriatic onycholysis, and PPP. PMID:28053373

  4. Extra-Articular Symptoms in Constellation with Selected Serum Cytokines and Disease Activity in Spondyloarthritis.

    PubMed

    Przepiera-Będzak, Hanna; Fischer, Katarzyna; Brzosko, Marek

    2016-01-01

    Objectives. In this study, we assessed the extra-articular symptoms in constellation with selected serum cytokines and disease activity in spondyloarthritis (SpA). Patients and Methods. We studied 287 SpA patients: 131 had AS, 110 had PsA, and 46 had SAPHO. We assessed extra-articular symptoms in all cases. In 191 SpA patients, we measured serum interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-23 (IL-23), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). Results. Patients with acute anterior uveitis (AAU) had higher VAS (P = 0.0008), BADSDAI (P = 0.0001), ASDAS-ESR (P = 0.04), CRP (P = 0.006), IL-6 (P = 0.02), and IL-18 (P = 0.03) levels. Patients with inflammatory bowel disease (IBD) had higher VAS (P = 0.03), CRP (P = 0.0009), and IL-6 (P = 0.0003) levels. Patients with skin psoriasis had lower VAS (P = 0.001) and BASDAI (P = 0.00007) levels. Patients with psoriatic onycholysis had lower VAS (P = 0.006), BASDAI (P = 0.00001), and CRP (P = 0.02) and higher IL-23 (P = 0.04) levels. Patients with PPP had lower BASDAI (P = 0.04) and higher ET-1 (P = 0.001) levels. Conclusions. SpA patients with increased serum IL-18 and decreased serum ET-1 had an increased risk of extra-articular symptoms. In SpA patients, increased disease activity was associated with an increased risk of AAU and IBD and a decreased risk of skin psoriasis, psoriatic onycholysis, and PPP.

  5. Serum platelet factor 4 is a reliable activity parameter in adult patients with inflammatory bowel disease

    PubMed Central

    Ye, Lei; Zhang, Yu-Ping; Yu, Na; Jia, Ya-Xu; Wan, Shu-Jun; Wang, Fang-Yu

    2017-01-01

    Abstract To investigate the diagnostic utility of serum platelet factor 4 (PF4) levels and to assess its accuracy in detecting inflammatory bowel disease activity. This study included 45 patients with ulcerative colitis (UC), 45 patients with Crohn disease (CD), and 30 control subjects at Jinling Hospital between May 2014 and July 2015. Laboratory tests measured white blood count, C-reactive protein, erythrocyte sedimentation rate, and platelet count. PF4 was examined by enzyme-linked immunosorbent assays. Patients were divided into 2 groups according to disease activity: active and inactive. Median PF4 values dramatically increased in UC and CD patients compared with the healthy group (UC: 26.64 [20.00–36.22] mg/mL vs 20.02 [14.63–26.83] mg/mL, P = 0.002; CD: 25.56 [18.57–36.36] mg/mL vs 20.02 [14.63–26.83] mg/mL, P = 0.014); however, the serum PF4 levels between UC and CD failed to show a significant difference (26.64 [20.00–36.22] mg/mL vs 25.56 [18.57–36.36] mg/mL, P = 0.521). Furthermore, serum PF4 levels were elevated in both UC and CD patients with active disease (UC: 20.19 [14.89–23.53] mg/mL vs 28.86 [22.57–37.29] mg/mL, P < 0.001; CD: 18.33 [16.72–25.77] mg/mL vs 34.38 [22.58–39.92] mg/mL, P < 0.001). Multivariate analysis revealed higher PF4 level as an independent predictor of disease activity in UC and CD patients (UC: odds ratio 30.375, P = 0.002; CD: odds ratio 54.167, P < 0.001). The cut-off level of PF4 for distinguishing active from inactive UC patients was 24.1 mg/mL. While in CD patients, the cut-off level of PF4 was 19.24 mg/mL. Serum PF4 levels could be a potential biomarker for monitoring the disease activity of inflammatory bowel disease. PMID:28296751

  6. Dimer formation in radiation-irradiated aqueous solution of lysozyme studied by light-scattering-intensity measurement.

    PubMed

    Hashimoto, S; Seki, H; Masuda, T; Imamura, M; Kondo, M

    1981-07-01

    The reaction of lysozyme with OH., Br.-2 and e-aq, produced in an aqueous solution by pulsed electrons and gamma-rays, were investigated. Irradiated enzymes showed an increase in the light scattering intensity (LSI) which is proportional to the absorbed dose. Results obtained from SDS gel electrophoresis confirm dimerization of lysozyme, which is considered to be responsible for the increase in LSI. It was found that the rate constant of the dimerization of protein radicals produced in the reaction with OH. is 2K=(1.0 +/- 0.3) X 10(6)M-1 s-1 and the yield of the dimerization is 0.6 in G. The enzymatic activity of the dimer is shown to be reduced to about 30 per cent of that of the intact enzyme. It is concluded that the radiation-induced inactivation of lysozyme is largely due to dimerization.

  7. Direct biomolecule binding on nonfouling surfaces via newly discovered supramolecular self-assembly of lysozyme under physiological conditions.

    PubMed

    Yang, Peng

    2012-08-01

    When lysozyme is dissolved in a neutral HEPES buffer solution (pH = 7.4) with 0.001-0.050 M TCEP added, a fast phase transition process occurs and the resulting novel fiber-like hierarchical supramolecular assemblies made by primary spherical-particle aggregation can function as a "superglue" that binds strongly and quickly onto non-fouling coatings. This binding is highly selective towards lysozyme, and excludes synthetic, chemical/physical activation/deactivation (blocking) steps. By using biotinylated lysozyme, such a phase transition quickly creates a perfect biotinylated surface on non-fouling surfaces for avidin binding, showing great potential for the development of low-cost and practical biochips.

  8. Active fragments of the antihemorrhagic protein HSF from serum of habu (Trimeresurus flavoviridis).

    PubMed

    Aoki, Narumi; Deshimaru, Masanobu; Terada, Shigeyuki

    2007-04-01

    Certain snakes have antihemorrhagic proteins in their sera. Habu serum factor (HSF), an antihemorrhagic protein isolated from the serum of the Japanese habu snake (Trimeresurus flavoviridis) is composed of two cystatin-like domains (D1 and D2) and a His-rich domain, and it inhibits several snake venom metalloproteinases (SVMPs). The activity of HSF can be abolished by trinitrophenylation of Lys residues with 2,4,6-trinitrobenzene sulphonic acid. Upon complex formation of HSF with SVMP, however, the loss of its inhibitory activity by the chemical modification was suppressed, and Lys(15), Lys(41), and Lys(103) residues in HSF were not trinitrophenylated. In order to identify the domain that is critical to the inhibitory activity on SVMPs, native HSF was digested with papain followed by cleavage with cyanogen bromide, yielding a low-molecular mass fragment that was composed of two peptide chains (residues 5-89 and 312-317) linked by a disulfide bond. This fragment inhibited several SVMPs and showed significant antihemorrhagic activity. This indicates that the N-terminal half of D1 is indispensable for the antihemorrhagic activity of HSF. Furthermore, a three-dimensional model of two cystatin-like domains constructed by the homology modeling has indicated that three Lys residues (15, 41, and 103) are exposed to the same surface of HSF molecule.

  9. Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

    SciTech Connect

    Mamontov, Eugene; O'Neill, Hugh Michael

    2014-01-01

    Recent studies have outlined the use of eutectic solution of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without the solvent crystallization. The eutectic point in (H2O)R(LiCl) system corresponds to R 7.3, and it is of interest to investigate whether less concentrated aqueous solutions of LiCl could be employed in low-temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low-temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins.

  10. Aging-Related Correlation between Serum Sirtuin 1 Activities and Basal Metabolic Rate in Women, but not in Men

    PubMed Central

    2017-01-01

    Sirtuin (SIRT) is a main regulator of metabolism and lifespan, and its importance has been implicated in the prevention against aging-related diseases. The purpose of this study was to identify the pattern of serum SIRT1 activity according to age and sex, and to investigate how serum SIRT1 activity is correlated with other metabolic parameters in Korean adults. The Biobank of Jeju National University Hospital, a member of the Korea Biobank Network, provided serum samples from 250 healthy adults. Aging- and metabolism-related factors were analyzed in serum, and the data were compared by the stratification of age and sex. Basal metabolic rate (BMR) decreased with age and was significantly lower in men in their fifties and older and in women in their forties and older compared with twenties in men and women, respectively. SIRT1 activities were altered by age and sex. Especially, women in their thirties showed the highest SIRT1 activities. Correlation analysis displayed that SIRT1 activity is positively correlated with serum triglyceride (TG) in men, and with waist circumference, systolic blood pressure, diastolic blood pressure, and serum TG in women. And, SIRT1 activity was negatively correlated with aspartate aminotransferase/alanine aminotransferase ratio in women (r = −0.183, p = 0.039). Positive correlation was observed between SIRT1 activity and BMR in women (r = 0.222, p = 0.027), but not in men. Taken together, these findings suggest the possibility that serum SIRT1 activities may be utilized as a biomarker of aging. In addition, positive correlation between SIRT1 activity and BMR in women suggests that serum SIRT1 activity may reflect energy expenditure well in human. PMID:28168178

  11. Structural characteristics of hydration sites in lysozyme.

    PubMed

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

    2011-06-01

    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.

  12. Serum and blister fluid pharmacokinetics and bactericidal activities of ampicillin-sulbactam, cefotetan, cefoxitin, ceftizoxime, and ticarcillin-clavulanate.

    PubMed

    Jaresko, G S; Barriere, S L; Johnson, B L

    1992-10-01

    Ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, cefotetan, and ceftizoxime are promoted for the treatment of mixed aerobic-anaerobic bacterial infections. Their activities have been compared in vitro but not in vivo. In order to assess the in vivo activities of these agents in serum and interstitial fluid, we administered single, intravenous doses of these antimicrobial agents to healthy subjects. Concentrations of the antimicrobial agents in serum and suction-induced blister fluid and bactericidal activity were measured by high-pressure liquid chromatography and the standard methodology of the National Committee for Clinical Laboratory Standards, respectively. The organisms used for bactericidal activity tests were one isolate each of Staphylococcus aureus, Klebsiella pneumoniae, and Bacteroides fragilis. Pharmacokinetic parameters in serum and blister fluid were similar to those derived in other investigations. Of note were the high and prolonged concentrations of ticarcillin and cefotetan in blister fluid, despite high-level serum protein binding. The bactericidal activities in serum and blister fluid reflected the relative in vitro activities and kinetic dispositions of the various antimicrobial agents except for the bactericidal activity of cefotetan, which was substantially lower in blister fluid than serum, despite a blister fluid:serum area under the concentration-time curve ratio of 1.5. Similarly, the activity of ticarcillin-clavulanate in blister fluid was also substantially less than would have been predicted by the blister fluid:serum ratio of the area under the concentration-time curve of 1.1, possibly because of the low concentrations of clavulanate in blister fluid. The rankings of the in vivo bactericidal activities of the five drugs were as follows: for S. aureus, ampicillin-sulbactam > ticarcillin-clavulanate > ceftizoxime > cefoxitin > cefotetan; for K. pneumoniae, ceftizoxime > cefotetan > ampicillin-sulbactam = ticarcillin

  13. Elevation of serum urokinase plasminogen activator receptor and liver stiffness in postoperative biliary atresia

    PubMed Central

    Udomsinprasert, Wanvisa; Honsawek, Sittisak; Jirathanathornnukul, Napaphat; Chongsrisawat, Voranush; Poovorawan, Yong

    2016-01-01

    AIM To investigate serum urokinase-type plasminogen activator receptor (uPAR) and liver stiffness in biliary atresia (BA) and examine the correlation of circulating uPAR, liver stiffness, and clinical outcomes in postoperative BA children. METHODS Eighty-five postKasai BA children and 24 control subjects were registered. Circulating uPAR was measured using enzyme-linked immunosorbent essay. Liver stiffness was analyzed using transient elastography. RESULTS BA children had significantly greater circulating uPAR and liver stiffness scores than control subjects (P < 0.001). Circulating uPAR and liver stiffness were substantially higher in jaundiced BA children than non-jaundiced BA children (P < 0.001). In addition, circulating uPAR was positively associated with serum aspartate aminotransferase (r = 0.507, P < 0.001), alanine aminotransferase (r = 0.364, P < 0.001), total bilirubin (r = 0.559, P < 0.001), alkaline phosphatase (r = 0.325, P < 0.001), and liver stiffness scores (r = 0.508, P < 0.001). CONCLUSION Circulating uPAR and liver stiffness values were greater in BA children than healthy controls. The increased circulating uPAR was associated with liver dysfunction in BA. As a consequence, serum uPAR and liver stiffness may be used as noninvasive biomarkers indicating the progression of liver fibrosis in postKasai BA. PMID:27957246

  14. Nanopore-based electrical and label-free sensing of enzyme activity in blood serum.

    PubMed

    Kukwikila, Mikiembo; Howorka, Stefan

    2015-09-15

    A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing.

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

    PubMed

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

    2015-01-01

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

  16. Carboxymethyl chitosan-poly(amidoamine) dendrimer core-shell nanoparticles for intracellular lysozyme delivery.

    PubMed

    Zhang, Xiaoyang; Zhao, Jun; Wen, Yan; Zhu, Chuanshun; Yang, Jun; Yao, Fanglian

    2013-11-06

    Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

  17. Formulation of antiacne serum based on lime peel essential oil and in vitro antibacterial activity test against Propionibacterium acnes

    NASA Astrophysics Data System (ADS)

    Fitri, Noor; Fatimah, Ifat; Chabib, Lutfi; Fajarwati, Febi Indah

    2017-03-01

    Propionibacterium acnes are a normal bacterium in human skin but it can become primary pathogens that can cause inflammation on the skin. Research about new antibacterial compounds is important because resistance of bacteria acne to antibiotics. Some of Essential oils have antibacterial properties. Lime peel essential oil and patchouli essential oil have some terpenoids that act as antibacterial compounds such as Linalool and Seychellene. The purpose of this research was to formulate anti acne serum based on lime peel essential oil and patchouli oil and to determine the zone of inhibition against of Propionibacterium acnes. This study made 21 variations of formulation of anti acne serum, consisted of lime peel essential oil, patchouli oil and olive oil. Anti acne serum was evaluated i.e. in vitro antibacterial activity test against Propionibacterium acnes for 5 days, organoleptic, stability test, pH test, viscosity test and GC-MS analysis. Nine serum formulations had been selected, which based on their most favorite order. Those favorite serums had antibacterial inhibitory against Propionibacterium acnes between 20.80 - 26.12 mm, whereas control positive only 12.47 mm and control negative 5.78 mm. The most favorite serum with the best antibacterial activity was serum formula A. The composition of serum A consist of lime peel essential oil: patchouli oil: olive oil (11:1:18).

  18. Serum β-Glucuronidase Activity in Response to Fruit and Vegetable Supplementation: A Controlled Feeding Study

    PubMed Central

    Maruti, Sonia S.; Chang, Jyh-Lurn; Prunty, JoAnn; Bigler, Jeannette; Schwarz, Yvonne; Li, Shuying S.; Li, Lin; King, Irena B.; Potter, John D.; Lampe, Johanna W.

    2008-01-01

    Background Fruit and vegetable intake may lower the risk of some cancers. One hypothesized, but understudied, chemopreventive mechanism is that plant food constituents inhibit β-glucuronidase, an acid hydrolase that deconjugates glucuronides. Methods We conducted a cross-over feeding trial in 63 healthy women and men aged 20−40 years, to examine the effect of diet on serum β-glucuronidase activity. Participants were randomized to 2 two-week experimental diets with an intervening washout period: a diet high in selected citrus fruit, crucifers, and soy (F&V) and a diet devoid of fruits, vegetables, and soy (basal). Serum β-glucuronidase activity was measured during the pre-intervention, F&V, and basal periods. Linear mixed models were used to obtain effect estimates and 95% confidence intervals (CI). Results We observed statistically significantly higher β-glucuronidase activity during the F&V than the basal diet (ratio, F&V versus basal diet, 1.09; 95% CI, 1.05−1.13; P <0.01). These results were probably due to decreased β-glucuronidase activity during the basal diet (ratio, basal period versus pre-intervention, 0.93; 95% CI, 0.87−0.98; P=0.01), rather than increased enzyme activity during the F&V diet (ratio, F&V period versus pre-intervention, 1.01; 95% CI, 0.96−1.06; P=0.64). The diet-enzyme activity relation did not differ by sex (P interaction=0.30), but there was a suggestion of a short-term diet effect at 8 days versus 15 days (P interaction=0.06). Conclusion This intervention of selected fruits and vegetables did not lower β-glucuronidase activity. Further investigation is needed regarding what other foods and phytochemicals may influence β-glucuronidase activity and effect modifiers of this relation PMID:18628435

  19. Hydrogen sulfide reduces serum triglyceride by activating liver autophagy via the AMPK-mTOR pathway.

    PubMed

    Sun, Li; Zhang, Song; Yu, Chengyuan; Pan, Zhenwei; Liu, Yang; Zhao, Jing; Wang, Xiaoyu; Yun, Fengxiang; Zhao, Hongwei; Yan, Sen; Yuan, Yue; Wang, Dingyu; Ding, Xue; Liu, Guangzhong; Li, Wenpeng; Zhao, Xuezhu; Liu, Zhaorui; Li, Yue

    2015-12-01

    Autophagy plays an important role in liver triglyceride (TG) metabolism. Inhibition of autophagy could reduce the clearance of TG in the liver. Hydrogen sulfide (H2S) is a potent stimulator of autophagic flux. Recent studies showed H2S is protective against hypertriglyceridemia (HTG) and noalcoholic fatty liver disease (NAFLD), while the mechanism remains to be explored. Here, we tested the hypothesis that H2S reduces serum TG level and ameliorates NAFLD by stimulating liver autophagic flux by the AMPK-mTOR pathway. The level of serum H2S in patients with HTG was lower than that of control subjects. Sodium hydrosulfide (NaHS, H2S donor) markedly reduced serum TG levels of male C57BL/6 mice fed a high-fat diet (HFD), which was abolished by coadministration of chloroquine (CQ), an inhibitor of autophagic flux. In HFD mice, administration of NaSH increased the LC3BII-to-LC3BI ratio and decreased the p62 protein level. Meanwhile, NaSH increased the phosphorylation of AMPK and thus reduced the phosphorylation of mTOR in a Western blot study. In cultured LO2 cells, high-fat treatment reduced the ratio of LC3BII to LC3BI and the phosphorylation of AMPK, which were reversed by the coadministration of NaSH. Knockdown of AMPK by siRNA in LO2 cells blocked the autophagic enhancing effects of NaSH. The same qualitative effect was observed in AMPKα2(-/-) mice. These results for the first time demonstrated that H2S could reduce serum TG level and ameliorate NAFLD by activating liver autophagy via the AMPK-mTOR pathway.

  20. Serum Vitamin D Level and Rheumatoid Arthritis Disease Activity: Review and Meta-Analysis

    PubMed Central

    Lin, Jin; Liu, Jian; Davies, Michael L.; Chen, Weiqian

    2016-01-01

    Background The evidence from epidemiological studies concerning the relationship between serum vitamin D concentrations and rheumatoid arthritis (RA) is inconsistent. This meta-analysis is aimed at determining the magnitude of the correlation between this common autoimmune disease and vitamin D, an important nutrient known to dampen adaptive immune responses. Methods Through multiple search strategies, relevant literature was identified and evaluated for quality before May 16 2015. Data extracted from eligible studies was synthesized to calculate pooled correlation coefficient (r), mean difference (MD) and odds ratio (OR). The Venice criteria were applied to assess the credibility of the evidence for each statistically significant association. Results A total of 24 reports involving 3489 patients were selected for analysis. RA patients had lower vitamin D levels than healthy controls (MD:-16.52 nmol/L, 95% confidence intervals [CI]:-18.85 to -14.19 nmol/L). There existed a negative relationship between serum 25-hydroxyvitamin D (25OHD) level and disease activity index, e.g. 25OHD vs. Disease Activity Score in 28 joints (DAS28): r = -0.13, 95% CI -0.16 to -0.09; 25OHD vs. C-reactive protein: r = -0.12, 95% CI -0.23 to -0.00. Additionally, latitude-stratified subgroup analysis yielded a relatively stronger negative correlation between 25OHD and DAS28 in low-latitude areas. This inverse relationship also appeared more significant in developing countries than in developed countries. No publication bias was detected. Conclusion RA patients had lower vitamin D values than healthy controls. There was a negative association between serum vitamin D and RA disease activity. However, more strictly controlled studies are needed to validate these findings. PMID:26751969

  1. Anaemia, Serum Iron Concentrations and δ-Aminolevulinate Dehydratase Activity in Laying Hens Infected Naturally by Salmonella Gallinarum.

    PubMed

    Machado, A C; Boiago, M M; do Carmo, G M; Bottari, N B; Araujo, D N; Giuriatti, J; Morsch, V M; Schetinger, M R C; Casagrande, R A; Wisser, C S; Stefani, L M; Alves, M S; Da Silva, A S

    2016-07-01

    The aim of this study was to evaluate anaemia, serum iron concentrations and δ-aminolevulinate dehydratase (ALA-D) activity in laying hens infected naturally by Salmonella Gallinarum and having severe hepatic lesions. Liver and serum samples were collected from 27 laying hens (20 infected and seven uninfected). The δ-ALA-D activity, haematocrit and serum iron concentrations were evaluated. There were significant decreases in δ-ALA-D activity, haematocrit and serum iron concentrations (P <0.01) in birds infected by S. Gallinarum when compared with uninfected birds. There was a positive correlation (P <0.001) between serum iron concentration, haematocrit (r(2) = 0.82) and δ-ALA-D activity (r(2) = 0.75). A positive correlation was also observed between δ-ALA-D activity and haematocrit (r(2) = 0.78; P <0.01). Liver samples showed moderate focal coagulative necrosis associated with infiltration of lymphoplasmacytic cells, macrophages and heterophils. The anaemia in the infected hens may be related to reduction in δ-ALA-D activity and serum iron concentrations, since both are important for haemopoiesis.

  2. [Bactericidal activity of serum and chemotherapy in sensitive and resistant exciter (author's transl)].

    PubMed

    Eyer, H; Metz, H; Preac-Mursic, V

    1975-11-21

    Comparing examinations with Ampicillin sensitive and resistant bacteria-strains show that the bactericidal activity of serum is dependent on the bacteria-strains, on the Ampicillin sensitivity of the particular exciter and on the number of bacteria/ml (germ count). Bactericide effect could always be obtained with sensitive strains as a result of additional chemotherapy. With several resistant strains a bactericide effect could not be obtained in this case the continuous optimal Ampicillin addition was the decisive factor. Because of the extremely complicated process of the bactericide one should not make general conclusions from the individual experimental results.

  3. Dielectric and gravimetric studies of water binding to lysozyme.

    PubMed

    Bone, S

    1996-08-01

    Time domain dielectric spectroscopy and hydration isotherm measurements as a function of temperature have been applied to hydrated lysozyme powder. Two dielectric dispersions were identified, the first centred at approximately 8 MHz and a second above 1 GHz. The higher dispersion is considered to be the result of rotational relaxation of water molecules bound to the enzyme. In this case the results indicate the existence of a population of 32 water molecules per lysozyme molecule which are irrotationally bound to the lysozyme structure. A larger population of water molecules is relatively free to respond to the electric field and exhibits a dipole moment close to that of vapour phase water molecules. Multi-temperature hydration isotherm measurements are used to calculate enthalpies and entropies associated with the binding of water to lysozyme. Discontinuities both in dielectric and in thermodynamic characteristics in the range 10-14% hydration are interpreted as a re-ordering of the water structure on the enzyme surface.

  4. Fabrication of polypyrrole nano-arrays in lysozyme single crystals

    NASA Astrophysics Data System (ADS)

    England, Matt W.; Lambert, Elizabeth M.; Li, Mei; Turyanska, Lyudmila; Patil, Avinash J.; Mann, Stephen

    2012-10-01

    A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals.A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals. Electronic supplementary information (ESI) available: Optical microscopy, SEM, TEM images, FTIR spectra and tables, conductivity plot. Experimental methods. See DOI: 10.1039/c2nr32413j

  5. Self-assembly of beta-casein and lysozyme.

    PubMed

    Pan, Xiaoyun; Yu, Shaoyong; Yao, Ping; Shao, Zhengzhong

    2007-12-15

    The self-assembly of beta-casein and lysozyme, a linear and a globular protein with isoelectric point of pH 5.0 and 10.7, respectively, was studied. Polydisperse electrostatic complex micelles formed when mixing beta-casein and lysozyme aqueous solutions. After the micelle solution was heated, lysozyme gelated and beta-casein was trapped in the gel, producing narrowly dispersed nanoparticles. The nanoparticles were characterized with laser light scattering, zeta-potential, steady state fluorescence, atomic force microscopy, and transmission electron microscopy. The nanoparticles have spherical shape and their sizes depend on the pH of the heat treatment and the molar ratio of beta-casein to lysozyme. The nanoparticles display amphoteric property and are relatively hydrophobic at pH around 5 and around 10. The net charges on the surface stabilize the nanoparticles in the solution.

  6. Location of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Lerbret, A.; Affouard, F.; Bordat, P.; Hédoux, A.; Guinet, Y.; Descamps, M.

    2008-04-01

    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.

  8. The Process of Separating Bovine Serum Albumin Using Hydroxyapatite and Active Babassu Coal (Orbignya martiana)

    PubMed Central

    Zuñiga, Abraham Damian Giraldo; Sousa, Rita de Cássia Superbi; Zacchi Scolforo, Carmelita

    2016-01-01

    Bovine serum albumin is one of the major serum proteins; it plays an important role as a result of its functional and nutritional properties which have bioactive peptides. Adsorption method was used to separate protein, which involves hydroxyapatite, synthetic hydroxyapatite, and active babassu coal. Initially, characterization was carried out using the zeta potential of the adsorbents. Kinetic pseudo-first- and pseudo-second-order models were applied. For isotherms, equilibrium data studies were carried out using the Langmuir and Freundlich models, in addition to determining the efficiency of adsorptive process. The results of the zeta potential showed loads ranging from +6.9 to −42.8 mV. The kinetic data were better represented in the pseudo-second-order model with chemisorption characteristics. The adsorption capacity of the adsorbents decreased as pH increased, indicating that the electrostatic bonds and some functional groups of active babassu coal contributed to the reduction of adsorption, especially oxygen linked to carbon atoms. The value of pH 4.0 showed the best results of adsorption, being obtained as the maximum adsorption capacity (qm) and yield (%) (where qm = 87.95 mg g−1 and 74.2%; 68.26 mg g−1 and 68.6%; and 36.18 mg g−1, 37.4%) of hydroxyapatite, synthetic hydroxyapatite, and active babassu coal, respectively. PMID:27376149

  9. Blood and colostrum/milk serum gamma-glutamyltransferase activity as a predictor of passive transfer status in lambs.

    PubMed

    Maden, M; Altunok, V; Birdane, F M; Aslan, V; Nizamlioglu, M

    2003-04-01

    The importance of blood and colostrum/milk serum gamma-glutamyl transferase (gamma-GT) enzyme activity was evaluated to assess passive transfer status in healthy lambs. Thirty Akkaraman sheep (3-6 years old) were used which had normal pregnancy period and the same conditions, and the age of the lambs ranged between 0 and 15 days. Blood and colostrum/milk samples were collected from sheep and lambs after birth, before suckling (0) and after on 1st, 3rd, 7th and 15th days. Serum immunoglobulin G (IgG) concentration was determined by the use of Single Radial Immunodiffusion method. Serum gamma-GT activity was measured, using a commercially available kit in blood and colostrum/milk samples. Correlations were carried out between immunoglobulin and gamma-GT levels. Regression models (simple and multiple) were calculated with significant data. Linear correlation was determined between colostrum/milk gamma-GT activity and IgG concentrations and between serum gamma-GT activity and IgG concentrations in lambs on the 0 day. (r: 0.607, P: 0.001), 1st (r: 0.768, P: 0.001) and the 3rd (r: 0.603, P: 0.001) days and on the 1st (r: 0.637, P: 0.001) and 3rd (r: 0.478, P: 0.012) days in the experiment, respectively. Multivariate regression models were developed to estimate sample IgG concentration. Serum and colostrum/milk IgG concentration could be predicted using the formula: lamb serum IgG = 825 + 0.688 (lamb gamma-GT) + 52 (days); colostrum/milk IgG = 832 + 0.505 (colostrum/milk gamma-GT) - 167 (days). The regression models were moderately accurate in predicting serum IgG concentration (R2 = 0.51) and colostrum/milk IgG concentration (R2 = 0.55). Test sensitivity and positive predictive values for serum gamma-GT enzyme activity were found to be 96 and 100% and for colostrum/milk gamma-GT enzyme activity were found to be 100 and 68% to prediction IgG concentration. Serum and colostrum/milk gamma-GT activity can be used to assess passive transfer status of lambs. Along with this

  10. Destruction of single-species biofilms of Escherichia coli or Klebsiella pneumoniae subsp. pneumoniae by dextranase, lactoferrin, and lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. An intrinsically shielded hydrogel for the adsorptive recovery of lysozyme.

    PubMed

    Wang, Lu; Zhang, Rongsheng; Eisenthal, Robert; Hubble, John

    2006-07-01

    The present paper addresses the selective recovery of lysozyme from egg white using CM-dextran (carboxymethyldextran)-based hydrogels containing Cibacron Blue as an affinity ligand and co-immobilized BSA intended to act as a shielding agent to reduce non-specific adsorption. Initial studies using pure lysozyme were conducted that indicated that the adsorption capacity increased with ligand density and that adsorption was well described by a Langmuir-type isotherm. The inclusion of BSA as a putative shielding agent did not decrease the adsorption capacity for lysozyme in single-adsorbate experiments. To assess the effectiveness of the shielding strategy, subsequent experiments were conducted with both defined lysozyme/ovalbumin mixtures and hen's-egg white. From these studies, the optimal operating conditions for lysozyme recovery have been determined. These include: optimal initial egg-white concentration [a 10% (v/v) solution of native egg white in the chosen buffer], affinity-ligand density (1.86 mM) and ligand-to-shielding-agent ratio (4:1). The purity of lysozyme obtained from egg white was improved from 69% with a non-shielded hydrogel to 94% with an intrinsically shielded hydrogel. Finally, the possibility of using a protein, rather than dextran-backbone-based, hydrogel was investigated. It was found that BSA could take the place of CM-dextran as the gel backbone in a simplified synthesis, producing a gel which also proved effective for lysozyme recovery with a 30% lysozyme in egg-white solution purified to approx. 92% in a single adsorption-desorption cycle.

  12. Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

    PubMed Central

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

    2007-01-01

    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 ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_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. PMID:17785473

  13. Implications of serum paraoxonase activity in obesity, diabetes mellitus, and dyslipidemia

    PubMed Central

    Kota, Sunil K.; Meher, Lalit K.; Kota, Siva K.; Jammula, Sruti; Krishna, S. V. S.; Modi, Kirtikumar D.

    2013-01-01

    Human serum paraoxonase 1 (PON1) is an enzyme with esterase activity, and is physically bound to high-density lipoproteins (HDL). It plays a key role in the action of HDL toward protection of lipoprotein and biological membrane against oxidative damage. It may have a protective role against atherosclerosis by virtue of its action on hydrolyzing lipid peroxides and preventing accumulation of phospholipids in oxidized low-density lipoprotein (LDL). PON1 is hypothesized to be an indicator of the risk of atherosclerosis and coronary artery disease development. Numerous studies have implicated PON1 activity in relation to various endocrine disorders. The current article reviews the clinical perspectives of PON1 activity with regards to obesity, diabetes mellitus with its complications, and dyslipidemia. PMID:23869295

  14. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

    PubMed

    Yang, G; Cecconi, C; Baase, W A; Vetter, I R; Breyer, W A; Haack, J A; Matthews, B W; Dahlquist, F W; Bustamante, C

    2000-01-04

    Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

  15. Salt Taste Enhancing l-Arginyl Dipeptides from Casein and Lysozyme Released by Peptidases of Basidiomycota.

    PubMed

    Harth, Lisa; Krah, Ulrike; Linke, Diana; Dunkel, Andreas; Hofmann, Thomas; Berger, Ralf G

    2016-08-24

    Some l-arginyl dipeptides were recently identified as salt taste enhancers, thus opening the possibility to reduce dietary sodium uptake without compromising palatability. A screening of 15 basidiomycete fungi resulted in the identification of 5 species secreting a high peptidolytic activity (>3 kAU/mL; azocasein assay). PFP-LC-MS/MS and HILIC-MS/MS confirmed that l-arginyl dipeptides were liberated when casein or lysozyme served as substrate. Much higher yields of dipeptides (42-75 μmol/g substrate) were released from lysozyme than from casein. The lysozyme hydrolysate generated by the complex set of peptidases of Trametes versicolor showed the highest l-arginyl dipeptide yields and a significant salt taste enhancing effect in a model cheese matrix and in a curd cheese. With a broad spectrum of novel specific and nonspecific peptidases active in the slightly acidic pH range, T. versicolor might be a suitable enzyme source for low-salt dairy products.

  16. Effects of reduced levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate on levels of volatile and biogenic amines.

    PubMed

    Ancín-Azpilicueta, Carmen; Jiménez-Moreno, Nerea; Moler, José Antonio; Nieto-Rojo, Rodrigo; Urmeneta, Henar

    2016-10-01

    Sulphur dioxide (SO2) is an important preservative for wine, but its presence in foods can cause allergies and this has given impetus to the research for alternatives. The aim of this study was to reduce levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate and examine the influence on levels of volatile and biogenic amines. To do so, vinifications were carried out using lysozyme, dimethyl dicarbonate (DMDC) and mixtures of these with SO2 in different concentrations (25 and 50 mg l(-1)). Results were compared with a control vinification with only SO2 (50 mg l(-1)). Mixing low concentrations of SO2 with lysozyme and DMDC reduced the concentration of biogenic amines (histamine, tyramine, putrescine, cadaverine, phenylethylamine + spermidine and spermine). In general, the total concentration of volatile amines (dimethylamine, isopropylamine, isobutylamine, pyrrolidine, ethylamine, diethylamine, amylamine and hexylamine) was higher in the sample fermented only with SO2. The concentrations of amines with secondary amino groups (dimethylamine, diethylamine, pyrrolidine) were higher in the sample only fermented with SO2 than those fermented with DMDC and lysozyme or with a mixture of preservatives. When SO2 was the only preservative in wine, total amine concentration (biogenic and volatile amines) was higher than for the rest of the treatments. Lysozyme by itself, and lysozyme mixed with SO2, both reduced the formation of biogenic amines but given the antioxidant activity of SO2 the use of the preservative mixture seems more advisable.

  17. FORMATION OF PROTOPLASTS FROM STREPTOCOCCUS FAECALIS BY LYSOZYME1

    PubMed Central

    Bibb, William R.; Straughn, W. R.

    1962-01-01

    Bibb, William R. (University of North Carolina, Chapel Hill) and W. R. Straughn. Formation of protoplasts from Streptococcus faecalis by lysozyme. J. Bacteriol. 84:1094–1098. 1962.—Incubation of whole cells of Streptococcus faecalis F24 in the presence of the crystalline egg-white lysozyme and appropriate sucrose concentration resulted in the formation of discrete spherical structures. On dilution, these osmotically fragile structures lysed immediately. Methyl pentose determinations on isolated cell walls and protoplast membranes verified the presence of rhamnose in the cell walls and its essentially complete absence in protoplast membranes. Cell walls were rendered soluble by lysozyme. After lysozyme treatment of cell walls, 96% of the rhamnose present was not sedimented by centrifugation at 12,500 × g for 30 min. No cell-wall structures were recognized by phasecontrast or electron microscopy. After direct lysis of whole cells of S. faecalis F24 by lysozyme, protoplast membranes were isolated. It is concluded that, in the strain of group D streptococcus studied, lysozyme effectively removes the cell wall. Images PMID:13968087

  18. Penetration and fusion of phospholipid vesicles by lysozyme

    SciTech Connect

    Kim, J.; Kim, H.

    1989-10-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-(({sup 125}I)iodophenyl)diazirine (({sup 125}I)TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent ({sup 125}I)TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.

  19. Fluorescence study of the membrane effects of aggregated lysozyme.

    PubMed

    Kutsenko, Olga K; Trusova, Valeriya M; Gorbenko, Galyna P; Lipovaya, Anna S; Slobozhanina, Ekaterina I; Lukyanenko, Lyudmila M; Deligeorgiev, Todor; Vasilev, Aleksey

    2013-11-01

    The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp62 and Trp108 increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.

  20. Decreased serum cholesteryl-ester transfer activity in a patient with familial hyperalphalipoproteinemia.

    PubMed

    Takegoshi, T; Haba, T; Kitoh, C; Tokuda, T; Mabuchi, H

    1988-08-01

    Lipoprotein patterns and cholesteryl-ester transfer activity (CETA) were examined in a patient with familial hyperalphalipoproteinemia (FHALP). The proband was a 41-year-old Japanese male. He was found to have hypercholesterolemia, with a serum total cholesterol level of 382 mg/dl and a HDL-cholesterol level of 177 mg/dl. HDL showed a high cholesterol/Apo AI ratio. His father, all of his siblings and one of his children showed high HDL-cholesterol levels (91, 100, 70, 108, 75 and 98 mg/dl, respectively). These data suggest that all members of his family were heterozygotes. He had neither cutaneous or tendinous xanthomas nor any clinical signs of atherosclerosis. The proband appears to have only one-tenth of the normal level of CETA. However, the level of lipid-transfer protein I (LTP-I) activity was near normal. Thus, this patient is most likely to have an exaggerated level of LTP-I inhibitor(s). Effects of probucol on serum lipoprotein and apolipoprotein levels were studied in our patient. Treatment with 250 mg of probucol twice daily reduced total serum cholesterol, low density lipoprotein (LDL) and HDL-cholesterol levels by 33.32 and 33%, respectively. Apo AI, B and E levels decreased by 22, 16 and 35% respectively. HDL-cholesterol/Apo AI ratio decreased from 0.9 to 0.76. CETA showed no significant changes. However, cholesterol ester mass transfer increased from 10.8 to 14.9% after treatment with probucol. These results suggest that probucol appears to be a useful drug for FHALP.

  1. Effects of three reputed carboxylesterase inhibitors upon rat serum esterase activity.

    PubMed

    Chambers, J P; Hartgraves, S L; Murphy, M R; Wayner, M J; Kumar, N; Valdes, J J

    1991-01-01

    Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

  2. The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

    PubMed

    Miętka, K; Brzostek, K; Guz-Regner, K; Bugla-Płoskońska, G

    2016-01-01

    Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

  3. Increased Serum Activity of Matrix Metalloproteinase-9 in Patients with Acute Variceal Bleeding

    PubMed Central

    Kwon, Oh Sang; Jung, Hyuk Sang; Bae, Kyung Sook; Jung, Young Kul; Kim, Yeon Suk; Choi, Duck Joo; Kim, Yun Soo

    2012-01-01

    Background/Aims Matrix metalloproteinases (MMP)-2 and -9 can degrade essential components of vascular integrity. The aim of this study was to investigate the association between those MMPs and variceal bleeding (VB). Methods Fifteen controls, 12 patients with acute ulcer bleeding (UB) group, 37 patients with varix (V group), and 35 patients with acute VB group were enrolled. Serum was obtained to measure MMP-2 and -9 activity by zymogram protease assays. Results The activity levels of these compounds were compared with the controls' median value. The median MMP-9 activity was 1.0 in controls, 1.05 in the UB group, 0.43 in the V group, and 0.96 in the VB group. The level of MMP-9 activity was higher in the VB group than in the V group (p<0.001). In the VB group, there was a signifi cant decrease in MMP-9 activity over time after bleeding (p<0.001). The median MMP-2 activity level was 1.0 in controls, 1.01 in the UB group, 1.50 in the V group, and 1.55 in the VB group. The level of MMP-2 activity was similar in the VB and V groups. Conclusions The level of MMP-9 activity increased in association with VB. The role of MMP-9 in the pathogenesis of VB should be verified. PMID:22570756

  4. Chitotriosidase activity in the blood serum and organs of mice of various strains under the influence of chitin.

    PubMed

    Monoszon, A A; Cherkanova, M S; Duzhak, A B; Korolenko, T A

    2012-11-01

    Mouse chitotriosidase cleaving chitin belongs to the family of mammalian chitinases, whose biological functions are poorly understood. Chitotriosidase activity in mouse serum was shown to be much higher than in humans. The following interstrain differences were revealed in mouse chitotriosidase activity: GR>C57Bl/6>BALB/c>A/Sn>CBA. Chitotriosidase activity in CBA mice was lowest and practically did not differ from that in C3H/He and ICR mice. No sex-related differences were found in enzyme activity. Hybrids of opposite strains CBA and C57Bl/6 were characterized by dominant inheritance of this sign (elevated activity of chitotriosidase in the serum). Intragastric administration of chitin in a single dose of 100 mg/kg was followed by a decrease in chitotriosidase activity in the lungs, but not in the blood serum and homogenate of gastric cells from CBA mice. These data indicate that intragastric administration of chitin does not induce chitotriosidase in mice.

  5. X-ray structure of Glu 53 human lysozyme.

    PubMed Central

    Harata, K.; Muraki, M.; Hayashi, Y.; Jigami, Y.

    1992-01-01

    The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction. PMID:1363898

  6. Serum thyroid hormones and tissue 5'-monodeiodinase activity in acutely thyroidectomized newborn lambs

    SciTech Connect

    Polk, D.H.; Wu, S.Y.; Fisher, D.A.

    1986-08-01

    After either total thyroidectomy or sham operation in full-term fetal sheep, fetuses were delivered and serial blood samples were obtained for measurements of thyroxine (T4), triiodothyronine (T3), and catecholamines. Despite comparable serum T4 values, serum T3 values were lower in the thyroidectomized animals. Four hours after birth, the animals were killed with an intravenous overdose of barbiturate. Brain, thyroid, liver, kidney, and brown adipose tissues were dissected and analyzed for thyroxine 5'-monodeiodinase (5'-MDI) activity in vitro. 5'-MDI activity was comparable in all tissues from sham-operated and thyroidectomized lambs. Plasma epinephrine and norepinehprine concentrations, mean arterial pressure, mean pulse, rectal temperature, and arterial blood gas values were similar in the two groups of animals. These data support the hypothesis that the thyroid gland is the major source of T3 for the T3 surge in the immediate newborn period. They also indicate that the neonatal T3 surge has limited immediate metabolic significance in euthyroid newborns.

  7. Lysozyme dimer association: Similarities and differences compared with lysozyme monomer association

    NASA Astrophysics Data System (ADS)

    Onuma, Kazuo; Inaka, Koji

    2008-03-01

    The protein with a molecular weight of 28.6 kDa in lysozyme solution, which has been recognized as a lysozyme dimer, was purified and its association was observed using time-resolved static light scattering and dynamic light scattering under the same buffer condition as that used in lysozyme monomer association. The chromatography results and SDS-PAGE analysis showed that the bonding state of each molecule in a dimer unit was not uniform, i.e., there were at least two kinds of bonds, strong and weak. Some of the weak-bonded dimmers dissociated to monomers (molecular weight: 14.3 kDa) in the SDS-PAGE process. The relative amount of weak-bonded dimers greatly affected the association kinetics. With a 99% pure dimer solution (1% monomers in SDS-PAGE), association proceeded in the same manner as that of a monomer solution: the Zimm-square plot had a concave shape with a maximum at a particular q2 for apparent protein concentrations, up to 2.4 mg/mL. The dynamic light-scattering data showed clear bimodal (dimer and aggregate), distributions. With a 95% pure dimer solution, the association behavior drastically changed when the apparent concentration exceeded 2.0 mg/mL. The Zimm-square plot had a bending point at a low q2, and two discrete lines fitted the plot. The particles in the solution were either oligomers or large aggregates, both of which had polydispersity distributions, and an amorphous phase formed from the aggregates. This was not observed for monomer association.

  8. Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Ni, Yuyang; Li, Jun; Huang, Zhenzhen; He, Ke; Zhuang, Jiaqi; Yang, Wensheng

    2013-11-01

    The using of macromolecular additives is known to be a simple and effective way to improve the activity of immobilized enzymes on solid support, yet the mechanism has not been well understood. Taking horseradish peroxidase (HRP) as an example, only 30 % of its catalytic activity was kept after being immobilized on the surface of 25-nm Au nanoparticles, mainly attributed to the conformational change of the heme-containing active site. The catalytic activity of HRP was significantly improved to 80 % when a certain amount of bovine serum albumin (BSA) was added at the initial stage of the immobilization. Systematic spectral investigation indicated that the addition of BSA inhibited the tertiary structure change around the active site, which was a prerequisite for improved activity of the immobilized HRP. Steady-state kinetic analyses revealed that the introduction of BSA could effectively improve the turnover rate of substrate to product in spite of slight reduced affinity to substrates, which also contributed to the improved catalytic activity.

  9. Serum 25-Hydroxyvitamin D3 and BAFF Levels Are Associated with Disease Activity in Primary Sjogren's Syndrome

    PubMed Central

    Lee, Sang Jin; Oh, Hye Jin; Choi, Byoong Yong; Jang, Yu Jin; Lee, Joo Youn; Park, Jin Kyun

    2016-01-01

    The study investigated the association between disease activity and serum 25-hydroxyvitamin D3 (25(OH)-D3), B cell activation of the tumor necrosis factor family (BAFF), or β2 microglobulin in patients with primary Sjogren's syndrome (SS). Sixty-nine primary SS patients and 22 sicca control patients were included in the study. Disease activity was measured with EULAR Sjogren's syndrome disease activity index (ESSDAI). Serum levels of 25(OH)-D3 and β2 microglobulin were measured by radioimmunoassay and BAFF was measured by an enzyme-linked immunosorbent assay. Serum levels of 25(OH)-D3 were significantly lower in SS patients compared to the sicca controls (p = 0.036). Serum levels of BAFF tended to be higher (p = 0.225) and those of β2 microglobulin were significantly higher in patients with SS than in sicca controls (p = 0.023). In univariate regression analyses, ESSDAI was significantly associated with serum levels of 25(OH)-D3, BAFF, and β2 microglobulin. After stepwise backward multivariate linear regression analyses including age and acute phase reactants, ESSDAI was associated with 25(OH)-D3 (β = −0.042, p = 0.015) and BAFF (β = 0.001, p = 0.015) in SS patients. In SS patients, ESSDAI is negatively associated with serum levels of 25(OH)-D3 and positively associated with BAFF. PMID:28074193

  10. Calcium-binding and structural stability of echidna and canine milk lysozymes.

    PubMed

    Kikuchi, M; Kawano, K; Nitta, K

    1998-10-01

    For echidna and canine milk lysozymes, which were presumed to be the calcium-binding lysozymes by their amino acid sequences, we have quantitated their calcium-binding strength and examined their guanidine unfolding profiles. The calcium-binding constants of echidna and canine lysozymes were determined to be 8.6 x 10(6) M(-1) and 8.9 x 10(6) M(-1) in 0.1 M KCl at pH 7.1 and 20 C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of alpha-lactalbumin, through a stable molten globule intermediate. However, neither calcium-bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg-white lysozyme, avian calcium-binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel-Boston-Berlin: Birkhauzer Verlag. pp 9-31) that the lineage of avian and echidna calcium-binding lysozymes and that of eutherian calcium-binding lysozymes diverged separately from that of conventional lysozymes.

  11. Biological effects of static electric field: Plasma/serum proteome analysis of rats.

    PubMed

    Harutyunyan, Hayk; Artsruni, Gagik

    2013-03-01

    The external static electric field (SEF) of man-made origin brings to the substantially increased SEF background in a human environment the biological activity of which is a moot question. The paper reports on rats blood plasma/serum proteome modifications by means of 1D polyacrilamide gel electrophoresis and clotting process alterations after the short- and long-term SEF exposures of 200 kV/m. The results indicate decrease of fast α1 and α2 globular proteins in plasma coinciding with clotting acceleration after the short-term SEF, and attenuation of clotting-dependent proteome modifications reflected with incomplete coagulation after the long-term SEF exposure. Increased lysozyme activity in serum unlike plasma was observed after both SEF exposures. Applied model of the high-voltage SEF environment indicates dependence of biological systems functioning on the external SEF.

  12. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  13. [SERUM LEVEL OF ENDOTHELIAL MONOCYTE ACTIVATING POLYPEPTIDE-II IN CHILDHOOD-ONSET TYPE 1 DIABETIC PATIENTS AND OBESE ADOLESCENTS].

    PubMed

    Mogylnytska, L A

    2015-01-01

    The atherosclerotic process begins in adolescence, and its progression is determined by the same risk factors as in adults. Endothelial monocyte activating polypeptide-II (EMAP-II) is a multifunctional cytokine with proinflammatory and antiangiogenetic activity that may play a pathogenic role in the development of endothelial dysfunction and atherosclerosis. The aim of our study was to determine the serum level of EMAP-II in childhood-onset type 1 diabetic patients and obese adolescents. We found increased of serum level of EMAP-II in childhood-onset type 1 diabetic patients and in patients with obesity that do not suffer from diabetes. Also, the level of EMAP-II correlated with the serum level of glycosylated hemoglobin and blood glucose, and key markers of lipid metabolism, body mass index. Increased serum level of EMAP-II may be one of the pathway of endothelial dysfunction in type 1 diabetes.

  14. Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs

    PubMed Central

    Wohlkönig, Alexandre; Huet, Joëlle; Looze, Yvan; Wintjens, René

    2010-01-01

    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. PMID:21085702

  15. Development of an affinity cryogel for one step purification of lysozyme from chicken egg white.

    PubMed

    Mól, Paula Chequer Gouveia; Veríssimo, Lizzy Ayra Alcântara; Eller, Monique Renon; Minim, Valéria Paula Rodrigues; Minim, Luis Antonio

    2017-02-15

    In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20-80μm and swelling capacity of 19.6±0.9g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04cm to 0.8cm. Adsorption isotherms were measured at 15°C and 35°C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285mg/g at 15°C and 363mg/g at 35°C. Thermodynamic analysis based on the Van't Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.

  16. Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.

    PubMed

    Leontiev, V V; Uversky, V N; Permyakov, E A; Murzin, A G

    1993-03-05

    The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

  17. Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method.

    PubMed

    Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X

    2016-07-03

    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.

  18. Comparison of two codon optimization strategies enhancing recombinant Sus scrofa lysozyme production in Pichia pastoris.

    PubMed

    Zhu, D; Cai, G; Wu, D; Lu, J

    2015-05-16

    Lysozyme has played an important role in animal feed additive industry, food additive industry and biological engineering. For improving expression efficiency of recombinant lysozyme from Sus scrofa, two genes respectively designed by the most used codon optimization strategies, "one amino acid one codon" and "codon randomization", were synthesized and expressed in Pichia pastoris X—33. At shaking flask level, Sus scrofa lysozyme (SSL) under two conditions had a highest activity of 153.33±10.41 and 538.33±15.18 U/mL after a 5 days induction of 1% methanol, with secreted protein concentration 80.03±1.94 and 239.60±4.16 mg/L, respectively. Compared with the original SSL gene, the expression of optimized SSL gene by the second strategy showed a 2.6 fold higher level, while the first method had no obvious improvement in production. In total secreted protein, the proportions of recombinant SSL encoded by the original gene, first method optimized gene and the second—strategy optimized one were 75.06±0.25%, 74.56±0.14% and 79.00±0.14%, respectively, with the same molecular weight about 18 kDa, optimum acidity pH 6.0 and optimum temperature 35degC.

  19. In vitro squelching of activated transcription by serum response factor: evidence for a common coactivator used by multiple transcriptional activators.

    PubMed Central

    Prywes, R; Zhu, H

    1992-01-01

    Low amounts of serum response factor (SRF) activate transcription in vitro from a fos promoter construct containing an SRF binding site. Using this human HeLa cell-derived in vitro transcription system, we have found that high amounts of SRF inhibited, or 'squelched', transcription from this construct. Transcription from several other promoters activated by different gene-specific factors, including CREB and the acidic activator VP16, was also inhibited by high amounts of SRF. Basal transcription, from TATA-only promoters, however, was not inhibited. These results suggest that SRF binds to a common factor(s) (termed coactivator) required for activated transcription by a diverse group of transcriptional activators. Inhibition of transcription by SRF could be blocked by a double stranded oligonucleotide containing an SRF binding site. Mutations in SRF which abolished its DNA binding activity also reduced its ability to inhibit transcription. In addition, a C-terminal truncation of SRF which reduced its ability to activate transcription also reduced SRF's ability to inhibit transcription. These results suggest that activation and inhibition of transcription may be mediated by SRF binding to the same factor and that SRF can only bind to this factor when SRF is bound to plasmid DNA. Images PMID:1531519

  20. Impurity effects on orientation of lysozyme crystals nucleated on fatty acid thin films

    NASA Astrophysics Data System (ADS)

    Kubo, T.; Hondoh, H.; Nakada, T.

    2008-04-01

    Commercially available lysozyme samples that have different lot numbers (E02Z04 and E05802) were crystallized on fatty acid thin films. The orientation of lysozyme crystals nucleated on the films was investigated by atomic force microscopy and optical microscopy. The numbers of lysozyme crystals with specific planes parallel to the films are different. In other words, the impurities contained in commercial lysozyme significantly affect the orientation of lysozyme crystals. Detailed analysis of the orientation distribution of the lysozyme crystals nucleated from the purified sample showed that acetic acid is one of the substances promote the epitaxy.

  1. The effect of cold on serum thyroid hormones and hepatic 5 prime mono-deiodinase activity

    SciTech Connect

    Hesslink, R.L. Jr.; Quesada, M.; D'Alesandro, M.; Homer, L.D.; Reed, J.L.; Christopherson, R.; Young, B.A. Univ. of Alberta, Edmonton )

    1991-03-11

    Cold exposed swine have an increases serum concentration of triiodothyronine (T{sub 3}) and increased T{sub 3} production rate. It is thought that hepatic thyroxine (T{sub 4}) deiodination (5DI) contributes to circulating T{sub 3} concentrations. The authors investigated the effects of cold exposure (14 days) on energy intake, serum free T{sub 3} (FT{sub 3}) and free T{sub 4} (FT{sub 4}) levels; and 5DI in 5-month boars. Hepatic 5DI activity was determined by measuring the {sup 125}I generated from trace amounts of {sup 125}I T{sub 4}. FT{sub 3} and FT{sub 4} were assayed by RIA. Swine were housed in either 20C (control; n = 5) or 4C (cold; n = 7) chambers and given food ad libitum. Cold exposure increased energy intake by 42%. The increase (93%) in hepatic 5DI V{sub max} after cold exposure parallels the increase in whole animal T{sub 3} production and may account for FT{sub 3} values found after cold exposure.

  2. Serum from aged F344 rats conditions the activation of young macrophages.

    PubMed

    Gómez, Christian R; Acuña-Castillo, Claudio; Nishimura, Sumiyo; Pérez, Viviana; Escobar, Alejandro; Salazar-Onfray, Flavio; Sabaj, Valeria; Torres, Claudio; Walter, Robin; Sierra, Felipe

    2006-03-01

    There is considerable controversy about the molecular mechanisms responsible for the variations in innate immunity associated with age. While in vivo, aged animals and humans react to an inflammatory signal with an excessive production of pro-inflammatory cytokines, studies in vitro generally show that this response is attenuated in macrophages from old individuals. In an effort to examine possible extrinsic factors that might affect the response of macrophages to lipopolysaccharide (LPS), we have challenged peritoneal macrophages obtained from young rats with sera obtained from rats of different ages. Our results indicate that the serum from aged rats significantly impairs the capacity of young macrophages to induce tumor necrosis factor-alpha (TNF-alpha) production, while at the same time it increases the basal levels of interleukin-6 (IL-6). The effect of serum from aged donors on TNF-alpha secretion requires pre-incubation and is sensitive to heat inactivation. In contrast, the stimulating effect on IL-6 is resistant to heat, and thus should not be due to a protein factor. Therefore, our results indicate that the age-related changes in macrophage activity are not only the consequence of intrinsic changes, but there also appears to be a modulatory effect imparted by the external milieu.

  3. Concomitant Prevalence of Low Serum Diamine Oxidase Activity and Carbohydrate Malabsorption

    PubMed Central

    Meinitzer, Andreas; Mangge, Harald; Kriegshäuser, Gernot; Halwachs-Baumann, Gabriele; Reininghaus, Eva Z.; Bengesser, Susanne A.; Schnedl, Wolfgang J.

    2016-01-01

    The aim of this retrospective study was to analyze the concomitant prevalence rates for lactose malabsorption (LM), fructose malabsorption (FM), and histamine intolerance (HI) in patients with so far unexplained gastrointestinal (GI) symptoms. A total of 439 outpatients, who presented unclear abdominal discomfort, underwent lactose (50 g) and fructose (25 g) hydrogen (H2) breath tests. Additionally, serum diamine oxidase (DAO) measurements were performed. Individuals with low serum DAO activity (<10 U/mL), GI symptoms, and response to histamine-free diet were diagnosed with HI. Of all 439 patients, 341 (77.7%) were found with 7 various GI conditions. In total, 94 (21.4%), 31 (7.1%), and 100 (22.8%) individuals presented LM, FM, or HI only, whereas 116 (26.4%) patients showed an overlap of GI entities investigated here. Interestingly, 89 out of 241 (36.9%) individuals with carbohydrate malabsorption were also diagnosed with HI (LM + HI: 52 [11.8%], FM + HI: 23 [5.2%], and LM + FM + HI 14 [3.2%] individuals). In conclusion different combinations of LM, FM, and HI are present in individuals with unclear abdominal discomfort/pain. In clinical practice we suggest testing for LM, FM, and additional HI in the diagnostic work-up of these patients. Depending on these various diagnoses possible, patients should get an individualized dietary advice. PMID:28042564

  4. Low serum somatomedin activity in celiac disease. A misleading aspect in growth failure from asymptomatic celiac disease.

    PubMed

    Lecornu, M; David, L; François, R

    1978-12-01

    In 4 children with celiac disease, aged 7 months to 11 years, serum somatomedin activities (SMA) were consistently low: less than 0.40 U/ml (N greater than 0.50 U/ml). Basal plasma growth hormone (GH) levels were not elevated and increased normally under arginine-insulin stimulation in 3 patients. Human GH administration at a dosage which usually determines an increase of serum SMA in children with GH deficiency (4 mg/day/2 days) did not modify significantly the low serum SMA. However, in 1 child a clear-cut increase of serum SMA (0.22-0.82 U/ml) was noted under a higher dosage of human GH (8 mg/day/2 days). In 3 patients serum SMA was studied 3 weeks to 4 months after starting the gluten-free diet and was found to be normal. A limitation of the somatomedin generation unrelated to a deficit in GH secretion and probably resistant to GH appears therefore to be present in celiac disease. The rapid normalization of serum SMA under gluten-free diet suggests that the low serum SMA is induced through some unknown hormonal or metabolic signal by the protein malabsorption and/or the nutritional deficiency present in celiac disease.

  5. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    PubMed

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Impact of antibacterial drugs on human serum paraoxonase-1 (hPON1) activity: an in vitro study

    PubMed Central

    Söyüt, Hakan; Kaya, Elif Duygu; Beydemir, Şükrü

    2014-01-01

    Objective To investigate the in vitro effects of the antibacterial drugs, meropenem trihydrate, piperacillin sodium, and cefoperazone sodium, on the activity of human serum paraoxonase (hPON1). Methods hPON1 was purified from human serum using simple chromatographic methods, including DEAE-Sephadex anion exchange and Sephadex G-200 gel filtration chromatography. Results The three antibacterial drugs decreased in vitro hPON1 activity. Inhibition mechanisms meropenem trihydrate was noncompetitive while piperacillin sodium and cefoperazone sodium were competitive. Conclusions Our results showed that antibacterial drugs significantly inhibit hPON1 activity, both in vitro, with rank order meropenem trihydrate piperacillin sodium cefoperazone sodium in vitro. PMID:25183328

  7. Effect of Antiviral Therapy on Serum Activity of Angiotensin Converting Enzyme in Patients with Chronic Hepatitis C

    PubMed Central

    Husic-Selimovic, Azra; Sofic, Amela; Huskic, Jasminko; Bulja, Deniz

    2016-01-01

    Introduction: Renin-angiotenzin system (RAS) is frequently activated in patients with chronic liver disease. Angiotenzin - II (AT-II), produced by angiotenzin converting enzyme (ACE), has many physiological effects, including an important role in liver fibrogenesis. Combined antiviral therapy with PEG-IFN and ribavirin besides its antiviral effect also leads to a reduction in liver parenchyma fibrosis. Aim of the study: Determining the value of ACE in serum of patients with chronic hepatitis C before and after combined antiviral therapy, as well as the value of ACE activities in sera of the control group. Materials and methods: We studied 50 patients treated at Gastroenterohepatology Department, in the time-period of four years. Value of ACE in serum was determined by Olympus AU 400 device, with application of kit “Infinity TN ACE Liquid Stable Reagent”. HCV RNA levels in sera were measured by real time PCR. HCV RNA test was performed with modular analysis of AMPLICOR and COBAS AMPLICOR HCV MONITOR test v2.0, which has proved infection and was used for quantification of the viruses and monitoring of the patients’ response to therapy. Liver histology was evaluated in accordance with the level of necroinflammation activity and stage of fibrosis. Results: Serum activities of ACE in chronic hepatitis C patients is statistically higher than the values in the control group (p=0.02). Antiviral therapy in chronic hepatitis C patients statistically decreases serum activities of ACE (p= 0.02) and indirectly affects fibrogenesis of the liver parenchyma. Correlation between ACE and ALT activity after the therapy was proved (0.3934). Conclusion: Our findings suggest that the activity of ACE in serum is a good indirect parameter of the liver damage, and could be used as an indirect prognostic factor of the level of liver parenchyma damage. Serum activity of ACE can be used as a parameter for non-invasive assessment of intensity of liver damage. PMID:27147779

  8. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    PubMed

    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

    2017-03-01

    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

  9. Effects of lead shot ingestion on delta-aminolevulinic acid dehydratase activity, hemoglobin concentration, and serum chemistry in bald eagles

    USGS Publications Warehouse

    Hoffman, D.J.; Pattee, O.H.; Wiemeyer, Stanley N.; Mulhern, B.

    1981-01-01

    Lead shot ingestion by bald eagles (Haliaeetus leucocephalus) is considered to be widespread and has been implicated in the death of eagles in nature. It was recently demonstrated under experimental conditions that ingestion of as few as 10 lead shot resulted in death within 12 to 20 days. In the present study hematological responses to lead toxicity including red blood cell ALAD activity, hemoglobin concentration and 23 different blood serum chemistries were examined in five captive bald eagles that were unsuitable for rehabilitation and release. Eagles were dosed by force-feeding with 10 lead shot; they were redosed if regurgitation occurred. Red blood cell ALAD activity was inhibited by nearly 80% within 24 hours when mean blood lead concentration had increased to 0.8 parts per million (ppm). By the end of 1 week there was a significant decrease (20-25%) in hematocrit and hemoglobin, and the mean blood lead concentration was over 3 ppm. Within as little as 1-2 weeks after dosing, significant elevations in serum creatinine and serum alanine aminotransferase occurred, as well as a significant decrease in the ratio of serum aspartic aminotransferase to serum alanine aminotransferase. The mean blood lead concentration was over 5 ppm by the end of 2 weeks. These changes in serum chemistry may be indicative of kidney and liver alterations.

  10. Elevated serum level of IL-27 and VEGF in patients with ankylosing spondylitis and associate with disease activity.

    PubMed

    Lin, Tian-Tian; Lu, Jing; Qi, Chen-Yue; Yuan, Lin; Li, Xiao-Lin; Xia, Li-Ping; Shen, Hui

    2015-05-01

    Interleukin (IL)-27 is an IL-12 family cytokine and exerts a critical role in immune regulation in the context of infection, autoimmunity, and angiogenesis. In this study, we aimed to investigate the possible pathophysiological role of IL-27 and vascular endothelial growth factor (VEGF) in ankylosing spondylitis (AS). One hundred and forty AS patients and 90 healthy controls were included in the current study. The levels of IL-27 and VEGF in serum and synovial fluid (SF) samples were measured by enzyme-linked immunosorbent assay. Erythrocyte sedimentation rate, C-reactive protein, and human leukocyte antigen (HLA)-B27 were measured by standard laboratory techniques. Disease activity in AS was scored with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Hip involvement, peripheral arthritis, and eye involvement were also recorded. The serum levels of IL-27 were remarkably higher in AS patients than healthy groups and significantly correlated with serum levels of VEGF. Furthermore, the serum levels of IL-27 were correlated with BASDAI independent of other markers of inflammation. Elevated serum levels of IL-27 and VEGF were detected in AS patients with peripheral arthritis and HLA-B27 positive. The SF levels of IL-27 and VEGF were significantly higher than serum levels in AS patients with peripheral arthritis. By contrast, levels of IL-27 and VEGF were not increased in AS patients with hip involvement and eye involvement. IL-27 may regulate the immunological or inflammatory process of AS.

  11. Interaction mechanism between berberine and the enzyme lysozyme

    NASA Astrophysics Data System (ADS)

    Cheng, Ling-Li; Wang, Mei; Wu, Ming-Hong; Yao, Si-De; Jiao, Zheng; Wang, Shi-Long

    2012-11-01

    In the present paper, the interaction between model protein lysozyme (Lys) and antitumorigenic berberine (BBR) was investigated by spectroscopic methods, for finding an efficient and safe photosensitizer with highly active transient products using in photodynamic therapy study. The fluorescence data shows that the binding of BBR could change the environment of the tryptophan (Trp) residues of Lys, and form a new complex. Static quenching is the main fluorescence quenching mechanism between Lys and BBR, and there is one binding site in Lys for BBR and the type of binding force between them was determined to be hydrophobic interaction. Furthermore, the possible interaction mechanism between BBR and Lys under the photoexcitation was studied by laser flash photolysis method, the results demonstrated that BBR neutral radicals (BBR(-H)•) react with Trp (K = 3.4 × 109 M-1 s-1) via electron transfer to give the radical cation (Trp/NH•+) and neutral radical of Trp (TrpN•). Additionally BBR selectively oxidize the Trp residues of Lys was also observed by comparing the transient absorption spectra of their reaction products. Through thermodynamic calculation, the reaction mechanisms between 3BBR∗ and Trp or Lys were determined to be electron transfer process.

  12. Scaling and self-organized criticality in proteins: Lysozyme c

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2009-11-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein functionality is often dominated by long-range hydro(phobic/philic) interactions, which both drive protein compaction and mediate protein-protein interactions. In contrast to previous reductionist short-range hydrophobicity scales, the holistic Moret-Zebende hydrophobicity scale [Phys. Rev. E 75, 011920 (2007)] represents a hydroanalytic tool that bioinformatically quantifies SOC in a way fully compatible with evolution. Hydroprofiling identifies chemical trends in the activities and substrate binding abilities of model enzymes and antibiotic animal lysozymes c , as well as defensins, which have been the subject of tens of thousands of experimental studies. The analysis is simple and easily performed and immediately yields insights not obtainable by traditional methods based on short-range real-space interactions, as described either by classical force fields used in molecular-dynamics simulations, or hydrophobicity scales based on transference energies from water to organic solvents or solvent-accessible areas.

  13. Correlation of disease activity and serum level of carcinoembryonic antigen in acquired idiopathic generalized anhidrosis: A case report.

    PubMed

    Honma, Masaru; Iinuma, Shin; Kanno, Kyoko; Komatsu, Shigetsuna; Minami-Hori, Masako; Ishida-Yamamoto, Akemi

    2015-09-01

    Hypohidrosis and anhidrosis are congenital or acquired conditions which are characterized by inadequate sweating. Acquired idiopathic generalized hypohidrosis/anhidrosis (AIGA) includes idiopathic pure sudomotor failure (IPSF), which has the following distinct features: sudden onset in youth, increased serum immunoglobulin E and responds favorably to systemic corticosteroid. No clinical markers reflecting the disease severity or activity have been established. Here, we report a case of AIGA in a Japanese patient successfully treated with repeated methylprednisolone pulse therapy. In this case, serum carcinoembryonic antigen (CEA) levels increased up to 19.8 ng/mL along with aberrant CEA immunoreactivity of eccrine sweat glands. Interestingly, the serum CEA level normalized as sweating improved with repeated methylprednisolone pulse therapy. Therefore, serum CEA level may serve as a useful clinical marker of hypohidrosis or anhidrosis.

  14. Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities.

    PubMed

    Boopathy, R; Balasubramanian, A S

    1985-09-02

    The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to

  15. Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum.

    PubMed Central

    Lonsdale-Eccles, J D; Mpimbaza, G W; Nkhungulu, Z R; Olobo, J; Smith, L; Tosomba, O M; Grab, D J

    1995-01-01

    African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition

  16. Marathon runners presented lower serum cholesteryl ester transfer activity than sedentary subjects.

    PubMed

    Serrat-Serrat, J; Ordóñez-Llanos, J; Serra-Grima, R; Gómez-Gerique, J A; Pellicer-Thoma, E; Payés-Romero, A; González-Sastre, F

    1993-06-01

    Acute exercise promotes raised HDL cholesterol concentrations by lipolysis stimulation, but this effect is insufficient to explain the more permanent HDL increases seen during regular exercise. During training periods in a group of marathon runners, we measured lipid transfer protein I (LTP-I)-mediated cholesteryl ester transfer activity (CETA) and its relationship to their HDL concentrations. Runners of both sexes showed significantly lower CETA values than those of sedentary controls. Male runners also had significantly lower serum concentrations of triglyceride, VLDL cholesterol and apolipoprotein B, and significantly higher concentrations of HDL cholesterol and apolipoprotein A-I than male controls. Results indicate that regular practice of aerobic exercise promotes modifications of lipoprotein metabolism related not only to lipolysis, but also to lower CETA. Such modifications are associated with reduced risk of atherosclerosis.

  17. Microgravity decreases c-fos induction and serum response element activity.

    PubMed

    de Groot, R P; Rijken, P J; den Hertog, J; Boonstra, J; Verkleij, A J; de Laat, S W; Kruijer, W

    1990-09-01

    Several studies have shown that altered gravity conditions influence mammalian cell growth and differentiation. The molecular mechanisms underlying these effects, however, remain relatively obscure. In this paper we show that microgravity reached in a sounding rocket strongly decreases epidermal growth factor (EGF)-induced expression of the proto-oncogenes c-fos and c-jun, which are both implicated in the regulation of proliferation and differentiation. Decreased activity of the serum response element (SRE), present in the c-fos promoter-enhancer region, is probably responsible for the decrease in EGF-induced c-fos expression. In addition, we show that gravity alterations differentially modulate distinctive signal transduction pathways, indicating that gravity-dependent modulations of mammalian cell proliferation are unlikely to be caused by a nonspecific stress response of the cell.

  18. Evaluating the protective effects of vitamin C on serum and erythrocyte cholinesterase activity of male rats exposed to malathion

    PubMed Central

    Taherdehi, Faezeh Ghorbani; Nikravesh, Mohammad Reza; Jalali, Mehdi; Fazel, Alireza

    2016-01-01

    Introduction Malathion is one of organophosphate poisons (OPPs) that inhibit cholinesterase activity and induce oxidative stress in target organs, such as the reproductive system. The aim of this study was to assess the effects of Malathion on serum and erythrocyte cholinesterase activity in male rats and also to assess the protective effects of vitamin C in this regard. Methods This experimental study was performed in the Pharmacology Laboratory of the Pharmacy Faculty and in the Advanced Histology Techniques Laboratory of the Medical Faculty of Mashhad University of Medical Sciences (MUMS) in January 2014. Thirty male wistar rats, weighting 200–250 g, were divided into five groups of six. The different groups were exposed as follows: group 1: Malathion 50 mg/kg; group 2: Vitamin C; group 3: Malathion plus Vitamin C with the specified doses; sham group: normal saline; and control group: no exposure. After six weeks, 3 ml blood samples were taken from the rats, and titrimetric and Ellman methods were used to assess serum and erythrocyte cholinesterase activity, respectively. The data was analyzed by SPSS 16, and p < 0.05 was considered significant. Results The activities of serum and erythrocyte cholinesterase were inhibited significantly in the Malathion exposed group compared to the control group (p < 0.001). The administration of Vitamin C alone significantly increased the activities of serum and erythrocyte cholinesterase. The serum and erythrocyte cholinesterase inhibition showed improvement in the group that received both Malathion and Vitamin C. Conclusion Malathion reduced the activities of serum and erythrocyte cholinesterase in exposed animals. It probably has the same intoxication effects on people who are exposed. Improvement of cholinesterase activity by antioxidant effects of Vitamin C suggests that Vitamin C supplementation can be used to decrease side effects of OPP exposure. PMID:27648190

  19. E-ADA activity in serum of lambs experimentally infected with Haemonchus contortus.

    PubMed

    Da Silva, Aleksandro S; Fausto, Guilherme C; Grando, Thirssa H; Cadore, Carlos A; Pimentel, Victor C; Jaques, Jeandre A; Schetinger, Maria R C; Monteiro, Silvia G; Leal, Marta L R

    2013-08-01

    The aim of this study was to evaluate adenosine deaminase (E-ADA) activity in sera of lambs experimentally infected with Haemonchus contortus. We used 12 lambs divided into 2 groups; Group A had 5 healthy, non-infected animals (control) and Group B had 7 healthy animals infected with H. contortus . Lambs were infected orally with 500 larvae (L3) per animal every 2 days, for a period of 20 days, and later the infection was confirmed by examination of feces (eggs per gram [EPG] via fecal egg count). Blood collection was performed at days 0, 20, 40, 60, and 80 post-infection (PI) for analysis of E-ADA activity. Animals in Group A showed negative EPG throughout the experiment unlike those from Group B that had elevated EPG counts. E-ADA activity was reduced in the serum of animals infected with H. contortus when compared to non-infected controls at days 20, 40, 60, and 80 PI. Therefore, it is concluded that infection with H. contortus influences the E-ADA activity in lambs.

  20. A three-way junction aptasensor for lysozyme detection.

    PubMed

    Xia, Yunfeng; Gan, Siwen; Xu, Qinghao; Qiu, Xiaowen; Gao, Peiyi; Huang, Shasheng

    2013-01-15

    A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.

  1. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  2. Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia.

    PubMed

    Koizumi, J; Mabuchi, H; Yoshimura, A; Michishita, I; Takeda, M; Itoh, H; Sakai, Y; Sakai, T; Ueda, K; Takeda, R

    1985-12-01

    Lipoprotein patterns and cholesteryl ester transfer activity (CETA) were examined in 2 patients with familial hyperalphalipoproteinaemia (FHALP). The proband was a healthy 58-year-old Japanese male who had an HDL cholesterol of 7.83 mmol/l (301 mg/dl). His sister's HDL cholesterol was 4.52 mmol/l (174 mg/dl), which suggested that both were homozygous carriers of FHALP. In both subjects HDL showed a high cholesterol/apo A-I ratio and appeared to be a larger-sized particle than normal HDL on agarose gel chromatography. Two of the proband's children showed higher HDL cholesterol levels (1.74 mmol/l, 2.16 mmol/l) than normal, but another 2 children showed normal levels (1.48 mmol/l, 1.40 mmol/l). However, the ratios of HDL cholesterol to total cholesterol and to apo A-I in all children were higher than normal. These data suggest, but do not prove, that all his children were heterozygotes. Apo B levels in all of the family members studied were lower than normal (47-80 mg/dl). Deceased members of the same family had not died from cardiovascular disease. Cholesteryl-ester transfer activity was studied in both patients. When serum or lipoprotein deficient serum (d greater than 1.21) and [3H]cholesteryl ester labelled HDL3 were incubated in the presence of an LCAT inhibitor, there was no evidence of cholesteryl ester transfer from HDL to VLDL and/or LDL, unlike normal subjects. The deficiency of CETA in these patients with FHALP presumably accounted for the increase in particle size and cholesterol enrichment of HDL.

  3. Association of serum paraoxonase enzyme activity and oxidative stress markers with dyslipidemia in obese adolescents

    PubMed Central

    Zaki, Moushira Erfan; El-Bassyouni, Hala; Kamal, Sanaa; El-Gammal, Mona; Youness, Eman

    2014-01-01

    Objectives: The aim of the present study was to investigate the serum paraoxonase 1 (PON1) concentration and oxidative stress markers and assess its relations with the biochemical parameters in obese adolescents. Materials and Methods: One hundred and fifty obese adolescents (range 16-18 years) and 150 healthy age- and sex-matched controls were enrolled in the study. The data were extracted from a project entitled “Obesity among Youth: Lifestyle and Genetic Factors” funded by the Science and Technology Development Fund, Egypt. Serum paraoxonase 1 (PON1), nitric oxide (NO), and malonaldehyde were measured. Anthropometry, fasting glucose, insulin concentrations, total cholesterol, high density lipoprotein–cholesterol, low density lipoprotein–cholesterol, triglycerides, systolic and diastolic blood pressure (BP) were measured. Insulin resistance was determined by Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Diagnostic accuracy of oxidative markers to identify dyslipidemia was calculated with ROC analysis. Results: The study showed that PON1 activity was significantly lower in obese adolescents than controls. Obese adolescents had significant lower NO level and significant increased MA values as compared to controls. PON1 was negatively correlated with MAD and body mass index in obese subjects. Obese adolescents showed dyslipidemia and increased blood pressure and HOMA-IR values. PON1 had high area under the curve in ROC analysis for identifying dyslipidemia in obese subjects. Conclusions: Our results indicate that obese subjects have increased oxidative stress and decreased PON1 activity. The lower paraoxonase level might contribute to the greater risk of dyslipidemia, insulin resistance, high blood pressure that are considered as important components in the pathogenesis of the metabolic syndrome in obese adolescents. PMID:24944928

  4. Fever and increased serum IL-1 activity as a systemic manifestation of acute phototoxicity in New Zealand White rabbits.

    PubMed

    Ansel, J C; Luger, T A; Green, I

    1987-07-01

    Ultraviolet (UV) radiation is a significant environmental hazard for humans and animals. Although the clinical effect of an acute UV exposure such as cutaneous inflammation, malaise, somnolence, chills, and fever have been appreciated for many years, the underlying mechanisms mediating these effects are poorly understood. Since chills and fever are the most dramatic systemic sequelae after a prolonged exposure to UV, we specifically examined the effect of whole-body UV irradiation on core body temperature and serum endogenous pyrogen activity of New Zealand White rabbits, correlating this with serum interleukin 1 (IL-1) activity and alterations of serum divalent cation levels. We found that an acute dose of UV irradiation (Westinghouse FS-40 lamps, 0.2 mJ/cm2/s X 8 h) resulted in a significant increase in the core body temperature 2 h post UV (0.8 degree C), peaking 5 h post UV (1.8 degree C), and returning to normal 24 h post UV. Likewise, the sera from the UV-irradiated rabbits had significant endogenous pyrogen activity when transferred into naive recipient animals, causing an increase in core body temperature within 45 min (0.65 +/- 0.12 degree C), decreasing over the next 2 h, and returning to normal 6 h post injection. No endotoxin contamination was detected in any serum samples. This post-UV febrile response was accompanied by a prolonged increase in serum IL-1 activity (5-10 X) and a significant alteration in serum divalent cation levels, with the rabbits becoming euthermic even as the serum IL-1 levels remained elevated. These findings provide new information concerning the pathogenesis and kinetics of these systemic effects after an acute dose of UV irradiation.

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

    PubMed

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

    2005-08-01

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

  6. Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

    PubMed Central

    Shephard, M D; Peake, M J; Walmsley, R N

    1986-01-01

    A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made. PMID:3760234

  7. Adenosine deaminase activity in serum and lymphocytes of rats infected with Sporothrix schenckii.

    PubMed

    Castro, Verônica S P; Pimentel, Victor C; Da Silva, Aleksandro S; Thomé, Gustavo R; Wolkmer, Patrícia; Castro, Jorge L C; Costa, Márcio M; da Silva, Cássia B; Oliveira, Daniele C; Alves, Sydney H; Schetinger, Maria R C; Lopes, Sonia T A; Mazzanti, Cinthia M

    2012-07-01

    Sporotrichosis is a fungal infection of subcutaneous or chronic evolution, inflammatory lesions characterized by their pyogranulomatous aspect, caused by the dimorphic fungus Sporothrix schenckii. Adenosine deaminase (ADA) is a "key" enzyme in the purine metabolism, promoting the deamination of adenosine, an important anti-inflammatory molecule. The increase in ADA activity has been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this fungal infection. The objective of this study was to evaluate the activity of serum ADA (S-ADA) and lymphocytes (L-ADA) of rats infected with S. schenckii. We used seventy-eight rats divided into two groups. In the first experiment, rats were infected subcutaneously and in the second experiment, infected intraperitoneally. Blood samples for hematologic evaluation and activities of S-ADA and L-ADA were performed at days 15, 30, and 40 post-infection (PI) to assess disease progression. In the second experiment, it was observed an acute decrease in activity of S-ADA and L-ADA (P < 0.05), suggesting a compensatory mechanism in an attempt to protect the host from excessive tissue damage. With chronicity of disease the rats in the first and second experiment at 30 days PI showed an increased activity of L-ADA (P < 0.05), promoting an inflammatory response in an attempt to combat the spread of the agent. Thus, it is suggested that infection with S. schenckii alters the activities of S-ADA in experimentally infected rats, demonstrating the involvement of this enzyme in the pathogenesis of sporotrichosis.

  8. Comparison of Antibodies with Amylase Activity from Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

    PubMed Central

    Doronin, Vasilii B.; Parkhomenko, Taisiya A.; Castellazzi, Massimiliano; Cesnik, Edward; Buneva, Valentina N.; Granieri, Enrico; Nevinsky, Georgy A.

    2016-01-01

    We have recently shown that IgGs from serum and cerebrospinal fluid (CSF) of MS patients are active in hydrolysis of DNA and myelin basic protein. According to literature data, anti-DNA and anti-MBP abzymes may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development. At the same time, the involvement of antibodies with amylase activity in the pathogenesis of any autoimmune disease has not yet been identified. Electrophoretically and immunologically homogeneous IgGs were obtained by a sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We are able to present the first unpredictable evidence showing that IgGs from CSF possess amylase activity and efficiently hydrolyze maltoheptaose; their average specific Ab activity is ~30-fold higher than that of antibodies from sera of the same MS patients. Specific average RA (SAA) for IgGs from healthy volunteers was approximately ~1000 lower than that for MS patients. In addition, it was shown that a relative SAA of total proteins of CSF (including Abs) ~15-fold lower than that for purified IgGs, while the relative SAA of the total sera protein is higher than that of sera IgGs by a factor of 1033. This result speaks in favor of the fact that amylolytic activity of CSF proteins is mainly caused by the activity of amylase abzymes. One cannot exclude, that amylase abzymes of CSF can play a, as yet unknown, role in the pathogenesis of MS. Some possible reasons of these findings are discussed. PMID:27196086

  9. Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

    PubMed

    Lyulcheva, Ekaterina; Taylor, Eleanor; Michael, Magdalene; Vehlow, Anne; Tan, Shengjiang; Fletcher, Adam; Krause, Matthias; Bennett, Daimark

    2008-11-01

    MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

  10. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    PubMed Central

    Arfilli, Valentina; Carnicelli, Domenica; Ardissino, Gianluigi; Torresani, Erminio; Scavia, Gaia; Brigotti, Maurizio

    2015-01-01

    Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins). PMID:26556372

  11. Bovine serum albumin with glycated carboxyl groups shows membrane-perturbing activities.

    PubMed

    Yang, Shin-Yi; Chen, Ying-Jung; Kao, Pei-Hsiu; Chang, Long-Sen

    2014-12-15

    The aim of the present study aimed to investigate whether glycated bovine serum albumin (BSA) showed novel activities on the lipid-water interface. Mannosylated BSA (Man-BSA) was prepared by modification of the carboxyl groups with p-aminophenyl α-d-mannopyranoside. In contrast to BSA, Man-BSA notably induced membrane permeability of egg yolk phosphatidylcholine (EYPC)/egg yolk sphingomyelin (EYSM)/cholesterol (Chol) and EYPC/EYSM vesicles. Noticeably, Man-BSA induced the fusion of EYPC/EYSM/Chol vesicles, but not of EYPC/EYSM vesicles. Although BSA and Man-BSA showed similar binding affinity for lipid vesicles, the lipid-bound conformation of Man-BSA was distinct from that of BSA. Moreover, Man-BSA adopted distinct structure upon binding with the EYPC/EYSM/Chol and EYPC/EYSM vesicles. Man-BSA could induce the fusion of EYPC/EYSM/Chol vesicles with K562 and MCF-7 cells, while Man-BSA greatly induced the leakage of Chol-depleted K562 and MCF-7 cells. The modified BSA prepared by conjugating carboxyl groups with p-aminophenyl α-d-glucopyranoside also showed membrane-perturbing activities. Collectively, our data indicate that conjugation of carboxyl groups with monosaccharide generates functional BSA with membrane-perturbing activities on the lipid-water interface.

  12. Analysis of Monomer Aggregation and Crystal Growth Rates of Lysozyme

    NASA Technical Reports Server (NTRS)

    Nadarajah, Arunan

    1996-01-01

    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.

  13. Activity of cholinesterases and adenosine deaminase in blood and serum of rats experimentally infected with Trypanosoma cruzi

    PubMed Central

    DA SILVA, A S; PIMENTEL, V C; FIORENZA, A M; FRANÇA, R T; TONIN, A A; JAQUES, J A; LEAL, C A M; DA SILVA, C B; MORSCH, V; SCHETINGER, M R C; LOPES, S T A; MONTEIRO, S G

    2011-01-01

    This study aimed to evaluate the activity of cholinesterases and adenosine deaminase (ADA) in blood and serum of rats infected with Trypanosoma cruzi. Twelve adult rats were used in the experiment divided into two uniform groups. Rodents from group A (control group) were non-infected and animals from group B served as infected, receiving intraperitoneally 3.3×107 trypomastigotes/each. Blood collection was performed at days 60 and 120 post-infection (PI) in order to evaluate the hemogram, blood activity of acetylcholinesterase, and serum butyrylcholinesterase and ADA activities. Hematological parameters did not differ between groups. A significant increase (P<0.05) of acetylcholinesterase activity was observed in blood while butyrylcholinesterase had a significant reduction (P<0.01) in serum of infected rats at days 60 and 120 PI. ADA activity in serum showed an inhibition in infected animals when compared to non-infected at day 120 PI. Based on these results, it is possible to conclude that the activity of cholinesterases and ADA were changed in animals infected with T. cruzi. The possible causes of these alterations will be discussed in this paper. PMID:21929880

  14. Exosomal microRNA miR-92a concentration in serum reflects human brown fat activity

    PubMed Central

    Chen, Yong; Buyel, Joschka J.; Hanssen, Mark J. W.; Siegel, Franziska; Pan, Ruping; Naumann, Jennifer; Schell, Michael; van der Lans, Anouk; Schlein, Christian; Froehlich, Holger; Heeren, Joerg; Virtanen, Kirsi A.; van Marken Lichtenbelt, Wouter; Pfeifer, Alexander

    2016-01-01

    Brown adipose tissue (BAT) dissipates energy and its activity correlates with leanness in human adults. 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography coupled with computer tomography (PET/CT) is still the standard for measuring BAT activity, but exposes subjects to ionizing radiation. To study BAT function in large human cohorts, novel diagnostic tools are needed. Here we show that brown adipocytes release exosomes and that BAT activation increases exosome release. Profiling miRNAs in exosomes released from brown adipocytes, and in exosomes isolated from mouse serum, we show that levels of miRNAs change after BAT activation in vitro and in vivo. One of these exosomal miRNAs, miR-92a, is also present in human serum exosomes. Importantly, serum concentrations of exosomal miR-92a inversely correlate with human BAT activity measured by 18F-FDG PET/CT in two unique and independent cohorts comprising 41 healthy individuals. Thus, exosomal miR-92a represents a potential serum biomarker for BAT activity in mice and humans. PMID:27117818

  15. [Activity of various serum enzymes in calves suffering from nutritionally-induced muscular dystrophy].

    PubMed

    Kursa, J

    1975-08-01

    The paper described the findings of the activity of aspartate amino transferase (GOT) and alanine amino transferase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and aldolase in the blood serum of calves examined for white-muscle disease (WMD). Relapsing mass accurrence of the disease was reported from various agricultural enterprises where calves were fed a milk replacer without vitamin E. In comparison with clinically healthy calves fed a feed mixture with vitamin E, calves suffering from the clinical form of WMD showed an alkaline phosphatase level decrease from 32.3 +/- 7.6 u. K. A. to 15.1 +/- 8.2 U. K. A. On the other hand, the activities of ALD, GOT, GPT, and LDH showed a statistically significant increase. The acute and subacute course of the disease increased enzyme activities as follows: ALD from 4.2 +/- 1.1 mumol (= 70.0 +/- 17.0 i. u.) to 9.7 +/- 2.1 mumol (= 163.0 +/- 33.2 i. u.), GOT from 0.9 +/- 0.5 mumol (= 68.0 +/- 5.8 i. u.) to 16.7 +/- 11.7 mumol (= 567.0 +/- 40.0 i. u.), GPT from 0.2 +/- 0.8 mumol (= 5.0 +/- 12.4 i. u.) to 9.8 +/- 2.8 mumol (= 330.0 +/- 40.4 i. u.), LDH from 46.1 +/- 5.4 mumol (= 765.0 +/- 40.0 i. u.) to 72.7 +/- 24.3 mumol (= 1,207.0 +/- 403.0 i. u.). In WMD-affected herds, similar enzyme activity fluctuations were observed even in calves showing no clinical signs of the disease. It follows from the study that the examination of serum enzymes provides a method to demonstrate the clinical and pre-clinical forms of white-muscle disease and that it can be included in the set of tests for the diagnosis of diseases in calves. The significant differences in all calves in the affected herds show that the disease is a danger to all animals in the herd fed a deficient mixture.

  16. Adenosine triphosphate prevents serum deprivation-induced apoptosis in human mesenchymal stem cells via activation of the MAPK signaling pathways.

    PubMed

    Berlier, Jessica L; Rigutto, Sabrina; Dalla Valle, Antoine; Lechanteur, Jessica; Soyfoo, Muhammad S; Gangji, Valerie; Rasschaert, Joanne

    2015-01-01

    Human mesenchymal stem cells (hMSC) are multipotent cells derived from various sources including adipose and placental tissues as well as bone marrow. Owing to their regenerative and immunomodulatory properties, their use as a potential therapeutic tool is being extensively tested. However, one of the major hurdles in using cell-based therapy is the use of fetal bovine serum that can trigger immune responses, viral and prion diseases. The development of a culture medium devoid of serum while preserving cell viability is therefore a major challenge. In this study, we demonstrated that adenosine triphosphate (ATP) restrained serum deprivation-induced cell death in hMSC by preventing caspases 3/7 activation and modulating ERK1/2 and p38 MAPK signaling pathways. We also showed that serum deprivation conditions triggered dephosphorylation of the proapoptotic protein Bad leading to cell death. Adjunction of ATP restored the phosphorylation state of Bad. Furthermore, ATP significantly modulated the expression of proapoptopic and antiapoptotic genes, in favor of an antiapoptotic profile expression. Finally, we established that hMSC released a high amount of ATP in the extracellular medium when cultured in a serum-free medium. Collectively, our results demonstrate that ATP favors hMSC viability in serum deprivation conditions. Moreover, they shed light on the cardinal role of the MAPK pathways, ERK1/2 and p38 MAPK, in promoting hMSC survival.

  17. Probing lysozyme conformation with light reveals a new folding intermediate.

    PubMed

    Hamill, Andrea C; Wang, Shao-Chun; Lee, C Ted

    2005-11-22

    A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a

  18. The solubility of hen egg-white lysozyme

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

    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.

  19. Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme.

    PubMed

    Morgenstern, Josefine; Baumann, Pascal; Brunner, Carina; Hubbuch, Jürgen

    2017-03-15

    During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2kDa, 5kDa and 10kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved.

  20. Mg deficiency results in modulation of serum lipids, glutathione, and NO synthase isozyme activation in cardiovascular tissues: relevance to de novo synthesis of ceramide, serum Mg and atherogenesis.

    PubMed

    Shah, Nilank C; Liu, Jian-Ping; Iqbal, Jahangir; Hussain, Mahmood; Jiang, Xian-Cheng; Li, Zhiqiang; Li, Yan; Zheng, Tao; Li, Wenyan; Sica, Anthony C; Perez-Albela, Jose Luis; Altura, Bella T; Altura, Burton M

    2011-01-01

    The present work tested the hypothesis that short-term (S-T) dietary deficiency of magnesium (Mg) (21 days) in rats would: 1) result in reduction in serum(s) sphingomyelin (SM) and changes in several blood lipids, HDL-cholesterol (HDL-C) and phosphatidylcholine (PC) concomitant with elevations in s cholesterol (chol), s LDL+VLDL and trigycerides (TG), as well as reduction in the PC/cholesterol ratio; 2) lead to oxidative stress, characterized by reductions in glutathione (glut) content in the various chambers of the heart and activation of e-NOS and n-NOS in the atria, ventricles and aortic smooth muscle (ASM); 3) produce early cardiac damage characterized by leakage of creatine kinase (CK) and lactic dehydrogenase (LDH); and 4) demonstrate that these pathophysiological changes are a result of profound reductions in s ionized Mg (Mg(2+)) and activation of the SM-ceramide pathway. In addition, we hypothesized that: 1) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg(2+) would lead to de novo synthesis of ceramide and activation of NO synthase with reduction in glut, both of which would be attenuated by inhibition of sphingomyelinase (SMase) and serine palmitoyl CoA transferase (SPT); and 2) low levels of Mg(2+)added to the drinking water would either prevent or ameliorate these manifestations. Our data indicate that S-T Mg deficiency resulted in reductions in s Mg(2+), SM, PC, HDL-C and the PC/chol ratio concomitant with decreases in tissue levels of glut, leakage of cardiac CK and LDH, as well as activation of e-NOS and n-NOS in all chambers of the heart and ASM. The greater the reduction in s Mg(2+), the greater the effects on all parameters analyzed; very significant correlations to levels of s SM and Mg(2+) were found with all of the serum and tissue biochemical -molecular analytes measured. Our experiments also showed that VSMCs exposed to low Mg(2+)resulted in activation of NO synthase, loss of glut and de novo

  1. Mushroom toxicosis in dogs in general practice causing gastroenteritis, ptyalism and elevated serum lipase activity.

    PubMed

    Hall, J; Barton, L

    2013-05-01

    Mushroom toxicosis is rarely diagnosed in dogs and is poorly reported in the veterinary literature. This report suggests that mushroom toxicosis is a potentially under-diagnosed condition in first opinion practice in the UK. Nine dogs with clinical signs consistent with mushroom toxicosis were identified from the records of an out-of-hours emergency service between August 2010 and January 2011. Four dogs were later excluded because of clinical inconsistencies. Clinical signs included acute profuse ptyalism (5/5), diarrhoea (5/5), vomiting (4/5), hypovolaemia (4/5), stuporous (3/5) or obtunded mentation (1/5), miosis (2/5) and hypothermia (2/5). Serum lipase activity was elevated in 4/4 dogs; canine-specific pancreatic lipase was elevated in the remaining dog. Four dogs recovered with aggressive intravenous fluid therapy, analgesia and supportive care; the remaining dog was euthanased due to severe clinical signs and financial constraints. Mushroom toxicosis is an important differential diagnosis for acute gastroenteritis and one possible cause of some cases of "Seasonal Canine Illness". Affected dogs may demonstrate elevated pancreatic enzymes and mushroom toxicosis should be considered in cases of elevated lipase or abnormal semi-quantitative canine-specific pancreatic lipase activities.

  2. Serum thymic factor, FTS, attenuates cisplatin nephrotoxicity by suppressing cisplatin-induced ERK activation.

    PubMed

    Kohda, Yuka; Kawai, Yoshiko; Iwamoto, Noriaki; Matsunaga, Yoshiko; Aiga, Hiromi; Awaya, Akira; Gemba, Munekazu

    2005-11-01

    Serum thymic factor (FTS), a thymic peptide hormone, has been reported to attenuate the bleomycin-induced pulmonary injury and also experimental pancreatitis and diabetes. In the present study, we investigated the effect of FTS on cis-diamminedichloroplatinum II (cisplatin)-induced nephrotoxicity. We have already demonstrated that cephaloridine, a nephrotoxic antibiotic, leads to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney, which probably contributes to cephaloridine-induced renal dysfunction. The aim of this study was to examine the effect of cisplatin on ERK activation in the rat kidney and also the effect of FTS on cisplatin-induced nephrotoxicity in rats. In vitro treatment of LLC-PK1 cells with FTS significantly ameliorated cisplatin-induced cell injury. Treatment of rats with intravenous cisplatin for 3 days markedly induced renal dysfunction and increased platinum contents in the kidney cortex. An increase in pERK was detected in the nuclear fraction prepared from the rat kidney cortex from days 1 to 3 after injection of cisplatin. FTS suppressed cisplatin-induced renal dysfunction and ERK activation in the kidney. FTS did not influence any Pt contents in the kidney after cisplatin administration. FTS has been shown to enhance the in vivo expression of heat shock protein (HSP) 70 in the kidney cortex. The beneficial role of FTS against cisplatin nephrotoxicity may be mediated in part by HSP70, as suggested by its up-regulation in the kidney cortex treated with FTS alone. Our results suggest that FTS participates in protection from cisplatin-induced nephrotoxicity by suppressing ERK activation caused by cisplatin.

  3. Pharmacokinetics and serum bactericidal activities of quinolones in combination with clindamycin, metronidazole, and ornidazole.

    PubMed Central

    Boeckh, M; Lode, H; Deppermann, K M; Grineisen, S; Shokry, F; Held, R; Wernicke, K; Koeppe, P; Wagner, J; Krasemann, C

    1990-01-01

    To enhance the antimicrobial spectrum of the quinolones against anaerobic organisms and gram-positive bacteria, we investigated in two studies the parenteral combinations of ciprofloxacin (200 mg) and ofloxacin (200 mg) with metronidazole (500 mg) or clindamycin (600 mg) and the oral combinations of enoxacin (400 mg) and fleroxacin (400 mg) with metronidazole (400 mg), clindamycin (300 mg), or ornidazole (500 mg) (only with fleroxacin). The pharmacokinetics and serum bactericidal activities (SBAs) against 5 aerobic and 2 anaerobic species (total, 58 strains) were determined in two groups of 10 healthy volunteers by using a randomized crossover study design. The additions of metronidazole, clindamycin, and ornidazole did not affect the pharmacokinetics of the quinolones. The combination of clindamycin with ciprofloxacin, ofloxacin, and, to a lesser extent, fleroxacin resulted in an increase of the SBA against gram-positive strains (mean peak titers): Staphylococcus aureus, ciprofloxacin alone, 1:5.5; ciprofloxacin-clindamycin, 1:19.9; ofloxacin alone, 1:3.6; ofloxacin-clindamycin, 1:17.5; fleroxacin alone, 1:4.3; fleroxacin-clindamycin, 1:8.1; Streptococcus pneumoniae (fleroxacin and enoxacin were not tested), ciprofloxacin alone, 1:2.0; ciprofloxacin-clindamycin, 1:53; ofloxacin alone, 1:2.6; and ofloxacin-clindamycin, 1:49.2. The high SBA of quinolones against gram-negative bacteria was not affected by the combinations; however, relatively low activities against Pseudomonas aeruginosa were detected. In general, against anaerobic bacteria, low bactericidal activities were determined in both studies (mean peak titers ranged from 1:2.1 to 1:3.1; mean trough titers range from 1:2.0 to 1:2.9). In clinical settings with severe mixed infections, a parenteral therapy consisting of modern quinolones together with clindamycin or imidazole derivatives seems to be active and offers no obvious interactions. PMID:2088195

  4. THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

    EPA Science Inventory

    THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

    One measure of th...

  5. (-)-Epicatechin gallate prevents alkali-salt mediated fibrillogenesis of hen egg white lysozyme.

    PubMed

    Ghosh, Sudeshna; Pandey, Nitin K; Dasgupta, Swagata

    2013-03-01

    Green tea polyphenols (GTPs) are found to be potent inhibitors of amyloid fibril formation. We report the effective inhibitory property of (-)-epicatechin gallate (ECG) during the alkali-salt induced fibrillogenesis of hen egg white lysozyme (HEWL) at 37 °C. Spectroscopic techniques such as fluorescence, circular dichroism and microscopic images show that (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) show moderate inhibition of fibrillation with ECG as the most potent polyphenol. Aromatic interactions, hydrophobic interactions, the radical scavenging activity and autoxidation of polyphenols are likely to be the major reasons for ECG being the most effective inhibitor.

  6. Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

    PubMed Central

    Bishop, Christopher A.; Best, Michael; Rich, Celeste B.; Stone, Phillip J.

    2017-01-01

    Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis. PMID:28257481

  7. Detection of active matrix metalloproteinase-3 in serum and fibroblast-like synoviocytes of collagen-induced arthritis mice.

    PubMed

    Lee, Aeju; Choi, Sung-Jae; Park, Kyeongsoon; Park, Jong Woong; Kim, Kwangmeyung; Choi, Kuiwon; Yoon, Soo-Young; Youn, Inchan

    2013-06-19

    The activity of rheumatoid arthritis (RA) correlates with the expression of proteases. Among several proteases, matrix metalloproteinase-3 (MMP-3) is one of the biological markers used to diagnose RA. The active form of MMP-3 is a key enzyme involved in RA-associated destruction of cartilage and bone. Thus, detection of active MMP-3 in serum or in vivo is very important for early diagnosis of RA. In this study, a soluble MMP-3 probe was prepared to monitor RA progression by detecting expression of active MMP-3 in collagen-induced arthritis (CIA) mice in vivo in both serum and fibroblast-like synoviocytes (FLSs). The MMP-3 probe exhibited strong sensitivity to MMP-3 and moderate sensitivity to MMP-7 at nanomolecular concentrations, but was not sensitive to other MMPs such as MMP-2, MMP-9, and MMP-13. In an optical imaging study, the MMP-3 probe produced early and strong NIR fluorescence signals prior to observation of erythema and swelling in CIA mice. The MMP-3 probe was able to rapidly and selectively detect and monitor active MMP-3 in diluted serum from CIA mice. Furthermore, histological data demonstrated that activated FLSs in arthritic knee joints expressed active MMP-3. Together, our results demonstrated that the MMP-3 probe may be useful for detecting active MMP-3 for diagnosis of RA. More importantly, the MMP-3 probe was able to detect active MMP-3 in diluted serum with high sensitivity. Therefore, the MMP-3 probe developed in this study may be a very promising probe, useful as a biomarker for early detection and diagnosis of RA.

  8. Serum free estradiol and estrogen receptor-α mediated activity are related to decreased incident hip fractures in older women

    PubMed Central

    Lim, Vanessa; Li, Jun; Gong, Yinhan; Yuan, Jian-Min; Wu, Tsung Sheng; Hammond, Geoffrey; Jin, Aizhen; Koh, Woon-Puay; Yong, E.L.

    2012-01-01

    There is paucity of data from Asian women on the association between serum estrogens and osteoporotic hip fracture risk. We conducted a case-control study nested within a population-based prospective cohort, The Singapore Chinese Health Study, to evaluate serum estrogens levels, ERα-mediated estrogenic activity and hip fracture risk in postmenopausal Asian women. Among 35,298 women who were recruited between 1993 and 1998, 15,410 women donated blood for research between 1999 and 2004. From this subcohort, we identified 140 cases who subsequently suffered hip fracture after blood donation, and 278 age-matched controls. Serum levels of total estrone, estradiol and sex hormone binding globulin levels were measured in a blinded fashion among cases and controls. ERα-mediated estrogenic activity of serum samples was quantified using a sensitive ERα-driven cell bioassay. Women with hip fracture had lower serum estrogens than control women. Compared to the lowest quintile, women in the highest quintile of free estradiol exhibited a statistically significant 57% reduction in risk of hip fracture (95% confidence interval (CI), 6%–80%), with a dose-dependent relationship (p for trend = 0.021). High levels of ERα-mediated estrogenic activity was also associated with decreased risk of hip fracture (p for trend=0.048). Overall, women with relatively high levels of both free estradiol and ERα-mediated estrogenic activity had a 55% reduction in hip fracture risk (95% CI, 17%–76%) compared to women with low levels of both. High levels of free estradiol and ERα-mediated estrogen activity in sera were associated with reduced hip fracture risk in Chinese postmenopausal women. PMID:22445734

  9. Benefits of Omega-3 Fatty Acids Supplementation on Serum Paraoxonase 1 Activity and Lipids Ratios in Polycystic Ovary Syndrome

    PubMed Central

    Mohammadi, Elahe; Rafraf, Maryam

    2012-01-01

    Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with increased risk of cardiovascular disease. The purpose of this study was to investigate the ef¬fects of omega-3 fatty acids on serum paraoxonase 1 activity and lipids ratios in polycystic ovary syndrome. Methods: This double-blind randomized controlled clinical trial was conducted on 64 PCOS pa¬tients with 20-35 years old. Thirty two of the subjects had taken 4 g/day omega -3 fatty acids and 32 patients were given placebo for 8 weeks. Fasting blood samples, anthropometric measure¬ments and dietary intakes were collected at the beginning and the end of the study. Serum total cholesterol, triglyceride, and HDL-C were measured using the enzymatic methods. LDL-C con¬centration was calculated by the Friedewald formula and arylesterase activity of serum PON1 was measured. Data were analyzed using SPSS software. Results: Omega-3 fatty acids significantly decreased TC/HDL-C and LDL-C/HDL-C ratios (P = 0.009 for both) and significantly increased serum PON1 activity (P = 0.048) compared with placebo. Changes in TG/HDL-C ratio were not statistically significant in omega-3 fatty acids group at the end of the study in comparison to placebo group. Reduction in TC/HDL-C, LDL-C/HDL-C and TG/HDL-C ratios and increase in serum PON1 activity were also significant in omega-3 fatty acids group at the end of the study compared with baseline values (P <0.001, P < 0.001, P = 0.004, and P = 0.001, respectively). Conclusion: Omega-3 fatty acids may decrease the risk for cardiovascular disease through the improvement in paraxonase-1 activity and reduction in some lipids ratio in PCOS women. PMID:24688934

  10. Crossover assessment of serum bactericidal activity of grepafloxacin, ofloxacin and clarithromycin against respiratory pathogens after oral administration to healthy volunteers.

    PubMed

    Dan, M; Poch, F; Asherov, J

    2001-06-01

    Serum bactericidal activity was studied in a crossover manner in 10 volunteers, after 2-day administration of grepafloxacin 600 mg qd, ofloxacin 400 mg bid and clarithromycin 500 mg bid. Bactericidal activity against clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Klebsiella pneumoniae, was estimated using a standardized microdilution method. Grepafloxacin was highly active against Gram-negative organisms and adequate against pneumococci (mean, 1:13.3). Clarithromycin was very active against both penicillin-susceptible and penicillin-partially-resistant S. pneumoniae (1:102.5) but had poor activity against H. influenzae (1:3.1). Minor adverse effects were commonly associated with grepafloxacin.

  11. Locations of Bromide Ions in Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  12. Science Study Aids 6: Lysozyme - The Cooperative Enzyme.

    ERIC Educational Resources Information Center

    Boeschen, John; Alderton, Gordon

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

  13. Kinetic Roughening Transition and Energetics of Tetragonal Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

    Interpretation of lysozyme crystal growth rates using well-established physical theories enabled the discovery of a phenomenon possibly indicative of kinetic roughening. For example, lysozyme crystals grown above a critical supersaturation sigma, (where supersaturation sigma = ln c/c(sub eq), c = the protein concentration and c(sub eq) = the solubility concentration) exhibit microscopically rough surfaces due to the continuous addition of growth units anywhere on the surface of a crystal. The rate of crystal growth, V(sub c), for the continuous growth process is determined by the continuous flux of macromolecules onto a unit area of the crystal surface, a, from a distance, xi, per unit time due to diffusion, and a probability of attachment onto the crystal surface, expressed. Based upon models applied, the energetics of lysozyme crystal growth was determined. The magnitudes of the energy barriers of crystal growth for both the (110) and (101) faces of tetragonal lysozyme crystals are compared. Finally, evidence supportive of the kinetic roughening hypothesis is presented.

  14. Direct AFM observations of impurity effects on a lysozyme crystal

    NASA Astrophysics Data System (ADS)

    Nakada, Toshitaka; Sazaki, Gen; Miyashita, Satoru; Durbin, Stephen D.; Komatsu, Hiroshi

    1999-01-01

    Impurity effects on the growth of tetragonal lysozyme crystals have been studied using in situ atomic force microscopy. Commercially available hen egg white lysozyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining, and purified by re-crystallization and successive high pressure liquid chromatography. On the (1 1 0) crystal surface, there was no significant difference in morphology between crystals grown in commercial and in purified solutions. On the (1 0 1) surface, however, a large number of small particles were found when the crystal was grown in the commercial solution, while the surface grown in the purified solution was quite smooth. Among the typical residual impurities contained in commercial lysozyme, only covalently bound lysozyme dimer yielded such particles. From measurements of particle separation and an estimate of the critical nucleation size, we infer that the particles reduced the step velocity according to the mechanism described by Cabrera et al. [N. Cabrera, D.A. Vermilyea, in: R.H. Doremus et al. (Eds.), Growth and Perfection of Crystals, 1958, P. 393].

  15. The folding-unfolding transition of equine lysozyme

    NASA Astrophysics Data System (ADS)

    Haezebrouck, P.; Van Dael, H.

    1993-03-01

    A detailed study of the chemical and thermal unfolding transition of equine lysozyme in the presence and in the absence of Ca 2+ gives evidence for a two-step unfolding process. The pretransition can be related to the transfer of exposed Trp groups to the protein interior.

  16. Resonance energy transfer study of lysozyme-lipid interactions.

    PubMed

    Gorbenko, Galyna P; Ioffe, Valeriya M; Molotkovsky, Julian G; Kinnunen, Paavo K J

    2008-05-01

    Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.

  17. Relation between low serum cholesteryl-ester transfer activity and abdominal aortic calcification in normolipidemic elderly subjects.

    PubMed

    Miyashita, Y; Morimoto, S; Fukuo, K; Masuyama, T; Yasuda, O; Koh, E; Tamatani, M; Nakahashi, T; Ogihara, T

    1993-01-01

    We studied the relation between cholesteryl-ester transfer activity (CETA) and abdominal aortic calcification in elderly subjects. Compared with 10 young healthy subjects (mean +/- S.D. age, 27 +/-2 years) and to 26 elderly subjects without abdominal aortic calcification (79 +/- 7 years), 16 elderly patients with abdominal aortic calcification (82 +/- 6 years) had significantly lower levels of serum CETA. However, there were no differences in the levels of serum lipids and apolipoproteins, including total cholesterol, triglycerides, high density lipoprotein cholesterol, low density lipoprotein cholesterol and apolipoproteins A-I, A-II, B, C-II and E, between the two elderly groups. When the two groups of elderly subjects were considered together, the level of serum CETA did not correlate significantly with any lipids and apolipoproteins. These results provide evidence that CETA may prevent the development of aortic calcification in normolipidemic elderly people.

  18. Peroxidase-coupled method for kinetic colorimetry of total creatine kinase activity in serum.

    PubMed

    Wimmer, M C; Artiss, J D; Zak, B

    1985-10-01

    We describe a peroxidase-coupled method involving a colorimetric indicator reaction for determining the total activity of creatine kinase (EC 2.7.3.2) in serum. The kinetically favorable reverse reaction is exploited to generate adenosine 5'-triphosphate, which is used in the glycerol kinase-catalyzed phosphorylation of glycerol. The glycerol 3-phosphate so generated is oxidized in the presence of alpha-glycerophosphate oxidase to produce hydrogen peroxide, which is reduced in the presence of peroxidase with the simultaneous oxidation and coupling of 4-aminoantipyrene and 2-hydroxy-3,5-dichlorobenzenesulfonate to produce an intensely colored red chromogen. Results of the proposed method (y) correlate well with those of the Boehringer-Mannheim "CK-NAC UV" method as applied to the Hitachi 705 chemistry analyzer (y = 1.025 chi - 18.1, r = 0.9985, n = 100, range = 19-4531 U/L). The sensitivity of the method, based on molar absorptivities, is nearly fourfold that of procedures involving the reduction of NADP+.

  19. Structural characteristics of activated carbons and ibuprofen adsorption affected by bovine serum albumin.

    PubMed

    Melillo, M; Gun'ko, V M; Tennison, S R; Mikhalovska, L I; Phillips, G J; Davies, J G; Lloyd, A W; Kozynchenko, O P; Malik, D J; Streat, M; Mikhalovsky, S V

    2004-03-30

    Structural characteristics of a series of MAST carbons were studied using scanning electron microscopy images and the nitrogen adsorption isotherms analyzed with several models of pores and different adsorption equations. A developed model of pores as a mixture of gaps between spherical nanoparticles and slitlike pores was found appropriate for MAST carbons. Adsorption of ibuprofen [2-(4-isobutylphenyl)propionic acid] on activated carbons possessing different pore size distributions in protein-free and bovine serum albumin (BSA)-containing aqueous solutions reveals the importance of the contribution of mesopores to the total porosity of adsorbents. The influence of the mesoporosity increases when considering the removal of the drug from the protein-containing solution. Cellulose-coated microporous carbon Norit RBX adsorbs significantly smaller amounts of ibuprofen than uncoated micro/mesoporous MAST carbons whose adsorption capability increases with increasing mesoporosity and specific surface area, burnoff dependent variable. A similar effect of broad pores is observed on adsorption of fibrinogen on the same carbons. Analysis of the ibuprofen adsorption data using Langmuir and D'Arcy-Watt equations as the kernel of the Fredholm integral equation shows that the nonuniformity of ibuprofen adsorption complexes diminishes with the presence of BSA. This effect may be explained by a partial adsorption of ibuprofen onto protein molecules immobilized on carbon particles and blocking of a portion of narrow pores.

  20. [Regularities of formation of chlorophyll-human serum albumin functionally active complexes in the aqueous medium].

    PubMed

    Semichaevskiĭ, V D

    1975-01-01

    In the system with constant content of the chlorophyll a and increasing amounts of human serum albumin, dependence of pigment incorporation into the complex upon interaction of its aqueous associates with protein solutions was studied by applying the gel filtration on Sephadex G-75 and by measuring light scattering and rate of sensitized photoreduction of the methyl red by ascorbic-acid. The curves were obtained after extraction of the chlorophyll by acetone from dry pigment-protein films formed after desiccation of the aqueous systems. Sigmoid character of the above dependences, their linearization in Hill's coordinates and the value of cooperativity coefficient close to 2 testifies in favour of the cooperative character of the complex formation, two pigment molecules reacting with a single protein molecule. Measurement of adsorption isotherms and their treatment with use of the Brunauer-Emmett-Teller theory of polymolecular adsorption make it possible to evaluate the maximum molar ratio of the pigment to the protein in the complex (close to 2). The pigment-pigment interaction suggests that the chlorophyll molecules adsorbed on the protein are in the state of loosely packed dimers. Deaggregation of aqueus pigment associates by the protein in the course of complex formation results in a considerable increase of the protosensitizing chlorophyll activity.

  1. Serum Response Factor Mediated Gene Activity in Physiological and Pathological Processes of Neuronal Motility

    PubMed Central

    Knöll, Bernd

    2011-01-01

    In recent years, the transcription factor serum response factor (SRF) was shown to contribute to various physiological processes linked to neuronal motility. The latter include cell migration, axon guidance, and, e.g., synapse function relying on cytoskeletal dynamics, neurite outgrowth, axonal and dendritic differentiation, growth cone motility, and neurite branching. SRF teams up with myocardin related transcription factors (MRTFs) and ternary complex factors (TCFs) to mediate cellular actin cytoskeletal dynamics and the immediate-early gene (IEG) response, a bona fide indicator of neuronal activation. Herein, I will discuss how SRF and cofactors might modulate physiological processes of neuronal motility. Further, potential mechanisms engaged by neurite growth promoting molecules and axon guidance cues to target SRF’s transcriptional machinery in physiological neuronal motility will be presented. Of note, altered cytoskeletal dynamics and rapid initiation of an IEG response are a hallmark of injured neurons in various neurological disorders. Thus, SRF and its MRTF and TCF cofactors might emerge as a novel trio modulating peripheral and central axon regeneration. PMID:22164132

  2. Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

    PubMed

    Griffiths, Natalie J; Hill, Darryl J; Borodina, Elena; Sessions, Richard B; Devos, Nathalie I; Feron, Christiane M; Poolman, Jan T; Virji, Mumtaz

    2011-12-01

    Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

  3. In Silico and Biochemical Characterization of Lysozyme-Like Proteins in the Rat

    PubMed Central

    Narmadha, Ganapathy; Yenugu, Suresh

    2016-01-01

    Background Spermatogenesis and sperm maturation in the male reproductive tract is dictated by a variety of proteins secreted in the testis and epididymis. Though the proteome of these tissues is known, the functional role of many of these proteins remains uncharacterized. In this study, we characterize the rat Lysozyme-like (Lyzl) genes and proteins. Methods In silico tools were used to predict the primary, secondary and tertiary structures. Reverse transcription PCR, immunofluorescence and immunoblotting were used to determine the expression pattern. Lysozyme like enzyme activity was assessed by standard assays. Results Six rat Lyzl genes namely Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7 were found to be highly conserved among the vertebrates with higher homology to mouse counterparts than with human counterparts. All the LYZL proteins contained the characteristic 4 disulfide bridges similar to c-type lysozyme. Only LYZL 1 and 6, conserved the active site amino acids of the lysozyme. Molecular modeling studies indicated that LYZL proteins exhibit strikingly similar three-dimensional structures among themselves. The secondary structure analysis of the recombinant LYZL proteins indicated the presence of α-helix, β-sheet and random coil with α-helix being the majority. Docking studies indicated the peptidoglycan binding nature of LYZL proteins. All the rat Lyzl mRNA transcripts (Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7) are predominantly expressed in testes though some of them are expressed in tissues other than reproductive tract. Their expression was androgen independent. The rat LYZL proteins are localized in the germinal epithelium and on the spermatozoa. Recombinant LYZL1 and 6 possessed muramidase, isopeptidase and antibacterial activities. The mechanism of antibacterial action of LYZL1 and LYZL6 involved bacterial membrane damage and leakage of cellular contents. Only LYZL1 and 6 possess peptidoglycan binding ability, whereas LYZL3, LYZL4 and LYZL5

  4. Sinusoidal 50-Hz magnetic fields depress rat pineal NAT activity and serum melatonin. Role of duration and intensity of exposure.

    PubMed

    Selmaoui, B; Touitou, Y

    1995-01-01

    The purpose of this study was to determine whether the exposure to a 50-Hz sinusoidal magnetic field could influence serum melatonin concentration and pineal enzymes activities in rats. The effects of both duration and intensity of exposure were also looked at. Two groups of Wistar male rats were exposed to 50-Hz magnetic fields of either 1, 10 or 100 microT. The first group was exposed for 12 hours and the second for 30 days (18 hours per day). During this time the animals were kept under a standard 12:12 light: dark cycle with a temperature of 25 degrees C and a relative humidity of 45 to 50%. Control (Sham-exposed) animals were kept in a similar environment but without exposure to a magnetic field. The animals were sacrificed under red dim light. Serum melatonin concentration and pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities were studied. Long-term exposure to a magnetic field (10 and 100 microT) significantly depressed the nocturne peak of serum melatonin concentration and pineal NAT activity whereas no effect was observed on HIOMT activity. Short-term exposure depressed both pineal NAT activity and nocturnal serum melatonin concentration but only with the highest intensity used (100 microT). Our results suggest that sinusoidal magnetic fields alter the production of melatonin through an inhibition of pineal NAT activity. Both duration and intensity of exposure play an important role in this effect. This work shows that, 1) sinusoidal magnetic field depresses NAT activity as static magnetic field does whereas HIOMT activity remains unaltered whatever the type of experiment and the intensity used, 2) the effect observed is related to both the duration of exposure and the intensity of magnetic fields, 3) the sensitivity threshold to magnetic fields vary with the duration of exposure which strongly suggests a cumulative effect of sinusoidal magnetic fields on pineal function.

  5. Plasma renin activity and serum aldosterone during prolonged physical strain. The significance of sleep and energy deprivation.

    PubMed

    Opstad, P K; Oktedalen, O; Aakvaag, A; Fonnum, F; Lund, P K

    1985-01-01

    Plasma renin activity (PRA), serum aldosterone and the serum and urinary levels of sodium and potassium have been investigated in 24 young men participating in a 5-day military training course with heavy continuous physical exercise, energy and sleep deprivation. The subjects were divided into three groups. Group 1 did not get any extra sleep or food, group 2 were compensated for the energy deficiency, and group 3 slept 3 h each night. The basic diet given to all the subjects was about 5,000 kJ and 2 g NaCl X 24 h-1 X cadet-1. The high calorie diet contained approximately 25,000-35,000 kJ and 20 g of NaCl X 24 h-1 X cadet-1. The study showed that serum aldosterone and PRA were extremely activated during such prolonged physical strain combined with lack of food and salt, whereas sleep deprivation did not seem to have any large influence. Only small variations were found in the serum levels of sodium and potassium and the urinary level of potassium during the course, whereas a decrease was seen in urinary sodium concentration. The fairly good correlations between the decrease in urinary sodium levels and the increase in PRA (r = 0.7) and further between PRA and serum aldosterone (r = 0.8) during the course indicate that there is a causal connection between the decrease in urinary sodium excretion and the increase in PRA and serum aldosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Seasonal variations of 25-OH vitamin D serum levels are associated with clinical disease activity in multiple sclerosis patients.

    PubMed

    Hartl, Christina; Obermeier, Viola; Gerdes, Lisa Ann; Brügel, Mathias; von Kries, Rüdiger; Kümpfel, Tania

    2017-04-15

    Low 25-hydroxy vitamin D (25-[OH]-D) serum concentrations have been associated with higher disease activity in multiple sclerosis (MS) patients. In a large cross-sectional study we assessed the vitamin D status in MS patients in relation to seasonality and relapse rate. 415 MS-patients (355 relapsing-remitting MS and 60 secondary-progressive, 282 female, mean age 39.1years) of whom 25-(OH)-D serum concentrations were determined at visits between 2010 and 2013 were included in the study. All clinical data including relapse at visit and expanded disability status scale were recorded in a standardized manner by an experienced neurologist. Seasonal variations of 25-(OH)-D serum concentrations were modelled by sinusoidal regression and seasonal variability in the prevalence of relapse by cubic regression. The mean 25-(OH)-D serum concentration was 24.8ng/ml (range 8.3-140ng/ml) with peak levels of 32.2ng/ml in July/August and nadir in January/February (17.2ng/ml). The lowest modelled prevalence of relapse was in September/October (28%) and the highest modelled prevalence in March/April (47%). The nadir of 25-(OH)-D serum concentrations preceded the peak in prevalence of relapses by two months. In summary, seasonal variation of 25-(OH)-D serum levels were inversely associated with clinical disease activity in MS patients. Future studies should investigate whether vitamin D supplementation in MS patients may decrease the seasonal risk for MS relapses.

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

    PubMed Central

    Biswas, Ashraf A.; Lee, Sung Sill; Mamuad, Lovelia L.; Kim, Seon-Ho; Choi, Yeon-Jae; Bae, Gui-Seck; Lee, Kichoon; Sung, Ha-Guyn; Lee, Sang-Suk

    2016-01-01

    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. PMID:27703130

  8. Effect of PEG end-group hydrophobicity on lysozyme interactions in solution characterized by light scattering.

    PubMed

    Priya, M Hamsa; Pratt, L R; Paulaitis, M E

    2011-11-15

    We compare protein-protein and protein-polymer osmotic virial coefficients measured by static light scattering for aqueous solutions of lysozyme with low-molecular-weight, hydroxy-terminated (hPEG) and methyl-terminated (mPEG) poly(ethylene glycol) at two solution conditions: pH 7.0 and 0.01 M ionic strength, and pH 6.2 and 0.8 M ionic strength. We find that adding PEG to aqueous lysozyme solutions makes a net repulsive contribution to lysozyme-lysozyme interactions, independent of ionic strength and PEG end-group hydrophobicity. PEG end-group hydrophobicity has a profound effect on the magnitude of this contribution, however, at low ionic strength where mPEG-lysozyme attractive interactions become significant. The enhanced attractions promote mPEG-lysozyme preferential interactions at the expense of lysozyme self-interactions, which