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 newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Expression of recombinant human lysozyme in bacterial artificial chromosome transgenic mice promotes the growth of Bifidobacterium and inhibits the growth of Salmonella in the intestine.

PubMed

Dan, Lu; Liu, Shen; Shang, Shengzhe; Zhang, Huihua; Zhang, Ran; Li, Ning

2018-04-20

Targeted gene modification is a novel intervention strategy to increase disease resistance more quickly than traditional animal breeding. Human lysozyme, a natural, non-specific immune factor, participates in innate immunity, exerts a wide range of antimicrobial activities against pathogens, and has immuneregulatory effects. Therefore, it is a candidate gene for improved disease resistance in animals. In this study, we successfully generated a transgenic mouse model by microinjecting a modified bacterial artificial chromosome containing a recombinant human lysozyme (rhLZ) gene into the pronuclei of fertilized mouse embryos. rhLZ was expressed in serum, liver, spleen, lung, kidney, stomach, small intestine, and large intestine but not in milk. rhLZ protein concentrations in the serum of transgenic mice ranged from 2.09 to 2.60 mg/l. To examine the effect of rhLZ on intestinal microbiota, total aerobes, total anaerobes, Clostridium, Enterococcus, Streptococcus, Salmonella, Escherichia coli, Staphylococcus, Bifidobacterium, and Lactobacillus were measured in the intestines of transgenic and wild type mice. Results showed that Bifidobacteria were significantly increased (p < 0.001), whereas Salmonella were significantly decreased (p < 0.001) in transgenic mice compared to wild type mice. Our study suggests that rhLZ expression is a potential strategy to increase animal disease resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

Nayduch, Dana; Joyner, Chester

2013-07-01

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

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

PubMed

Carvalho, Eunice B; Maga, Elizabeth A; Quetz, Josiane S; Lima, Ila F N; Magalhães, Hemerson Y F; Rodrigues, Felipe A R; Silva, Antônio V A; Prata, Mara M G; Cavalcante, Paloma A; Havt, Alexandre; Bertolini, Marcelo; Bertolini, Luciana R; Lima, Aldo A M

2012-08-11

Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

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

PubMed

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

2012-01-01

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

Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies.

PubMed

Koshiba, T; Tsumoto, K; Masaki, K; Kawano, K; Nitta, K; Kumagai, I

1998-08-01

During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92. To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92. These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies. For the preparation of lysozyme samples, a refolding system using thioredoxin was established. This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days. The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5. The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration. However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5. Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography. These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability. The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

PubMed

Sethi, Deepti; Mahajan, Sahil; Singh, Chaahat; Lama, Amrita; Hade, Mangesh Dattu; Gupta, Pawan; Dikshit, Kanak L

2016-02-05

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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.

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

A freeze-thaw method for disintegration of Escherichia coli cells producing T7 lysozyme used in pBAD expression systems.

PubMed

Wanarska, Marta; Hildebrandt, Piotr; Kur, Józef

2007-01-01

The pLysN plasmid containing the T7 lysozyme gene under control of the lac promoter was constructed to facilitate cell disintegration after expression of recombinant proteins in arabinose-induced expression systems. The usefulness of this plasmid was tested in Escherichia coli TOP10 and E. coli LMG194 cells carrying pBADMHADgeSSB plasmid containing Deinococcus geothermalis SSB protein gene under control of the araBAD promoter. The results showed that low-level expression of T7 lysozyme did not interfere with the target SSB protein production, and that the freezing-thawing treatment was sufficient for disruption of the E. coli cells producing low amounts of T7 lysozyme.

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

PubMed Central

Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

2015-01-01

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

Alterations in the host defense properties of human milk following prolonged storage or pasteurization.

PubMed

Akinbi, Henry; Meinzen-Derr, Jareen; Auer, Christine; Ma, Yan; Pullum, Derek; Kusano, Ryosuke; Reszka, Krzysztof J; Zimmerly, Kira

2010-09-01

Preterm infants are often fed pasteurized donor milk or mother's milk that has been stored frozen for up to 4 weeks. Our objectives were to assess the impact of pasteurization or prolonged storage at -20 degrees C on the immunologic components of human milk and the capability of the different forms of human milk to support bacterial proliferation. The concentrations and activities of major host defense proteins in the whey fractions of mother's milk stored for 4 weeks at -20 degrees C or pasteurized human donor milk were compared with freshly expressed human milk. Proliferation of bacteria incubated in the 3 forms of human milk was assessed. Relative to freshly expressed human milk, the concentrations of lysozyme, lactoferrin, lactoperoxidase, and secretory immunoglobulin A were reduced 50% to 82% in pasteurized donor milk and the activities of lysozyme and lactoperoxidase were 74% to 88% lower (P < 0.01). Proliferation of bacterial pathogens in pasteurized donor milk was enhanced 1.8- to 4.6-fold compared with fresh or frozen human milk (P < 0.01). The immunomodulatory proteins in human milk are reduced by pasteurization and, to a lesser extent, by frozen storage, resulting in decreased antibacterial capability. Stringent procedure to minimize bacterial contamination is essential during handling of pasteurized milk.

The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

PubMed Central

Hall, Chris; Flores, Maria Vega; Storm, Thilo; Crosier, Kathy; Crosier, Phil

2007-01-01

Background How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. Results We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. Conclusion These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation. PMID:17477879

Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

PubMed Central

Huber, M C; Bosch, F X; Sippel, A E; Bonifer, C

1994-01-01

The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion. Images PMID:7937145

[Fecal sIgS and lysozyme excretion in breast feeding and formula feeding].

PubMed

Eschenburg, G; Heine, W; Peters, E

1990-05-01

The bioavailability of sIgA and lysozyme from human milk was investigated in a total of 41 infants by radial immunodiffusion and by the Micrococcus lysodeicticus method, respectively. In four different pools of human milk used for balance studies the sIgA concentrations ranged between 2,200 and 17,850 mg/l. The lysozyme concentration varied from 64.5 to 283.5 mg/l. On human milk feeding the excretion of sIgA in 19 infants was 3,200 (0-8,200) mg per litre and 9.7 (0-131) mg lysozyme per litre, respectively. Corresponding values on formula feeding in 22 infants were 1030 (0-6400) and 2.6 (0-9) mg/l. Fecal sIgA excretion was significantly higher on human milk than on formula feeding. Balances of sIgA and lysozyme intake and excretion as performed in 9 infants revealed a less than 1% fecal excretion of both the protective substances. In vitro digestion of raw human milk with pepsin at pH 2 and 3 resulted in a rapid disappearance of immunologically reactive sIgA within 30 minutes after starting the incubation, while no changes in sIgA content were detectable at pH 4. Lysozyme proved to be resistant against peptic digestion. Tryptic digestion at pH 8 did not result in a decrease of human milk sIgA within 120 minutes of incubation at 37 degrees C while under analogous conditions lysozyme concentration approached to 0. These results point at the full bioavailability of both sIgA and lysozyme from human milk. The differing resistance of these protective substances against pepsin and trypsin is apparently adapted to physiological particularities of the digestive tract in early infancy.

Arsenic alters transcriptional responses to Pseudomonas aeruginosa infection and decreases antimicrobial defense of human airway epithelial cells.

PubMed

Goodale, Britton C; Rayack, Erica J; Stanton, Bruce A

2017-09-15

Arsenic contamination of drinking water and food threatens the health of hundreds of millions of people worldwide by increasing the risk of numerous diseases. Arsenic exposure has been associated with infectious lung disease in epidemiological studies, but it is not yet understood how ingestion of low levels of arsenic increases susceptibility to bacterial infection. Accordingly, the goal of this study was to examine the effect of arsenic on gene expression in primary human bronchial epithelial (HBE) cells and to determine if arsenic altered epithelial cell responses to Pseudomonas aeruginosa, an opportunistic pathogen. Bronchial epithelial cells line the airway surface, providing a physical barrier and serving critical roles in antimicrobial defense and signaling to professional immune cells. We used RNA-seq to define the transcriptional response of HBE cells to Pseudomonas aeruginosa, and investigated how arsenic affected HBE gene networks in the presence and absence of the bacterial challenge. Environmentally relevant levels of arsenic significantly changed the expression of genes involved in cellular redox homeostasis and host defense to bacterial infection, and decreased genes that code for secreted antimicrobial factors such as lysozyme. Using pathway analysis, we identified Sox4 and Nrf2-regulated gene networks that are predicted to mediate the arsenic-induced decrease in lysozyme secretion. In addition, we demonstrated that arsenic decreased lysozyme in the airway surface liquid, resulting in reduced lysis of Microccocus luteus. Thus, arsenic alters the expression of genes and proteins in innate host defense pathways, thereby decreasing the ability of the lung epithelium to fight bacterial infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins.

PubMed

Chang, Jih-Chin; Chen, Chao-Huei; Fang, Li-Jung; Tsai, Chi-Ren; Chang, Yu-Chuan; Wang, Teh-Ming

2013-12-01

The bioactive proteins in human milk may be influenced by prolonged storage process, pasteurization, and heat treatment. This study was conducted to evaluate the effects of these procedures. Three forms of human milk - freshly expressed, frozen at -20°C for a prolonged duration, and pasteurized milk - were collected from 14 healthy lactating mothers and a milk bank. The concentrations of major bioactive proteins (secretory immunoglobulin A, lactoferrin, lysozyme, and leptin) were quantified using enzyme-linked immunosorbent assay kits. Changes in these proteins by heat treatment at 40°C or 60°C for 30 minutes were further evaluated. The mean concentrations of lactoferrin and secretory immunoglobulin A were significantly reduced by 66% and 25.9%, respectively, in pasteurized milk compared with those in freshly-expressed milk. Heat treatment at 40°C or 60°C did not cause significant changes in lactoferrin and secretory immunoglobulin A, but there was an apparent increase in lysozyme (p = 0.016). There were no significant differences in leptin level among these three forms of milk prior to (p = 0.153) or after heat treatment (p = 0.053). Various freezing/heating/pasteurization processes applied to human milk prior to delivery to neonates could affect the concentration of immunomodulatory proteins, especially lactoferrin, secretory immunoglobulin A, and lysozyme. Leptin was unaffected by the various handling processes tested. Fresh milk was found to be the best food for neonates. Further studies are warranted to evaluate the functional activity of these proteins and their effects on infants' immunological status. Copyright © 2013. Published by Elsevier B.V.

Effect of flash-heat treatment on antimicrobial activity of breastmilk.

PubMed

Chantry, Caroline J; Wiedeman, Jean; Buehring, Gertrude; Peerson, Janet M; Hayfron, Kweku; K'Aluoch, Okumu; Lonnerdal, Bo; Israel-Ballard, Kiersten; Coutsoudis, Anna; Abrams, Barbara

2011-06-01

The World Health Organization recommends human immunodeficiency virus (HIV)-positive mothers in resource-poor regions heat-treat expressed breastmilk during periods of increased maternal-to-child transmission risk. Flash-heat, a "low tech" pasteurization method, inactivates HIV, but effects on milk protein bioactivity are unknown. The objectives were to measure flash-heat's effect on antimicrobial properties of lactoferrin, lysozyme, and whole milk and on the digestive resistance of lactoferrin and lysozyme. Flash-heated and unheated breastmilk aliquots from HIV-positive mothers in South Africa were "spiked" with Staphylococcus aureus and Escherichia coli and then cultured for 0, 3, and 6 hours. Lysozyme and lactoferrin activities were determined by lysis of Micrococcus luteus cells and inhibition of enteropathogenic E. coli, respectively, measured spectrophotometrically. Percentages of proteins surviving in vitro digestion, lactoferrin and lysozyme activity, and bacteriostatic activity of whole milk in heated versus unheated samples were compared. There was no difference in rate of growth of E. coli or S. aureus in flash-heated versus unheated whole milk (p = 0.61 and p = 0.96, respectively). Mean (95% confidence interval) antibacterial activity of lactoferrin was diminished 11.1% (7.8%, 14.3%) and that of lysozyme by up to 56.6% (47.1%, 64.5%) by flash-heat. Digestion of lysozyme was unaffected (p = 0.12), but 25.4% less lactoferrin survived digestion (p < 0.0001). In summary, flash-heat resulted in minimally decreased lactoferrin and moderately decreased lysozyme bioactivity, but bacteriostatic activity of whole milk against representative bacteria was unaffected. This suggests flash-heated breastmilk likely has a similar profile of resistance to bacterial contamination as that of unheated milk. Clinical significance of the decreased bioactivity should be tested in clinical trials.

Effect of Flash-Heat Treatment on Antimicrobial Activity of Breastmilk

PubMed Central

Wiedeman, Jean; Buehring, Gertrude; Peerson, Janet M.; Hayfron, Kweku; K'Aluoch, Okumu; Lonnerdal, Bo; Israel-Ballard, Kiersten; Coutsoudis, Anna; Abrams, Barbara

2011-01-01

Abstract Background and Objectives The World Health Organization recommends human immunodeficiency virus (HIV)-positive mothers in resource-poor regions heat-treat expressed breastmilk during periods of increased maternal-to-child transmission risk. Flash-heat, a “low tech” pasteurization method, inactivates HIV, but effects on milk protein bioactivity are unknown. The objectives were to measure flash-heat's effect on antimicrobial properties of lactoferrin, lysozyme, and whole milk and on the digestive resistance of lactoferrin and lysozyme. Methods Flash-heated and unheated breastmilk aliquots from HIV-positive mothers in South Africa were “spiked” with Staphylococcus aureus and Escherichia coli and then cultured for 0, 3, and 6 hours. Lysozyme and lactoferrin activities were determined by lysis of Micrococcus luteus cells and inhibition of enteropathogenic E. coli, respectively, measured spectrophotometrically. Percentages of proteins surviving in vitro digestion, lactoferrin and lysozyme activity, and bacteriostatic activity of whole milk in heated versus unheated samples were compared. Results There was no difference in rate of growth of E. coli or S. aureus in flash-heated versus unheated whole milk (p = 0.61 and p = 0.96, respectively). Mean (95% confidence interval) antibacterial activity of lactoferrin was diminished 11.1% (7.8%, 14.3%) and that of lysozyme by up to 56.6% (47.1%, 64.5%) by flash-heat. Digestion of lysozyme was unaffected (p = 0.12), but 25.4% less lactoferrin survived digestion (p < 0.0001). Conclusions In summary, flash-heat resulted in minimally decreased lactoferrin and moderately decreased lysozyme bioactivity, but bacteriostatic activity of whole milk against representative bacteria was unaffected. This suggests flash-heated breastmilk likely has a similar profile of resistance to bacterial contamination as that of unheated milk. Clinical significance of the decreased bioactivity should be tested in clinical trials. PMID:21091243

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

NASA Astrophysics Data System (ADS)

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

2008-04-01

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

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.

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

PubMed

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

2005-11-07

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

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis*

PubMed Central

Sun, Huai-Chang; Xue, Fang-Ming; Qian, Ke; Fang, Hao-Xia; Qiu, Hua-Lei; Zhang, Xin-Yu; Yin, Zhao-Hua

2006-01-01

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis. PMID:16532537

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

PubMed

Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

2015-01-01

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Suppressive effects of lysozyme on polyphosphate-mediated vascular inflammatory responses

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

Chung, Jiwoo; Ku, Sae-Kwang; Lee, Suyeon

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

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

USDA-ARS?s Scientific Manuscript database

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...

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

USDA-ARS?s Scientific Manuscript database

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...

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

PubMed

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

2012-04-01

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

Characteristics of Factors of Protozoa Blastocystis hominis Persistence.

PubMed

Potaturkina-Nesterova, N I; Il'ina, N A; Bugero, N V; Nesterov, A S

2016-10-01

Persistence activity manifested in the expression of anti-lysozyme, anti-lactoferrin, and antihistone factors promoting inactivation of natural anti-infection resistance factors in the body was revealed in Blastocystis hominis protozoa. Activities of these factors were ranged. The frequency of these factors in clinical isolates of blastocyst decreased in the following order: anti-lactoferrin activity (84.5±3.7%)→anti-lysozyme activity (64.8±5.7%)→anti-histone activity (48.1±2.3%). In healthy humans, the corresponding parameters were 7.3±1.3, 5.3±0.9, and 3.3±0.4%, respectively (p<0.05). It was shown that the studied activities in highly virulent blastocysts were higher than in groups of medium-, low-, and avirulent protozoa.

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

PubMed Central

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

2017-01-01

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

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. Copyright © 2015. Published by Elsevier Inc.

Antimicrobial peptide lysozyme has the potential to promote mouse hair follicle growth in vitro.

PubMed

Su, Yongsheng; Liu, Hui; Wang, Jin; Lin, Bojie; Miao, Yong; Hu, Zhiqi

2015-10-01

Lysozyme is a well-known antimicrobial peptide that exists widely in mammalian skin and it is also expressed by pilosebaceous units. However, the exact location of lysozyme in hair follicles and whether it exerts any direct effects on hair follicle growth are unclear. To determine whether lysozyme affected hair growth in vitro, micro-dissected mouse vibrissae follicles (VFs) were treated in serum-free organ culture for 3 days with lysozyme (1-10μg/ml). After that, the effects of lysozyme on dermal papilla (DP) cells were also investigated. Lysozyme was mainly identified in DP and dermal sheath regions of VF by immunochemistry. In addition, 5-10μg/ml lysozyme had a promoting effect on shaft production. It was also associated with significant proliferation of matrix keratinocytes by immunofluorescence observation. Furthermore, lysozyme promoted hair growth by increasing the levels of alkaline phosphatase and lymphoid enhancer factor 1 in DP, as determined by Western blotting. These results indicate that lysozyme is a promoter of VF growth via enhancing the hair-inductive capacity of DP cells during organ culture. Copyright © 2015 Elsevier GmbH. All rights reserved.

Young Pigs Consuming Lysozyme Transgenic Goat Milk Are Protected from Clinical Symptoms of Enterotoxigenic Escherichia coli Infection.

PubMed

Garas, Lydia C; Cooper, Caitlin A; Dawson, Matthew W; Wang, Jane-Ling; Murray, James D; Maga, Elizabeth A

2017-11-01

Background: Diarrheal diseases in infancy and childhood are responsible for substantial morbidity and mortality in developing nations. Lysozyme, an antimicrobial component of human milk, is thought to play a role in establishing a healthy intestinal microbiota and immune system. Consumption of breast milk has been shown to prevent intestinal infections and is a recommended treatment for infants with diarrhea. Objective: This study aimed to examine the ability of lysozyme-rich goat milk to prevent intestinal infection. Methods: Six-week-old Hampshire-Yorkshire pigs were assigned to treatment groups balanced for weight, sex, and litter and were fed milk from nontransgenic control goats (GM group) or human lysozyme transgenic goats (hLZM group) for 2 wk before they were challenged with porcine-specific enterotoxigenic Escherichia coli (ETEC). Fecal consistency, complete blood counts, intestinal histology, and microbial populations were evaluated. Results: Pigs in the hLZM group had less severe diarrhea than did GM pigs at 24 and 48 h after ETEC infection ( P = 0.01 and 0.05, respectively), indicating a less severe clinical disease state. Relative to baseline, postmilk hLZM pigs had 19.9% and 137% enrichment in fecal Bacteroidetes ( P = 0.028) and Paraprevotellaceae ( P = 0.003), respectively, and a 93.8% reduction in Enterobacteriaceae ( P = 0.007), whereas GM pigs had a 60.9% decrease in Lactobacillales ( P = 0.003) and an 83.3% enrichment in Burkholderiales ( P = 0.010). After ETEC infection, hLZM pigs tended to have lower amounts (68.7% less) of fecal Enterobacteriaceae than did GM pigs ( P = 0.058). There were 83.1% fewer bacteria translocated into the mesenteric lymph nodes of hLZM pigs than into those of GM pigs ( P = 0.039), and hLZM pigs had 34% lower mucin 1 and 61% higher tumor necrosis factor-α expression in the ileum than did GM pigs ( P = 0.046 and 0.034, respectively). Conclusion: Results of this study indicate that human lysozyme milk consumption before and during ETEC infection has a positive effect on clinical disease, intestinal mucosa, and gut microbiota in young pigs. © 2017 American Society for Nutrition.

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

PubMed

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

2012-01-01

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

Consuming Transgenic Goats' Milk Containing the Antimicrobial Protein Lysozyme Helps Resolve Diarrhea in Young Pigs

PubMed Central

Cooper, Caitlin A.; Garas Klobas, Lydia C.; Maga, Elizabeth A.; Murray, James D.

2013-01-01

Childhood diarrhea is a significant problem in many developing countries and E. coli is a main causative agent of diarrhea in young children. Lysozyme is an antimicrobial protein highly expressed in human milk, but not ruminant milk, and is thought to help protect breastfeeding children against diarrheal diseases. We hypothesized that consumption of milk from transgenic goats which produce human lysozyme (hLZ-milk) in their milk would accelerate recovery from bacterial-induced diarrhea. Young pigs were used as a model for children and infected with enterotoxigenic E. coli. Once clinical signs of diarrhea developed, pigs were fed hLZ-milk or non-transgenic control goat milk three times a day for two days. Clinical observations and complete blood counts (CBC) were performed. Animals were euthanized and samples collected to assess differences in histology, cytokine expression and bacterial translocation into the mesenteric lymph node. Pigs consuming hLZ-milk recovered from clinical signs of infection faster than pigs consuming control milk, with significantly improved fecal consistency (p = 0.0190) and activity level (p = 0.0350). The CBC analysis showed circulating monocytes (p = 0.0413), neutrophils (p = 0.0219), and lymphocytes (p = 0.0222) returned faster to pre-infection proportions in hLZ-milk fed pigs, while control-fed pigs had significantly higher hematocrit (p = 0.027), indicating continuing dehydration. In the ileum, pigs fed hLZ-milk had significantly lower expression of pro-inflammatory cytokine IL-8 (p = 0.0271), longer intestinal villi (p<0.0001), deeper crypts (p = 0.0053), and a thinner lamina propria (p = 0.0004). These data demonstrate that consumption of hLZ-milk helped pigs recover from infection faster, making hLZ-milk an effective treatment of E. coli-induced diarrhea. PMID:23516474

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

PubMed

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

2018-05-09

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

Highly Resolved Sub-Terahertz Vibrational Spectroscopy of Biological Macromolecules and Bacteria Cells

DTIC Science & Technology

2016-07-01

between average background spectrum and chicken egg - white lysozyme protein spectrum...spectroscopic signatures were conducted using human insulin protein and chicken egg -white lysozyme protein. Proteins with different structures...the comparison between the average background THz spectrum (black line in Figure 13) and the chicken egg -white lysozyme THz spectrum (blue line

Computational study of aggregation mechanism in human lysozyme[D67H

PubMed Central

Patel, Dharmeshkumar

2017-01-01

Aggregation of proteins is an undesired phenomena that affects both human health and bioengineered products such as therapeutic proteins. Finding preventative measures could be facilitated by a molecular-level understanding of dimer formation, which is the first step in aggregation. Here we present a molecular dynamics (MD) study of dimer formation propensity in human lysozyme and its D67H variant. Because the latter protein aggregates while the former does not, they offer an ideal system for testing the feasibility of the proposed MD approach which comprises three stages: i) partially unfolded conformers involved in dimer formation are generated via high-temperature MD simulations, ii) potential dimer structures are searched using docking and refined with MD, iii) free energy calculations are performed to find the most stable dimer structure. Our results provide a detailed explanation for how a single mutation (D67H) turns human lysozyme from non-aggregating to an aggregating protein. Conversely, the proposed method can be used to identify the residues causing aggregation in a protein, which can be mutated to prevent it. PMID:28467454

Identification and Expression Profile Analysis of Antimicrobial Peptide/Protein in Asian Corn Borer, Ostrinia furnacalis (Guenée)

PubMed Central

Zhang, Mingming; Zhou, Fan; Chu, Yuan; Zhao, Zhangwu; An, Chunju

2013-01-01

Antimicrobial peptides/proteins (AMPs) are a group of immune proteins that exhibit strong antibiotic properties against numerous infectious bacterial strains. They are evolutionarily conserved and present in every kingdom and phylum, ranging from prokaryotes to humans. We analyzed the transciptome from the larvae of Asian corn borer, Ostrinia furnacalis (Guenée), and identified several putative AMP transcripts, OfgLys5, OfgLys6, OfgLys10, OfgAtt, and OfgIID. OfgLys5, OfgLys6, and OfgLys10 are all highly homologous with c-type lysozymes, and OfgAtt shows significant identities with Lepidoptera attacin. The amino acid sequence of OfgLys5 and OfgLys6 possessed all conserved features critical for fundamental structure and function of c-type lysozyme, including the two catalytic sites, Glu32 and Asp50. OfgAtt is a typical glycine-rich protein. The antimicrobial activity of O. furnacalis hemolymph increased significantly after injection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. OfgAtt, IDD, and Lys6 are expressed at low level prior to the challenge, but strongly induced against Gram-positive and negative bacteria, and fungi. Under the same inducement conditions, the transcripts of these three genes elevated most when fifth instar larvae were injected. Therefore, O. furnacalis larvae are induced to produce antimicrobial materials in the hemolymph after the infection, and increase of lysozyme and attacin may contribute to the antimicrobial activity. PMID:24155672

Bacteria and Bioactivity in Holder Pasteurized and Shelf-Stable Human Milk Products

PubMed Central

2017-01-01

Abstract Background: Historically, Holder pasteurization has been used to pasteurize donor human milk available in a hospital setting. There is extensive research that provides an overview of the impact of Holder pasteurization on bioactive components of human milk. A shelf-stable (SS) human milk product, created using retort processing, recently became available; however, to our knowledge, little has been published about the effect of retort processing on human milk. Objective: We aimed to assess the ability of retort processing to eliminate bacteria and to quantify the difference in lysozyme and secretory immunoglobulin A (sIgA) activity between Holder pasteurized (HP) and SS human milk. Methods: Milk samples from 60 mothers were pooled. From this pool, 36 samples were taken: 12 samples were kept raw, 12 samples were HP, and 12 samples were retort processed to create an SS product. All samples were analyzed for total aerobic bacteria, coliform bacteria, Bacillus cereus, sIgA activity, and lysozyme activity. Raw samples served as the control. Results: One raw sample and 3 HP samples contained B. cereus at the time of culture. There were no detectable bacteria in SS samples at the time of culture. Raw samples had significantly greater lysozyme and sIgA activity than HP and SS samples (P < 0.0001). HP samples retained significantly more lysozyme and sIgA activity (54% and 87%, respectively) than SS samples (0% and 11%, respectively). Conclusions: Human milk processed using Holder pasteurization should continue to be screened for the presence of B. cereus. Clinicians should be aware of the differences in the retention of lysozyme and sIgA activity in HP and SS products when making feeding decisions for medically fragile or immunocompromised infants to ensure that patients are receiving the maximum immune protection. PMID:29955718

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

PubMed

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

2015-02-01

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

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

PubMed

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

2015-11-01

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

Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris.

PubMed

Borovaya, Alena; Dombrowski, Yvonne; Zwicker, Stephanie; Olisova, Olga; Ruzicka, Thomas; Wolf, Ronald; Schauber, Jürgen; Sárdy, Miklós

2014-10-01

In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

PubMed

Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

2013-11-01

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog.

PubMed

Pérez, J; Bautista, M J; Rollón, E; de Lara, F C; Carrasco, L; Martin de las Mulas, J

1996-07-01

The immunohistochemical expression of muscle actin has been studied in 45 canine hemangiopericytomas (CHP) using a monoclonal antibody (HHF35) and formalin-fixed, paraffin-embedded specimens. The distribution of vimentin, desmin, cytokeratins, lysozyme, factor VIII-related antigen, S-100 protein, and glial fibrillary acidic protein was studied both in CHP and in some canine soft-tissue neoplasms (seven fibrosarcomas, seven benign schwannomas, seven benign fibrous histiocytomas, and six leiomyosarcomas) used as controls for differential diagnosis. All CHP and control tumors expressed vimentin. Twenty-three CHP expressed muscle actin, whereas all control tumors analyzed were muscle actin-negative, with the exception of leiomyosarcomas. Among muscle actin- and vimentin-positive CHP, one case could be reclassified as leiomyosarcoma because it was desmin-positive, two cases expressed lysozyme, and nine cases expressed S-100 protein. Among muscle actin-negative and vimentin-positive CHP, seven expressed S-100 protein. In addition, S-100 protein was detected in five schwannomas. All CHP and control tumors analyzed were negative for cytokeratins, factor VIII-related antigen, and glial fibrillary acidic protein. Our results support the hypothesis of a pericytic origin of CHP, and suggest that muscle actin, desmin, vimentin, and lysozyme could be useful for the differential diagnosis of canine spindle cell tumors, but not all these neoplasms can be identified with these tumor tissue markers.

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

PubMed

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

2018-05-01

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

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.

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.

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

Goven, A.J.; Chen, S.C.; Fitzpatrick, L.C.

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 formore » 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.« less

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

21 CFR 862.1490 - Lysozyme (muramidase) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lysozyme (muramidase) test system. 862.1490 Section 862.1490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

[The estimation of systemic chemotherapy treatment administered in breast cancer on lysozyme activity in tears--preliminary report].

PubMed

Wojciechowska, Katarzyna; Jurowski, Piotr; Wieckowska-Szakiel, Marzena; Rózalska, Barbara

2012-01-01

Estimation of cytostatics influence used in breast cancer treatment on lysozyme activity in human tears depend on time of treatment. 8 women were treated at the base of chemotherapy schema: docetaxel with doxorubicin and 4 women treated with schema CMF: cyclophosphamide, methotrexate, 5-fluorouracil. Lysozyme activity in tears was assessed by measurement of diameter zone of Micrococcus lysodeicticus growth inhibition. It was revealed that both chemotherapy schema caused statistically significant reduction of diameter zone of M. lysodeicticus growth inhibition, after first and second course of chemotherapy treatment. After second chemotherapy course CMF schema induced loss of lysozyme activity in patient's tears (zero mm of M. lysodeicticus diameter zone growth inhibition). Systemic chemotherapy administered in breast cancer induce reduction of lysozyme activity in tears, that may cause higher morbidity of ocular surface infections caused by Gram-positive bacteria.

Study on the interactions between toxic nitroanilines and lysozyme by spectroscopic approaches and molecular modeling.

PubMed

Gu, Yunlan; Wang, Yanqing; Zhang, Hongmei

2018-05-05

Being exogenous environmental pollutants, nitroanilines (NAs) are highly toxic and have mutagenic and carcinogenic activity. Being lack of studies on interactions between NAs and lysozyme at molecular level, the binding interactions of lysozyme with o-nitroaniline (oNA), m-nitroaniline (mNA) and p-nitroaniline (pNA) were investigated by means of steady-state fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy, as well as molecular modeling. The experimental results revealed that the fluorescence of lysozyme is quenched by oNA and mNA through a static quenching, while the fluorescence quenching triggered by pNA is a combined dynamic and static quenching. The number of binding sites (n) and the binding constant (K b ) corresponding thermodynamic parameters ΔH ⊖ , ΔS ⊖ , ΔG ⊖ at different temperatures were calculated. The reactions between NAs and lysozyme were spontaneous and entropy driven and the binding of NAs to lysozyme induced conformation changes of lysozyme. The difference of the position of -NO 2 group affected the binding and the binding constants K b decreased in the following pattern: K b (pNA) >K b (mNA) >K b (oNA). Molecular docking studies were performed to reveal the most favorable binding sites of NAs on lysozyme. Our recently results could offer mechanistic insights into the nature of the binding interactions between NAs and lysozyme and provide information about the toxicity risk of NAs to human health. Copyright © 2018 Elsevier B.V. All rights reserved.

Interaction and inhibitory influence of the azo dye carmoisine on lysozyme amyloid fibrillogenesis.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-07-25

The binding of the common food colorant carmoisine and its inhibitory effect on amyloid fibrillation in lysozyme have been investigated. Since humans are increasingly exposed to various food colorants like carmoisine, such studies are highly relevant. In the presence of lysozyme, the carmoisine absorption spectrum exhibited hypochromic changes. The intrinsic fluorescence of lysozyme was also quenched on interaction. Time-resolved fluorescence results suggested that the binding mechanism involved ground state complexation. The binding was predominantly dominated by non-polyelectrolytic forces. The molecular distance between the donor (lysozyme) and the acceptor (carmoisine), calculated from FRET theory, was found to be 3.37 nm, indicating that carmoisine binds close to Trp-62/63 residues in the β-domain of the protein. Information on alterations in the microenvironment surrounding the Trp-residues was also obtained from synchronous fluorescence data. Carmoisine binding induced significant loss in the alpha helical organization of lysozyme. The binding, nevertheless, did not influence the thermal stability of lysozyme significantly. The binding reaction was exothermic and driven by large negative enthalpy and small but favourable entropic contributions. Thioflavin T assay, far-UV circular dichroism studies and AFM imaging profiles testified that carmoisine had a significant inhibitory effect on amyloid fibrillogenesis in lysozyme. Carmoisine also had a definitive defibrillating effect on existing fibrils. The results may provide new insights for designing new small molecule inhibitors for amyloid related diseases.

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

PubMed

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

2007-08-29

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

The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

PubMed Central

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

2007-01-01

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

Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis

NASA Astrophysics Data System (ADS)

Booth, David R.; Sunde, Margaret; Bellotti, Vittorio; Robinson, Carol V.; Hutchinson, Winston L.; Fraser, Paul E.; Hawkins, Philip N.; Dobson, Christopher M.; Radford, Sheena E.; Blake, Colin C. F.; Pepys, Mark B.

1997-02-01

Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-β fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

PubMed

Cooper, Caitlin A; Maga, Elizabeth A; Murray, James D

2015-08-01

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

Influence of lysozyme on the precipitation of calcium carbonate: a kinetic and morphologic study

NASA Astrophysics Data System (ADS)

Jimenez-Lopez, Concepcion; Rodriguez-Navarro, Alejandro; Dominguez-Vera, Jose M.; Garcia-Ruiz, Juan M.

2003-05-01

Several mechanisms have been proposed to explain the interactions between proteins and mineral surfaces, among them a combination of electrostatic, stereochemical interactions and molecular recognition between the protein and the crystal surface. To identify the mechanisms of interaction in the lysozyme-calcium carbonate model system, the effect of this protein on the precipitation kinetics and morphology of calcite crystals was examined. The solution chemistry and morphology of the solid were monitored over time in a set of time-series free-drift experiments in which CaCO 3 was precipitated from solution in a closed system at 25°C and 1 atm total pressure, in the presence and absence of lysozyme. The precipitation of calcite was preceded by the precipitation of a metastable phase that later dissolved and gave rise to calcite as the sole phase. With increasing lysozyme concentration, the nucleation of both the metastable phase and calcite occurred at lower Ω calcite, indicating that lysozyme favored the nucleation of both phases. Calcite growth rate was not affected by the presence of lysozyme, at least at protein concentrations ranging from 0 mg/mL to 10 mg/mL. Lysozyme modified the habit of calcite crystals. The degree of habit modification changed with protein concentration. At lower concentrations of lysozyme, the typical rhombohedral habit of calcite crystals was modified by the expression of {110} faces, which resulted from the preferential adsorption of protein on these faces. With increasing lysozyme concentration, the growth of {110}, {100}, and finally {001} faces was sequentially inhibited. This adsorption sequence may be explained by an electrostatic interaction between lysozyme and calcite, in which the inhibition of the growth of {110}, {100}, and {001} faces could be explained by a combined effect of the density of carbonate groups in the calcite face and the specific orientation (perpendicular) of these carbonate groups with respect to the calcite surface. Overgrowth of calcite in the presence of lysozyme demonstrated that the protein favored and controlled the nucleation on the calcite substrate. Overgrowth crystals nucleated epitaxially in lines which run diagonal to rhombohedral {104} faces.

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

PubMed Central

Williams, Amanda D.; Korolkova, Olga Y.; Sakwe, Amos M.; Geiger, Timothy M.; James, Samuel D.; Muldoon, Roberta L.; Herline, Alan J.; Goodwin, J. Shawn; Izban, Michael G.; Washington, Mary K.; Smoot, Duane T.; Ballard, Billy R.; Gazouli, Maria

2017-01-01

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis. PMID:28817680

A Molecular Dynamics Simulation of the Human Lysozyme – Camelid VHH HL6 Antibody System

PubMed Central

Su, Zhi-Yuan; Wang, Yeng-Tseng

2009-01-01

Amyloid diseases such as Alzheimer’s and thrombosis are characterized by an aberrant assembly of specific proteins or protein fragments into fibrils and plaques that are deposited in various tissues and organs. The single-domain fragment of a camelid antibody was reported to be able to combat against wild-type human lysozyme for inhibiting in-vitro aggregations of the amyloidogenic variant (D67H). The present study is aimed at elucidating the unbinding mechanics between the D67H lysozyme and VHH HL6 antibody fragment by using steered molecular dynamics (SMD) simulations on a nanosecond scale with different pulling velocities. The results of the simulation indicated that stretching forces of more than two nano Newton (nN) were required to dissociate the proteinantibody system, and the hydrogen bond dissociation pathways were computed. PMID:19468335

Ultrasensitive detection of lysozyme in droplet-based microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Cigliana, Giovanni; Spoto, Giuseppe

2018-05-01

Lysozyme (LYS) is a bacteriolytic enzyme, available in secretions such as saliva, tears and human milk. LYS is an important defence molecule of the innate immune system, and its overexpression can be a consequence of diseases such as leukemia, kidney disease and sarcoidosis. This paper reports on a digital microfluidic-based approach that combines the gold nanoparticle-enhanced chemiluminescence with aptamer interaction to detect human lysozyme into droplets 20 nanoliters in volume. The described method allows identifying LYS with a 44.6 femtomolar limit of detection, using sample volume as low as 1μL and detection time in the range of 10min. We used luminol to generate the chemiluminescence and demonstrated that the compartmentalization of LYS in droplets also comprising gold nanoparticles provided enhanced luminescence. We functionalized the gold nanoparticles with a thiolated aptamer to achieve the required selectivity that allowed us to detect LYS in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

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

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

Heat-denatured lysozyme could be a novel disinfectant for reducing hepatitis A virus and murine norovirus on berry fruit.

PubMed

Takahashi, Michiko; Okakura, Yumiko; Takahashi, Hajime; Imamura, Minami; Takeuchi, Akira; Shidara, Hiroyuki; Kuda, Takashi; Kimura, Bon

2018-02-02

Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning. Copyright © 2017 Elsevier B.V. All rights reserved.

High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

PubMed

Madadkar, Pedram; Nino, Sergio Luna; Ghosh, Raja

2016-11-01

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution. Copyright © 2016 Elsevier B.V. All rights reserved.

Chicken-type lysozyme functions in the antibacterial immunity in red swamp crayfish, Procambarus clarkii.

PubMed

Liao, Tian-Jiang; Gao, Jie; Wang, Jin-Xing; Wang, Xian-Wei

2018-08-01

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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

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

PubMed

Brundige, Dottie R; Maga, Elizabeth A; Klasing, Kirk C; Murray, James D

2010-08-01

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.

Fusion Partner Toolchest for the Stabilization and Crystallization of G Protein-Coupled Receptors

PubMed Central

Chun, Eugene; Thompson, Aaron A.; Liu, Wei; Roth, Christopher B.; Griffith, Mark T.; Katritch, Vsevolod; Kunken, Joshua; Xu, Fei; Cherezov, Vadim; Hanson, Michael A.; Stevens, Raymond C.

2012-01-01

SUMMARY Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of the T4 lysozyme fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs. In particular, the use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over the previously utilized T4 lysozyme, resulting in a significant improvement in stability and structure in GPCR-fusion constructs. PMID:22681902

[Inactivating Effect of Heat-Denatured Lysozyme on Murine Norovirus in Bread Fillings].

PubMed

Takahashi, Michiko; Yasuda, Yuka; Takahashi, Hajime; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

2018-01-01

In this study, we investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in bread fillings used for making stuffed buns and pastries. The inactivating effect of heat-denatured lysozyme, which was recently reported to have an antiviral effect, on MNV-1 contaminating the bread fillings was also examined. MNV-1 was inoculated into two types of fillings (chocolate cream, marmalade jam) at 4.5 log PFU/g, and the bread fillings were stored at 4℃ for 5 days. MNV-1 remained viable in the bread fillings during storage. However, addition of 1% heat-denatured lysozyme to the fillings resulted in a decrease of MNV-1 infectivity immediately after inoculation, in both fillings. On the fifth day of storage, MNV-1 infectivity was decreased by 1.2 log PFU/g in chocolate cream and by 0.9 log PFU/g in marmalade jam. Although the mechanism underlying the anti-norovirus effect of heat-denatured lysozyme has not been clarified, our results suggest that heat-denatured lysozyme can be used as an inactivating agent against norovirus in bread fillings.

Ammonia concentration and relative humidity in poultry houses affect the immune response of broilers.

PubMed

Wei, F X; Hu, X F; Xu, B; Zhang, M H; Li, S Y; Sun, Q Y; Lin, P

2015-04-10

To investigate the effect of ammonia (NH3) and humidity on the immune response of broilers, broilers were exposed to 30 or 70 mg/kg atmospheric NH3 for 21 days. Additionally, birds were exposed to 35, 60, and 85% relative humidity (RH). The relative weights of lymphoid organs, serum total protein, serum globulin, serum albumin, serum lysozyme, proliferation index of peripheral blood lymphocytes, and splenic cytokine gene expression were determined. Exposure to 70 mg/kg NH3 decreased the relative weight of the spleen during the experimental period, serum lysozyme concentration in the first and second weeks, and serum globulin concentration in the third week. The proliferation of peripheral blood lymphocytes was reduced. High levels of NH3 caused increase in IL-1β gene expression in the experimental period and IL-4 gene expression in the first week. Birds exposed to 85% RH had lower thymus and bursa of Fabricius weights in the third week and serum lysozyme concentration in the first week; IL-1β and IL-4 expressions were higher in the second and third weeks and first and second weeks, respectively, than in birds exposed to 60% RH. IL-4 expression was lower during the first week, and IL-1β expression was higher during the second week with 35% RH than with 60% RH. In conclusion, high NH3 level in the poultry house suppressed the immune response of broiler chickens. Neither high nor low RH benefited the immune response of broilers. Furthermore, there was an interactive effect between NH3 and RH on the immune response of broilers.

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

2017-07-01

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. © 2017 Japanese Dermatological Association.

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

PubMed Central

Jeruzalmi, D; Steitz, T A

1998-01-01

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

The Effect of Simulated Flash-Heat Pasteurization on Immune Components of Human Milk.

PubMed

Daniels, Brodie; Schmidt, Stefan; King, Tracy; Israel-Ballard, Kiersten; Amundson Mansen, Kimberly; Coutsoudis, Anna

2017-02-22

A pasteurization temperature monitoring system has been designed using FoneAstra, a cellphone-based networked sensing system, to monitor simulated flash-heat (FH) pasteurization. This study compared the effect of the FoneAstra FH (F-FH) method with the Sterifeed Holder method currently used by human milk banks on human milk immune components (immunoglobulin A (IgA), lactoferrin activity, lysozyme activity, interleukin (IL)-8 and IL-10). Donor milk samples ( N = 50) were obtained from a human milk bank, and pasteurized. Concentrations of IgA, IL-8, IL-10, lysozyme activity and lactoferrin activity were compared to their controls using the Student's t -test. Both methods demonstrated no destruction of interleukins. While the Holder method retained all lysozyme activity, the F-FH method only retained 78.4% activity ( p < 0.0001), and both methods showed a decrease in lactoferrin activity (71.1% Holder vs. 38.6% F-FH; p < 0.0001) and a decrease in the retention of total IgA (78.9% Holder vs. 25.2% F-FH; p < 0.0001). Despite increased destruction of immune components compared to Holder pasteurization, the benefits of F-FH in terms of its low cost, feasibility, safety and retention of immune components make it a valuable resource in low-income countries for pasteurizing human milk, potentially saving infants' lives.

Temporospatial fate of bacteria and immune effector expression in house flies (Musca domestica L.) fed GFP-E. coli O157:H7

PubMed Central

Fleming, A.; Kumar, H.V.; Joyner, C.; Reynolds, A.; Nayduch, D.

2014-01-01

House flies (Diptera: Muscidae; Musca domestica L.) harbor and transmit a variety of human enteropathogens including E. coli O157:H7. Interactions between ingested bacteria and the fly gut directly impact bacterial persistence, survival and ultimately fly vector competence. We assessed the temporospatial fate of GFP-E. coli O157:H7 (GFP-ECO157) in house flies along with fly antimicrobial responses for 12 h post-ingestion. In flies fed GFP-ECO157, culture and microscopy revealed a steady decrease in bacterial load over 12 h, which was likely attributable to the combined effects of immobilization within the peritrophic matrix, lysis and peristaltic excretion. However, flies can putatively transmit this pathogen in excreta because intact bacteria were observed in the crop and rectum. qRT-PCR analysis of antimicrobial peptides (AMP) and lysozyme gene expression showed minimal upregulation in both the gut and carcass of house flies fed GFP-ECO157. However, these genes were upregulated in fly heads and salivary glands, and effector proteins were detected in the gut of some flies. Collectively, these data indicate that house flies can serve as reservoirs of E. coli O157:H7 for up to 12 h, and factors in addition to AMPs and lysozyme may contribute to bacteria destruction in the gut. PMID:24712451

Glucocorticoid dexamethasone down-regulates basal and vitamin D3 induced cathelicidin expression in human monocytes and bronchial epithelial cell line.

PubMed

Kulkarni, Nikhil Nitin; Gunnarsson, Hörður Ingi; Yi, Zhiqian; Gudmundsdottir, Steinunn; Sigurjonsson, Olafur E; Agerberth, Birgitta; Gudmundsson, Gudmundur H

2016-02-01

Glucocorticoids (GCs) have been extensively used as the mainstream treatment for chronic inflammatory disorders. The persistent use of steroids in the past decades and the association with secondary infections warrants for detailed investigation into their effects on the innate immune system and the therapeutic outcome. In this study, we analyse the effect of GCs on antimicrobial polypeptide (AMP) expression. We hypothesize that GC related side effects, including secondary infections are a result of compromised innate immune responses. Here, we show that treatment with dexamethasone (Dex) inhibits basal mRNA expression of the following AMPs; human cathelicidin, human beta defensin 1, lysozyme and secretory leukocyte peptidase 1 in the THP-1 monocytic cell-line (THP-1 monocytes). Furthermore, pre-treatment with Dex inhibits vitamin D3 induced cathelicidin expression in THP-1 monocytes, primary monocytes and in the human bronchial epithelial cell line BCi NS 1.1. We also demonstrate that treatment with the glucocorticoid receptor (GR) inhibitor RU486 counteracts Dex mediated down-regulation of basal and vitamin D3 induced cathelicidin expression in THP-1 monocytes. Moreover, we confirmed the anti-inflammatory effect of Dex. Pre-treatment with Dex inhibits dsRNA mimic poly IC induction of the inflammatory chemokine IP10 (CXCL10) and cytokine IL1B mRNA expression in THP-1 monocytes. These results suggest that GCs inhibit innate immune responses, in addition to exerting beneficial anti-inflammatory effects. Copyright © 2015 Elsevier GmbH. All rights reserved.

Histological and Immunological Description of the Leishmanin Skin Test in Ibizan Hounds.

PubMed

Ordeix, L; Silva, J E Dos S; Llull, J; Quirola, P; Montserrat-Sangrà, S; Martínez-Orellana, P; Solano-Gallego, L

2018-01-01

The leishmanin skin test (LST), a delayed-type hypersensitivity (DTH) reaction to Leishmania infantum, can specifically identify dogs that have made a cell-mediated immune response to L. infantum infection. The Ibizan hound appears to be more resistant to L. infantum infection than other breeds of dog. The aim of this study was to describe the histological and immunohistochemical changes induced by the LST in Ibizan hounds living in an area highly endemic for leishmaniosis. The majority of dogs were apparently healthy, lacked serum antibody to L. infantum and blood parasitaemia, but had marked specific interferon gamma production after in-vitro blood stimulation with L. infantum. Leishmanin (3 × 10 8 killed promastigotes of L. infantum/ml) was injected intradermally and biopsy samples were obtained from a positive reaction at 72 h from nine Ibizan hounds. A moderate to intense, perivascular to interstitial dermatitis and panniculitis characterized the inflammatory response at the injection site. In addition, three samples had diffuse inflammation in the deep dermis and panniculus. Oedema and necrosis were present in the deep dermis and panniculus. Congestion and haemorrhage were observed in five biopsies. T lymphocytes (CD3 + ) and large mononuclear cells (lysozyme - ) were the most prevalent cells. CD3 + cells were significantly more numerous than CD20 + B cells and lysozyme + cells. B cells were sparsely distributed, especially in the deep dermis and panniculus. Rare neutrophils and macrophages (lysozyme + ) were observed with few eosinophils. Toll-like receptor (TLR)-2 protein was expressed in large mononuclear cells mainly located in the superficial dermis. Leishmania immunohistochemistry was negative and quantitative polymerase chain reaction was positive in all cases. The intradermal injection of killed L. infantum promastigotes in Ibizan hounds causes similar histological and immunohistochemical findings to those described for human subjects and are indicative of a DTH response. Moreover, TLR2 protein is expressed in inflammatory cells similar to findings in clinically affected skin biopsy samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

PubMed

Yang, Chih-Chiu; Lu, Chung-Lun; Chen, Sherwin; Liao, Wen-Liang; Chen, Shiu-Nan

2015-05-01

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Copyright © 2015 Elsevier Ltd. All rights reserved.

The role of antimicrobial peptides in animal defenses

NASA Astrophysics Data System (ADS)

Hancock, Robert E. W.; Scott, Monisha G.

2000-08-01

It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.

Examining the inhibitory potency of food additive fast green FCF against amyloid fibrillogenesis under acidic conditions.

PubMed

How, Su-Chun; Yang, Szu-Ming; Hsin, Ai; Tseng, Chia-Ping; Hsueh, Shu-Shun; Lin, Ming-Shen; Chen, Rita P-Y; Chou, Wei-Lung; Wang, Steven S-S

2016-12-07

More than thirty human proteins and/or peptides can fold incorrectly to form amyloid deposits associated with several protein aggregation diseases. No cure is currently available for treating these diseases. This work is aimed at examining the inhibitory potency of fast green FCF, a biocompatible dye, toward the fibrillogenesis/aggregation of lysozyme. As verified by ThT binding assay along with transmission electron microscopy, fast green FCF was observed to suppress the generation of lysozyme fibrils in a concentration-dependent manner. We next used circular dichroism absorption spectroscopy, ANS fluorescence spectroscopy, and SDS-PAGE to characterize the structural alterations in lysozyme samples upon the addition of fast green FCF. Furthermore, experiments with the addition of fast green FCF at different time points of incubation showed that fast green FCF also exhibited disaggregating activity against the preformed/existing lysozyme fibrils. We believe that the results from this study suggest a potential therapeutic role of biocompatible molecules in treating or preventing protein aggregation diseases.

Chrysin and its phosphate ester inhibit cell proliferation and induce apoptosis in Hela cells.

PubMed

Zhang, Ting; Chen, Xiaolan; Qu, Lingbo; Wu, Jinglan; Cui, Ran; Zhao, Yufen

2004-12-01

To improve the biological activities of chrysin (CR), we synthesize Diethyl Chysin-7-yl phosphate (CPE: C(19)H(19)O(7)P) and tetraethyl bis-phosphoric ester of chrysin (CP: C(23)H(28)O(10)P(2)) through a simplified Atheron-Todd reaction. The interactions of the CR and CPE with lysozyme were explored by electrospray ionization mass spectrometry (ESI) and fluorescence spectrometry method. Experimental results indicate that CPE could form the noncovalent compound with lysozyme, while the interaction of the CR with lysozyme was not detected. In addition, whether and how the compounds CPE and CP affect proliferation and apoptosis in human cervical cancer Hela cells were investigated. Moreover, the effects of CPE and CP in Hela cells were compared with that of the nonmodified CR compound. The Hela cells were co-cultured with CR, CP, and CPE as experimental groups, respectively, and corresponding control groups treated without CR, CP, and CPE. The proliferation and apoptosis were detected using MTT assay, HCl denatured-methyl green-pyronin staining, PCNA immunohistochemistry and TUNEL techniques. The cell growth IC(50), relative absorbance (RA), proliferating index (PI), PCNA-IR (immunoreactivity IR) integration value (IV), and apoptosis index (AI) were calculated and their correlation was analyzed in each group. The results show that all CR, CP, and CPE could inhibit proliferation and induce apoptosis in Hela cells. Moreover, the effects of CP and CPE were more potent than that of CR. The CP and CPE were proved to be a kind of stronger apoptosis inducers than nonphosphated CR. There was a negative correlation between proliferation and apoptosis. In conclusion, the CR, CP, and CPE could effectively inhibit growth by down-regulated expression of PCNA, and induce apoptosis in Hela cells. The efficiency of the modified CP and CPE preceded nonmodified CR compounds. The CP and CPE may be a new potential anti-cancer drug for therapy of human cervical carcinoma.

The Effect of Simulated Flash-Heat Pasteurization on Immune Components of Human Milk

PubMed Central

Daniels, Brodie; Schmidt, Stefan; King, Tracy; Israel-Ballard, Kiersten; Amundson Mansen, Kimberly; Coutsoudis, Anna

2017-01-01

A pasteurization temperature monitoring system has been designed using FoneAstra, a cellphone-based networked sensing system, to monitor simulated flash-heat (FH) pasteurization. This study compared the effect of the FoneAstra FH (F-FH) method with the Sterifeed Holder method currently used by human milk banks on human milk immune components (immunoglobulin A (IgA), lactoferrin activity, lysozyme activity, interleukin (IL)-8 and IL-10). Donor milk samples (N = 50) were obtained from a human milk bank, and pasteurized. Concentrations of IgA, IL-8, IL-10, lysozyme activity and lactoferrin activity were compared to their controls using the Student’s t-test. Both methods demonstrated no destruction of interleukins. While the Holder method retained all lysozyme activity, the F-FH method only retained 78.4% activity (p < 0.0001), and both methods showed a decrease in lactoferrin activity (71.1% Holder vs. 38.6% F-FH; p < 0.0001) and a decrease in the retention of total IgA (78.9% Holder vs. 25.2% F-FH; p < 0.0001). Despite increased destruction of immune components compared to Holder pasteurization, the benefits of F-FH in terms of its low cost, feasibility, safety and retention of immune components make it a valuable resource in low-income countries for pasteurizing human milk, potentially saving infants’ lives. PMID:28241418

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

PubMed Central

Casali, Nicola; Clark, Simon O.; Hooper, Richard; Williams, Ann; Velji, Preya; Gonzalo, Ximena

2015-01-01

Virulence factors (VFs) contribute to the emergence of new human Mycobacterium tuberculosis strains, are lineage dependent, and are relevant to the development of M. tuberculosis drugs/vaccines. VFs were sought within M. tuberculosis lineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity. Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX, caeA, and ponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozyme in vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across, M. tuberculosis lineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established. PMID:25776753

Pattern similarity study of functional sites in protein sequences: lysozymes and cystatins

PubMed Central

Nakai, Shuryo; Li-Chan, Eunice CY; Dou, Jinglie

2005-01-01

Background Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. Results Hydrophobicity and β-turn propensity of reference segments with 3–7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. Conclusion Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available. PMID:15904486

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

PubMed

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

2013-09-01

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

Agaricus bisporus powder improved cutaneous mucosal and serum immune parameters and up-regulated intestinal cytokines gene expression in common carp (Cyprinus carpio) fingerlings.

PubMed

Khodadadian Zou, Hassan; Hoseinifar, Seyed Hossein; Kolangi Miandare, Hamed; Hajimoradloo, Abdolmajid

2016-11-01

The aim of the present study was to investigate immunomodulatory effects of Agaricus bisporus, white bottom mushroom powder (WBMP) on common carp (Cyprinus carpio) fingerlings. Carps were fed on different levels of WBMP (0, 0.5, 1 and 2%) for 8 weeks and at the end of feeding trial, skin mucus immune parameters (total Ig, lysozyme and protease activity), cytokines gene expression (TNF-alpha, IL1b, IL8) in intestine as well as serum non-specific immune parameters (total Ig, lysozyme and ACH50) were measured. The results showed significant dose dependent increase of skin mucus immune parameters in carps fed WBMP (P < 0.05). While, no significant difference was observed between 0.5% WBMP and control group (P > 0.05). In case of serum non-specific immune parameters, except lysozyme activity, other parameters (Ig total and ACH50) were significantly affected by dietary inclusion of WBMP (P < 0.05). Also, evaluation of cytokines gene expression in the intestine of carps revealed remarkable up-regulation of TNF-alpha in fish fed 2% WBMP supplemented diet compared other treatment (P < 0.05). Likewise, IL1b gene expression was significantly increased in 1 and 2% WBMP treatments compared to the 0.5% WBMP and control groups (P < 0.05). IL8 gene expression was not affected by inclusion of WBMP in carp diet (P > 0.05). Furthermore, feeding on WBMP supplemented diet significantly improved growth performance (P < 0.05). These results indicated that WBMP can be considered as a promising immunostimulants in early stage of common carp culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Studies on immunocompetence status in two turkey varieties in India.

PubMed

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

2004-10-01

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

Production of biofunctionalized MoS2 flakes with rationally modified lysozyme: a biocompatible 2D hybrid material

NASA Astrophysics Data System (ADS)

Siepi, Marialuisa; Morales-Narváez, Eden; Domingo, Neus; Monti, Daria Maria; Notomista, Eugenio; Merkoçi, Arben

2017-09-01

Bioapplications of 2D materials embrace demanding features in terms of environmental impact, toxicity and biocompatibility. Here we report on the use of a rationally modified lysozyme to assist the exfoliation of MoS2 bulk crystals suspended in water through ultrasonic exfoliation. The design of the proposed lysozyme derivative provides this exfoliated 2D-materail with both, hydrophobic groups that interact with the surface of MoS2 and hydrophilic groups exposed to the aqueous medium, which hinders its re-aggregation. This approach, clarified also by molecular docking studies, leads to a stable material (ζ-potential, 27  ±  1 mV) with a yield of up to 430 µg ml-1. The bio-hybrid material was characterized in terms of number of layers and optical properties according to different slots separated by diverse centrifugal forces. Furthermore the obtained material was proved to be biocompatible using human normal keratinocytes and human cancer epithelial cells, whereas the method was demonstrated to be applicable to produce other 2D materials such as graphene. This approach is appealing for the advantageous production of high quality MoS2 flakes and their application in biomedicine and biosensing. Moreover, this method can be applied to different starting materials, making the denatured lysozyme a promising bio-tool for surface functionalization of 2D materials.

Resveratrol Interferes with an Early Step in the Fibrillization Pathway of Human Lysozyme and Modulates it towards Less-Toxic, Off-Pathway Aggregates.

PubMed

Kamal Zaidi, Fatima; Bhat, Rajiv

2018-01-18

The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time-resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol. Docking studies predicted the binding of resveratrol to aggregation-prone regions in HuL, and structure and activity analyses demonstrated the retention of much of the α-helical structure and activity of HuL in the presence of resveratrol. Resveratrol modulated the fibrillization pathway towards less-hydrophobic, less-toxic, off-pathway aggregates. These results demonstrate that binding of resveratrol to HuL could protect against the formation of pathogenic, cytotoxic aggregates formed in amyloidogenic disorders, such as systemic amyloidosis; thus suggesting its potential as a plausible therapeutic agent against lysozyme amyloidosis. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli

PubMed Central

Lewis, Steven B.; Prior, Alison; Ellis, Samuel J.; Cook, Vivienne; Chan, Simon S. M.; Gelson, William; Schüller, Stephanie

2016-01-01

Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8. PMID:27446815

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

Deoxyribonucleic Acid Replication and Expression of Early and Late Bacteriophage Functions in Bacillus subtilis

PubMed Central

Pène, Jacques J.; Marmur, Julius

1967-01-01

The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA. PMID:4990039

Two Goose-Type Lysozymes in Mytilus galloprovincialis: Possible Function Diversification and Adaptive Evolution

PubMed Central

Wang, Qing; Zhang, Linbao; Zhao, Jianmin; You, Liping; Wu, Huifeng

2012-01-01

Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2) were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC) of 0.95–1.91 µM and 1.20–2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4–5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y102, L200 and S202) detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses. PMID:23028813

Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil.

PubMed

Castro Gomes, Cláudia Maria; Sousa, Maria Gloria Teixeira; Menezes, Joyce Prieto Bezerra; Batista, Marliane Campos; Lima, Ana Carolina Stocco; Belda, Walter; Bradshaw, Daniel; Gama, Monica Elinor Alves; Laurenti, Márcia Dalastra; Silveira, Fernando Tobias; Corbett, Carlos Eduardo Pereira

2017-05-01

American cutaneous leishmaniasis (ACL) is a chronic infectious disease caused by different protozoan species of Leishmania, and it is endemic in both tropical and subtropical countries. Using immunohistochemistry, we investigate the density of CD68 + , lysozyme + , CD1a + , factor XIIIa + , CD4 + , CD8 + , CD56 + , interferon (IFN)-γ + , and inducible NO synthase (iNOS + ) cells. These cells were analyzed from 22 biopsy samples obtained from the lesions of ACL patients, whose infection was caused by Leishmania (Viannia) spp. Histopathological analysis showed dense mononuclear inflammatory infiltration in the dermis, which was composed of lymphocytes, macrophages, plasma cells, and discrete tissue parasitism. Granulomatous reactions were also present in the majority of cases. The density of the activated macrophages was higher than that of inactivated macrophages in the lesions. The density of Langerhans cells (CD1a + ) was lower than that of dermal dendrocytes (factor XIIIa + ). The density of CD8 + T lymphocytes was higher than that of CD4 + T lymphocytes. The cellular density of these immunological markers in relation to the species of Leishmania demonstrated that L. (Viannia) sp. lesions had higher IFN-γ expression than that Leishmania (Viania) braziliensis lesions. The evaluation of these markers, according to disease progression, did not reveal any significant differences. L. (Viannia) sp. infection leads to a favorable immune response in the host, as predominantly represented by lysozyme + , factor XIIIa + , CD8 + T cells, and the expression of (IFN)-γ + at the lesion site.

Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme

PubMed Central

Wu, Josephine W.; Liu, Kuan-Nan; How, Su-Chun; Chen, Wei-An; Lai, Chia-Min; Liu, Hwai-Shen; Hu, Chaur-Jong; Wang, Steven S. -S.

2013-01-01

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases. PMID:24349167

Parasite-specific immune response in adult Drosophila melanogaster: a genomic study

PubMed Central

Roxström-Lindquist, Katarina; Terenius, Olle; Faye, Ingrid

2004-01-01

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite-specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence. PMID:14749722

Kinetics of Competitive Adsorption between Lysozyme and Lactoferrin on Silicone Hydrogel Contact Lenses and the Effect on Lysozyme Activity.

PubMed

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

2015-05-01

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

A lysozyme and magnetic bead based method of separating intact bacteria.

PubMed

Diler, Ebru; Obst, Ursula; Schmitz, Katja; Schwartz, Thomas

2011-07-01

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Human Lysozyme Synergistically Enhances Bactericidal Dynamics and Lowers the Resistant Mutant Prevention Concentration for Metronidazole to Helicobacter pylori by Increasing Cell Permeability.

PubMed

Zhang, Xiaolin; Jiang, Anmin; Yu, Hao; Xiong, Youyi; Zhou, Guoliang; Qin, Meisong; Dou, Jinfeng; Wang, Jianfei

2016-10-28

Metronidazole (MNZ) is an effective agent that has been employed to eradicate Helicobacter pylori ( H. pylori ). The emergence of broad MNZ resistance in H. pylori has affected the efficacy of this therapeutic agent. The concentration of MNZ, especially the mutant prevention concentration (MPC), plays an important role in selecting or enriching resistant mutants and regulating therapeutic effects. A strategy to reduce the MPC that can not only effectively treat H. pylori but also prevent resistance mutations is needed. H. pylori is highly resistant to lysozyme. Lysozyme possesses a hydrolytic bacterial cell wall peptidoglycan and a cationic dependent mode. These effects can increase the permeability of bacterial cells and promote antibiotic absorption into bacterial cells. In this study, human lysozyme (hLYS) was used to probe its effects on the integrity of the H. pylori outer and inner membranes using as fluorescent probe hydrophobic 1- N -phenyl-naphthylamine (NPN) and the release of aspartate aminotransferase. Further studies using a propidium iodide staining method assessed whether hLYS could increase cell permeability and promote cell absorption. Finally, we determined the effects of hLYS on the bactericidal dynamics and MPC of MNZ in H. pylori . Our findings indicate that hLYS could dramatically increase cell permeability, reduce the MPC of MNZ for H. pylori , and enhance its bactericidal dynamic activity, demonstrating that hLYS could reduce the probability of MNZ inducing resistance mutations.

Host Defense Proteins in Breast Milk and Neonatal Yeast Colonization.

PubMed

Chow, Brian D W; Reardon, Juliann L; Perry, Emily O; Laforce-Nesbitt, Sonia S; Tucker, Richard; Bliss, Joseph M

2016-02-01

Colonization increases risk for invasive candidiasis in neonates. Breast milk host defense proteins may affect yeast colonization of infants. This study aimed to evaluate breast milk host defense proteins relative to yeast colonization in infants. Infants admitted for longer than 72 hours to the neonatal intensive care unit at Women & Infants Hospital in Providence, Rhode Island, were eligible. After consent, expressed breast milk and swabs from oral, rectal, and inguinal sites from infants were cultured weekly for 12 weeks, or until discharge, transfer, or death. Breast milk was tested for levels of human lactoferrin, lysozyme, apolipoprotein J, mucin-1, dermcidin, and soluble CD14 using commercial ELISA. Concentrations of these components were compared in breast milk received by infants who were colonized or not colonized with yeast. From an original cohort of 130, 61 infants had samples available for this subanalysis. A convenience sample of stored breast milk was analyzed. Median lactoferrin, apolipoprotein J, and mucin-1 did not differ between colonized and uncolonized groups. Soluble CD14 was higher in the surface-colonized group (1.8 μg/mL, n = 12) compared with the surface-uncolonized group (1.6 μg/mL, n = 12, P = .02). Median lysozyme levels were higher in the surface-uncolonized group (483.0 ng/mL, n = 12) versus the surface-colonized group (298.3 ng/mL, n = 12, P = .04). Median dermcidin levels were higher in the surface-uncolonized group (19.4 ng/mL, n = 12) versus the surface-colonized group (8.7 ng/mL, n = 12, P = .04). This study shows an association between colonization with Candida in neonates and lower levels of lysozyme and dermcidin in received breast milk. Further study is needed to confirm these findings. © The Author(s) 2015.

Structural basis for the appearance of a molten globule state in chimeric molecules derived from lysozyme and alpha-lactalbumin.

PubMed

Joniau, M; Haezebrouck, P; Noyelle, K; Van Dael, H

2001-07-01

The problem as to why alpha-lactalbumin, in the absence of Ca(2+), forms a molten globule intermediate, in contrast to its structural homologue lysozyme, has been addressed by the construction of chimeras of human lysozyme in which either the Ca(2+)-binding loop or a part of helix C of bovine alpha-lactalbumin were transplanted. Previously, we have shown that the introduction of both structural elements together in the lysozyme matrix causes the apo form of the resulting chimera to display molten globule behavior during the course of thermal denaturation. In this article, we demonstrate that this molten globule character is not correlated with the Ca(2+)-binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the alpha-lactalbumin conformation of the connecting loop between helix C and helix D, does not show a stable equilibrium intermediate. Rather, the molten globule character of the chimeras has to be related with a specific part of helix C. More particularly, attention is drawn to the four hydrophobic side-chains I93, V96, I99, and L100, the lysozyme counterparts of which are constituted of less bulky valines and alanine. Our observations are discussed in terms of decreased stability of the native form and increased stability of the intermediate molten globule. Copyright 2001 Wiley-Liss, Inc.

Bifidobacterium bifidum in a Rat Model of Necrotizing Enterocolitis: Antimicrobial Peptide and Protein Responses

PubMed Central

Underwood, Mark A.; Kananurak, Anchasa; Coursodon, Christine F.; Adkins-Reick, Camille K.; Chu, Hiutung; Bennett, Stephen H.; Wehkamp, Jan; Castillo, Patricia A.; Leonard, Brian C.; Tancredi, Daniel J.; Sherman, Michael P.; Dvorak, Bohuslav; Bevins, Charles L.

2013-01-01

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear. We report that like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF+BIF). All groups were exposed to asphyxia and cold stress. The expression of lysozyme, secretory phospholipase A2, pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF (NEC) group, compared to the DF and FF+BIF groups where disease was attenuated. We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut. PMID:22322385

The Natural Antimicrobial Enzyme Lysozyme is Up-Regulated in Gastrointestinal Inflammatory Conditions

PubMed Central

Rubio, Carlos A.

2014-01-01

The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum) are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014) exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days), by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett’s oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease), collagenous colitis, lymphocytic colitis, and Crohn’s colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention. PMID:25437608

Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide.

PubMed Central

Strunk, R C; Whitehead, A S; Cole, F S

1985-01-01

The third component of complement (C3) is a plasma glycoprotein with a variety of biologic functions in the initiation and maintenance of host response to infectious agents. While the hepatocyte is the primary source of plasma C3, mononuclear phagocytes contribute to the regulation of tissue availability of C3. Lipopolysaccharide (LPS), a constituent of cell walls of gram-negative bacteria, consists of a polysaccharide moiety (core polysaccharide and O antigen) covalently linked to a lipid portion (lipid A). Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis, we examined the effects of LPS on synthesis of C3 by human mononuclear phagocytes as well as synthesis of the second component of complement (C2), factor B, lysozyme, and total protein. LPS increased C3 synthesis 5-30-fold without affecting the kinetics of secretion of C3 or the synthesis of C2, lysozyme, or total protein. Factor B synthesis was consistently increased by LPS. Experiments with lipid A-inactivated LPS (alkaline treated), LPS from a polysaccharide mutant strain, and lipid X (a lipid A precursor) indicated that the lipid A portion is the structural element required for this effect. Northern blot analysis demonstrated at least a fivefold increase in C3 mRNA in LPS-treated monolayers, which suggests that the regulation of the increase in C3 synthesis is pretranslational. C2 mRNA and factor B mRNA were increased approximately twofold. The availability of specific gene products in human mononuclear phagocytes that respond to LPS should permit understanding of the molecular regulation of more complex functions of these cells elicited by LPS in which multiple gene products are coordinately expressed. Images PMID:3900137

Gene Expression in Human Accessory Lacrimal Glands of Wolfring

PubMed Central

Ubels, John L.; Gipson, Ilene K.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Van Dyken, Rachel E.; Hatton, Mark P.

2012-01-01

Purpose. The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. Methods. Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. Results. Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. Conclusions. The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs. PMID:22956620

The immunophenotypic relationship between the submucosal gland unit, columnar metaplasia and squamous islands in the columnar-lined oesophagus.

PubMed

Lörinc, Ester; Mellblom, Lennart; Öberg, Stefan

2015-12-01

To characterize the immunophenotypic relationship between the squamous and the glandular compartments in the oesophagus of patients with columnar-lined oesophagus (CLO). Eight tissue blocks from three oesophageal resection specimens from patients who underwent oesophagectomy for adenocarcinoma of the oesophagus were selected for immunohistochemical analysis. The markers of intestinal differentiation [CK20, CDX2 and MUC2] were all expressed in the expected pattern, solely in the glandular compartment of the resection specimens. CK4, CK17 and lysozyme were expressed in both the glandular and the squamous compartments. In addition, CK17 expression was found on both the squamous and glandular margins of the squamocolumnar transformation zones and in the submucosal gland (SMG) intraglandular and excretory ducts. There is an immunophenotypic relationship between the squamous and the glandular compartments of the CLO, with expression of lysozyme, CK4 and CK17 in both squamous and columnar cells. These overlapping immunophenotypes indicate similar differentiation paths, and link the SMG unit with the columnar metaplasia and the neosquamous islands in CLO. Our findings support the theory of a cellular origin of CLO and neosquamous islands from the SMG unit. © 2015 John Wiley & Sons Ltd.

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

PubMed

Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

2016-08-21

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

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

PubMed

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

2006-10-01

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

[Synergistic effects of lysozyme with EDTA-2Na on antibacterial activity].

PubMed

Li, Xiao-man; Wang, Xiao-yan; Gao, Xue-jun

2015-02-18

To evaluate the synergistic antibacterial effects of lysozyme with ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) on Enterococcus faecalis (E. faecalis) and Porphyromonas endodontalis (P. endodontalis). E. faecalis and P. endodontalis were cultured and adjusted to 10(8) CFU/mL. Then 0.3, 0.5, 1, 2, 5, 10, 50, 100, 150 and 300 g/L of lysozyme were prepared with deionized water; and the lysozyme solutions were mixed with 0.5, 1.0, 2.0 g/L of EDTA-2Na, respectively. The bacteria and lysosome with/without EDTA-2Na interacted for 15 min, then water-soluble tetrazolium (WST) working solution was added and the activity of the bacteria was calculated by measuring optical densities at 450 nm and 630 nm with microplate spectrophotometer. Regarding the pure lysozyme from 0.5 g/L to 150 g/L, more E. faecalis and P. endodontalis were inhibited when the concentration of lysozyme was higher, especially for E. faecalis. There was synergistic effect of lysozyme with EDTA-2Na on antibacterial activity, which was related to the concentration of lysozyme. On E. faecalis, the antibacterial activity of lysozyme with EDTA-2Na was 1.2-3.7 folds than the pure lysozyme when the concentration of lysozyme was 0.5-50 g/L (P<0.05), and on P. endodontalis, the antibacterial activity of lysozyme with EDTA-2Na was 1.3-3.5 folds than the pure lysozyme when the concentration of lysozyme was 0.5-10 g/L (P<0.05). When the concentration of lysozyme was higher than 100 g/L, EDTA-2Na did not show synergistic effect on the antibacterial activity (P>0.05). For E. faecalis and P. endodontalis, a low concentration of lysozyme with EDTA-2Na showed significant synergistic antibacterial activity, while a high concentration of lysozyme with EDTA-2Na did not.

Lysozyme-immobilized electrospun PAMA/PVA and PSSA-MA/PVA ion-exchange nanofiber for wound healing.

PubMed

Tonglairoum, Prasopchai; Ngawhirunpat, Tanasait; Rojanarata, Theerasak; Opanasopit, Praneet

2014-08-27

Abstract This research was aimed to develop the lysozyme immobilized ion-exchange nanofiber mats for wound healing. To promote the healing process, the PSSA-MA/PVA and PAMA ion-exchange nanofiber mats were fabricated to mimic the extracellular matrix structure using electrospinning process followed by thermally crosslinked. Lysozyme was immobilized on the ion-exchane nanofibers by an adsorption method. The ion-exchange nanofibers were investigated using SEM, FTIR and XRPD. Moreover, the lysozyme-immobilized ion-exchange nanofibers were further investigated for lysozyme content and activity, lysozyme release and wound healing activity. The fiber diameters of the mats were in the nanometer range. Lysozyme was gradually absorbed into the PSSA-MA/PVA nanofiber with higher extend than that is absorbed on the PAMA/PVA nanofiber and exhibited higher activity than lysozyme-immobilized PAMA/PVA nanofiber. The total contents of lysozyme on the PSSA-MA/PVA and PAMA/PVA nanofiber were 648 and 166 µg/g, respectively. FTIR and lysozyme activity results confirmed the presence of lysozyme on the nanofiber mats. The lysozyme was released from the PSSA-MA/PVA and PAMA/PVA nanofiber in the same manner. The lysozyme-immobilized PSSA-MA/PVA nanofiber mats and lysozyme-immobilized PAMA/PVA nanofiber mats exhibited significantly faster healing rate than gauze and similar to the commercial antibacterial gauze dressing. These results suggest that these nanofiber mats could provide the promising candidate for wound healing application.

Effects of melatonin and green-wavelength LED light on the physiological stress and immunity of goldfish, Carassius auratus, exposed to high water temperature.

PubMed

Jung, Seo Jin; Kim, Na Na; Choi, Young Jae; Choi, Ji Yong; Choi, Young-Ung; Heo, Youn Seong; Choi, Cheol Young

2016-10-01

This study investigated the effects of increasing water temperature (22-30 °C) on the physiological stress response and immunity of goldfish, Carassius auratus, and the ability of green light-emitting diode (LED) irradiation or melatonin injections to mitigate this temperature-induced stress. To evaluate the effects of either green-wavelength LED light or melatonin on stress in goldfish, we measured plasma triiodothyronine (T3), thyroxine (T4), and thyroid hormone receptor (TR) mRNA expression; plasma cortisol and glucose; and immunoglobulin M (IgM) and lysozyme mRNA expression. The thyroid hormone activities, TR mRNA expression, and plasma cortisol and glucose were higher in goldfish exposed to high-temperature water, but were lower after exposure to melatonin or green-wavelength LED light. Lysozyme mRNA expression and plasma IgM activity and protein expression were lower after exposure to high water temperatures and higher after melatonin or green-wavelength LED light treatments. Therefore, high water temperature induced stress and decreased immunity; however, green-wavelength LED light and melatonin treatments mitigated the effects of stress and enhanced immunity. The benefits of melatonin decreased with time, whereas those of green-wavelength LED treatment did not.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent--application to a human single-chain Fv fragment.

PubMed

Tsumoto, K; Shinoki, K; Kondo, H; Uchikawa, M; Juji, T; Kumagai, I

1998-10-01

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.

Structural changes and cellular localization of resuscitation-promoting factor in environmental isolates of Micrococcus luteus.

PubMed

Koltunov, Viktoria; Greenblatt, Charles L; Goncharenko, Anna V; Demina, Galya R; Klein, Benjamin Y; Young, Michael; Kaprelyants, Arseny S

2010-02-01

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

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.

The Effect of UV-C Pasteurization on Bacteriostatic Properties and Immunological Proteins of Donor Human Milk

PubMed Central

Christen, Lukas; Lai, Ching Tat; Hartmann, Ben; Hartmann, Peter E.; Geddes, Donna T.

2013-01-01

Background Human milk possesses bacteriostatic properties, largely due to the presence of immunological proteins. Heat treatments such as Holder pasteurization reduce the concentration of immunological proteins in human milk and consequently increase the bacterial growth rate. This study investigated the bacterial growth rate and the immunological protein concentration of ultraviolet (UV-C) irradiated, Holder pasteurized and untreated human milk. Methods Samples (n=10) of untreated, Holder pasteurized and UV-C irradiated human milk were inoculated with E. coli and S. aureus and the growth rate over 2 hours incubation time at 37°C was observed. Additionally, the concentration of sIgA, lactoferrin and lysozyme of untreated and treated human milk was analyzed. Results The bacterial growth rate of untreated and UV-C irradiated human milk was not significantly different. The bacterial growth rate of Holder pasteurized human milk was double compared to untreated human milk (p<0.001). The retention of sIgA, lactoferrin and lysozyme after UV-C irradiation was 89%, 87%, and 75% respectively, which were higher than Holder treated with 49%, 9%, and 41% respectively. Conclusion UV-C irradiation of human milk preserves significantly higher levels of immunological proteins than Holder pasteurization, resulting in bacteriostatic properties similar to those of untreated human milk. PMID:24376898

The effect of UV-C pasteurization on bacteriostatic properties and immunological proteins of donor human milk.

PubMed

Christen, Lukas; Lai, Ching Tat; Hartmann, Ben; Hartmann, Peter E; Geddes, Donna T

2013-01-01

Human milk possesses bacteriostatic properties, largely due to the presence of immunological proteins. Heat treatments such as Holder pasteurization reduce the concentration of immunological proteins in human milk and consequently increase the bacterial growth rate. This study investigated the bacterial growth rate and the immunological protein concentration of ultraviolet (UV-C) irradiated, Holder pasteurized and untreated human milk. Samples (n=10) of untreated, Holder pasteurized and UV-C irradiated human milk were inoculated with E. coli and S. aureus and the growth rate over 2 hours incubation time at 37°C was observed. Additionally, the concentration of sIgA, lactoferrin and lysozyme of untreated and treated human milk was analyzed. The bacterial growth rate of untreated and UV-C irradiated human milk was not significantly different. The bacterial growth rate of Holder pasteurized human milk was double compared to untreated human milk (p<0.001). The retention of sIgA, lactoferrin and lysozyme after UV-C irradiation was 89%, 87%, and 75% respectively, which were higher than Holder treated with 49%, 9%, and 41% respectively. UV-C irradiation of human milk preserves significantly higher levels of immunological proteins than Holder pasteurization, resulting in bacteriostatic properties similar to those of untreated human milk.

Protein Buffering in Model Systems and in Whole Human Saliva

PubMed Central

Lamanda, Andreas; Cheaib, Zeinab; Turgut, Melek Dilek; Lussi, Adrian

2007-01-01

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and α-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva. PMID:17327922

Evaluation of oriented lysozyme immobilized with monoclonal antibody

NASA Astrophysics Data System (ADS)

Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya

2008-12-01

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

PubMed

Kuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem

2017-12-16

To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.

PubMed Central

Smith, M D; Flickinger, J L; Lineberger, D W; Schmidt, B

1986-01-01

The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody. Images PMID:3008649

Principal Component Relaxation Mode Analysis of an All-Atom Molecular Dynamics Simulation of Human Lysozyme

NASA Astrophysics Data System (ADS)

Nagai, Toshiki; Mitsutake, Ayori; Takano, Hiroshi

2013-02-01

A new relaxation mode analysis method, which is referred to as the principal component relaxation mode analysis method, has been proposed to handle a large number of degrees of freedom of protein systems. In this method, principal component analysis is carried out first and then relaxation mode analysis is applied to a small number of principal components with large fluctuations. To reduce the contribution of fast relaxation modes in these principal components efficiently, we have also proposed a relaxation mode analysis method using multiple evolution times. The principal component relaxation mode analysis method using two evolution times has been applied to an all-atom molecular dynamics simulation of human lysozyme in aqueous solution. Slow relaxation modes and corresponding relaxation times have been appropriately estimated, demonstrating that the method is applicable to protein systems.

Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

PubMed

Wang, Quanfu; Hou, Yanhua; Yan, Peisheng

2012-06-01

Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes. © 2012 Institute of Food Technologists®

Diamond Nanogel-Embedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release

PubMed Central

2015-01-01

Temporarily implanted devices, such as drug-loaded contact lenses, are emerging as the preferred treatment method for ocular diseases like glaucoma. Localizing the delivery of glaucoma drugs, such as timolol maleate (TM), can minimize adverse effects caused by systemic administration. Although eye drops and drug-soaked lenses allow for local treatment, their utility is limited by burst release and a lack of sustained therapeutic delivery. Additionally, wet transportation and storage of drug-soaked lenses result in drug loss due to elution from the lenses. Here we present a nanodiamond (ND)-embedded contact lens capable of lysozyme-triggered release of TM for sustained therapy. We find that ND-embedded lenses composed of enzyme-cleavable polymers allow for controlled and sustained release of TM in the presence of lysozyme. Retention of drug activity is verified in primary human trabecular meshwork cells. These results demonstrate the translational potential of an ND-embedded lens capable of drug sequestration and enzyme activation. PMID:24506583

Antimicrobial polycaprolactone/polyethylene glycol embedded lysozyme coatings of Ti implants for osteoblast functional properties in tissue engineering

NASA Astrophysics Data System (ADS)

Visan, A.; Cristescu, R.; Stefan, N.; Miroiu, M.; Nita, C.; Socol, M.; Florica, C.; Rasoga, O.; Zgura, I.; Sima, L. E.; Chiritoiu, M.; Chifiriuc, M. C.; Holban, A. M.; Mihailescu, I. N.; Socol, G.

2017-09-01

In this study, coatings based on lysozyme embedded into a matrix of polyethylene glycol (PEG) and polycaprolactone (PCL) were fabricated by two different methods (Matrix Assisted Pulsed Laser Evaporation - MAPLE and Dip Coating) for obtaining antimicrobial coatings envisaged for long term medical applications. Coatings with different PEG:PCL compositions (3:1; 1:1; 1:3) were synthesized in order to evaluate the antimicrobial activity of lysozyme embedded into the polymeric matrix. The main surface features, such as roughness and wettability, with impact on the microbial adhesion as well as on the eukaryote cell function were measured. The obtained composite coatings exhibited a significant antibacterial activity against Escherichia coli, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus strains. As well, specific blended coatings showed appropriate viability, good spreading and normal cell morphology of SaOs2 human osteoblasts and mesenchymal stem cells (MSCs). These investigations highlight the suitability of biodegradable composites as implant coatings for decreasing the risk of bacterial contamination associated with prosthetic procedures.

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

PubMed

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

2015-04-01

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

A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis.

PubMed

Keith, D; Hong, B; Christensen, M

1997-05-01

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression.

PubMed

Cieslak, Jakub; Wodas, Lukasz; Borowska, Alicja; Sadoch, Jan; Pawlak, Piotr; Puppel, Kamila; Kuczynska, Beata; Mackowski, Mariusz

2017-05-01

Equine milk is considered to be an interesting product for human nutrition, mainly owing to its low allergenicity and significant amounts of bioactive proteins, including lysozyme (LYZ) and lactoferrin (LTF). The present study assessed the effect of genetic factors on LYZ and LTF concentration variability in mare's milk. Significant effects of horse breed and lactation stage on milk LYZ and LTF contents were observed. The highest level of LTF and the lowest concentration of LYZ were recorded for the Polish Warmblood Horse breed. The highest amounts of both proteins were found for the earliest investigated time point of lactation (5th week). Altogether 13 (nine novel) polymorphisms were found in the 5'-flanking regions of both genes, but they showed no significant relationship with milk LYZ and LTF contents. Several associations were found between selected SNPs and the LYZ gene relative transcript level. While the present study indicated the existence of intra- and interbreed variability of LYZ and LTF contents in mare's milk, this variation is rather unrelated to the 5'-flanking variants of genes encoding both proteins. This study is a good introduction for broader investigations focused on the genetic background for variability of bioactive protein contents in mare's milk. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus.

PubMed

Choi, Cheol Young; Shin, Hyun Suk; Choi, Young Jae; Kim, Na Na; Lee, Jehee; Kil, Gyung-Suk

2012-11-01

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630 nm), green (peak at 530 nm), and blue (peak at 450 nm) within a visible light. We investigated the oxidative stress induced by starvation for 12 days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish. Copyright © 2012 Elsevier Inc. All rights reserved.

Co-option of bacteriophage lysozyme genes by bivalve genomes.

PubMed

Ren, Qian; Wang, Chunyang; Jin, Min; Lan, Jiangfeng; Ye, Ting; Hui, Kaimin; Tan, Jingmin; Wang, Zheng; Wyckoff, Gerald J; 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. © 2017 The Authors.

Salivary pH, calcium, phosphorus and selected enzymes in healthy dogs: a pilot study.

PubMed

Iacopetti, Ilaria; Perazzi, Anna; Badon, Tamara; Bedin, Silvia; Contiero, Barbara; Ricci, Rebecca

2017-11-10

Saliva in dogs, as in humans, is a complex fluid secreted by different salivary glands in the oral cavity to protect the oral mucosa and teeth. The use of saliva as a substitute for blood in diagnosing and prognosticating disease in humans is widely accepted. Salivary biochemistry has also been used as a marker for periodontal disease in humans. No studies have as yet investigated the relation between salivary biochemistry and periodontal disease in dogs, however; neither has the salivary composition of healthy dogs with no oral disease been assessed. The purpose of this study was to obtain an overview on pH distribution and a set of salivary biochemical analytes (calcium, phosphorus, lactate dehydrogenase, lysozyme and amylase) commonly related to oral health in humans in a subset population of healthy young dogs with no periodontal disease or previous oral disease. Data were analyzed to gather salivary reference ranges for pH and each parameter and to assess a possible correlation between salivary and serum analytes. Twenty-nine adult client-owned dogs were recruited for the study. Lactate dehydrogenase and lysozyme showed higher concentrations in saliva than in serum, whereas amylase showed the contrary. Salivary biochemistry values did not differ between males and females or between non-neutered and neutered individuals. No significant correlations between salivary and serum calcium, phosphorus, lactate dehydrogenase, amylase and lysozyme were identified in this study. Data allowed intervals for the salivary pH and other analytes investigated to be obtained from healthy dogs with healthy oral conditions. These preliminary data can contribute to enlarge our understanding of the functional role of saliva and its relation to oral health in dogs.

A novel electrochemical aptamer-antibody sandwich assay for lysozyme detection.

PubMed

Ocaña, Cristina; Hayat, Akhtar; Mishra, Rupesh; Vasilescu, Alina; del Valle, Manel; Marty, Jean-Louis

2015-06-21

In this paper, we have reported a novel electrochemical aptamer-antibody based sandwich biosensor for the detection of lysozyme. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (lysozyme), a biotinylated antibody was used to complete the sandwich format. The subsequent additions of avidin-alkaline phosphatase as an enzyme label, and a 1-naphthyl phosphate substrate (1-NPP) allowed us to determine the concentration of lysozyme (Lys) via Differential Pulse Voltammetry (DPV) of the generated enzyme reaction product, 1-naphthol. Using this strategy, a wide detection range from 5 fM to 5 nM was obtained for a target lysozyme, with a detection limit of 4.3 fM. The control experiments were carried out by using bovine serum albumin (BSA), cytochrome c and casein. The results showed that the proposed biosensor had good specificity, stability and reproducibility for lysozyme analysis. In addition, the biosensor was applied for detecting lysozyme in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for lysozyme detection. This implies that the proposed sandwich biosensor is a promising analytical tool for the analysis of lysozyme in real samples.

Effects of date palm fruit extracts on skin mucosal immunity, immune related genes expression and growth performance of common carp (Cyprinus carpio) fry.

PubMed

Hoseinifar, Seyed Hossein; Khalili, Mohsen; Rufchaei, Rudabeh; Raeisi, Mojtaba; Attar, Marzieh; Cordero, Héctor; Esteban, M Ángeles

2015-12-01

The aim of this study was to investigate the effects of date palm fruit extracts (DPFE) on skin mucosal immunity, immune related genes expression and growth performance of fry common carp (Cyprinus carpio). One hundred and twenty specimens (4.06 ± 0.13 g) were supplied and allocated into six aquaria; specimens in three aquaria were fed non-supplemented diet (control) while the fish in the other 3 aquaria were fed with DPFE at 200 ml kg(-1). At the end of feeding trial (8 weeks) skin mucus immune parameters (total immunoglobulins, lysozyme, protease and alkaline phosphatase activity) and immune related gene expression (tumor necrosis factor α [tnfa], lysozyme [ly] and interleukin-1-beta, [il1b]) in the head-kidney were studied. The results revealed that feeding carp fry with 200 ml kg(-1) DPFE remarkably elevated the three skin mucus immune parameters tested (P < 0.05). However, evaluation of immune related gene expression demonstrated that the expression of tnfa and il1b was considerably decreased (P < 0.05) in fish fed DPFE diet, while the expression of ly remained similar (P > 0.05) compared to control fish (fed control diet). Furthermore, growth performance parameters were significantly improved in fry fed DPFE (P < 0.05). More studies are needed to understand different aspects of DPFE administration in fry mucosal immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Magnetic fluid - a novel approach to treat amyloid-related diseases

NASA Astrophysics Data System (ADS)

Antosova, Andrea; Siposova, Katarina; Koneracka, Martina; Zavisova, Vlasta; Daxnerova, Zuzana; Vavra, Ivo; Fedunova, Diana; Bagelova, Jaroslava; Kopcansky, Peter; Gazova, Zuzana

Protein amyloid aggregates are associated with several human pathologies termed amyloid-related diseases. We have investigated effect of two magnetic fluids (MFs) - electrostatically stabilized Fe3O4 magnetic nanoparticles (MF1) and sterically stabilized Fe3O4 magnetic nanoparticles by sodium oleate with adsorbed BSA (MF2) on amyloid aggregation of two proteins - human insulin and hen egg lysozyme. We have found that both MF1 and MF2 are able to interact with amyloid fibrils in vitro resulting into decreasing of amyloid aggregates. The extent of fibril disruption depends on MF concentration with extensive reduction of amyloid aggregates, 90% for lysozyme and 70% for insulin (ratio protein: MF=1:5). The obtained results suggest that magnetite component of MF play significant role in the process of amyloid fibril depolymerisation. Our findings indicate that MF1 and MF2 have potential to be used for treatment of amyloid diseases.

Activity of chitosan-lysozyme nanoparticles on the growth, membrane integrity, and β-1,3-glucanase production by Aspergillus parasiticus.

PubMed

Hernández-Téllez, Cynthia Nazareth; Rodríguez-Córdova, Francisco Julián; Rosas-Burgos, Ema Carina; Cortez-Rocha, Mario Onofre; Burgos-Hernández, Armando; Lizardi-Mendoza, Jaime; Torres-Arreola, Wilfrido; Martínez-Higuera, Aarón; Plascencia-Jatomea, Maribel

2017-10-01

Synthesis of nanocomposites from antimicrobial biopolymers such as chitosan (CS) and lysozyme (LZ) is an important and promising area in bionanotechnology. Chitosan-lysozyme (CS-LZ) nanoparticles (NPs) were prepared by the nanoprecipitation method, using commercial chitosan of 153 kDa. TEM and dynamic light scattering (DLS) analysis were carried out to evaluate the morphology, size, dispersion, and Z potential. Association efficiency of lysozyme was determined using Coomassie blue assay. The antifungal activity of NPs against Aspergillus parasiticus was evaluated through cell viability (XTT), germination and morphometry of spores, and reducing sugars production; the effects on membrane integrity and cell wall were also analyzed. NPs' size were found in the range of 13.4 and 11.8 nm for CS-LZ and CS NPs, respectively, and high Z potential value was observed in both NPs. Also, high association of lysozyme was presented in the CS matrix. With respect to the biological responses, CS-LZ NPs reduced the viability of A. parasiticus and a strong inhibitory effect on the germination of spores (100% of inhibition) was observed at 24 h in in vitro assays. CS-LZ and CS NPs affected the membrane integrity and the cell wall of spores of fungi with respect to control, which is consistent with the low amount of reducing sugars detected. CS-LZ NPs prepared by nanoprecipitation promise to be a viable and safe alternative for use in biological systems, with a possible low or null impact to humans and biota. However, the potential benefits and the environmental and health implications of NPs need to be globally discussed due to its possible negative effects.

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

PubMed

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

2014-06-01

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

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins.

PubMed

Müller, Egbert; Josic, Djuro; Schröder, Tim; Moosmann, Anna

2010-07-09

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.

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 and bacteria present in oral biofilms. PMID:28138698

Surface versus bulk activity of lysozyme deposited on hydrogel contact lens materials in vitro.

PubMed

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

2018-04-30

To determine and compare the levels of surface versus bulk active lysozyme deposited on several commercially available hydrogel contact lens materials. Hydrogel contact lens materials [polymacon, omafilcon A, nelfilcon A, nesofilcon A, ocufilcon and etafilcon A with polyvinylpyrrolidone (PVP)] were incubated in an artificial tear solution for 16 h. Total activity was determined using a standard turbidity assay. The surface activity of the deposited lysozyme was determined using a modified turbidity assay. The amount of active lysozyme present within the bulk of the lens material was calculated by determining the difference between the total and surface active lysozyme. The etafilcon A materials showed the highest amount of total lysozyme activity (519 ± 8 μg/lens, average of Moist and Define), followed by the ocufilcon material (200 ± 5 μg/lens) and these two were significantly different from each other (p < 0.05). The amount of surface active lysozyme on etafilcon and ocufilcon lens materials was significantly higher than that found on all other lenses (p < 0.05). There was no active lysozyme quantified in the bulk of the nelfilcon material, as all of the active lysozyme was found on the surface (1.7 ± 0.3 μg/lens). In contrast, no active lysozyme was quantified on the surface of polymacon, with all of the active lysozyme found in the bulk of the lens material (0.6 ± 0.6 μg/lens). The surface and bulk activity of lysozyme deposited on contact lenses is material dependent. Lysozyme deposited on ionic, high water content lens materials such as etafilcon A show significantly higher surface and bulk activity than many other hydrogel lens materials. Copyright © 2018 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-10-01

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

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

PubMed

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

2009-09-01

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

Microbial community related to lysozyme digestion process for boosting waste activated sludge biodegradability.

PubMed

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

2015-01-01

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

Hereditary Lysozyme Amyloidosis Variant p.Leu102Ser Associates with Unique Phenotype

PubMed Central

Nasr, Samih H.; Dasari, Surendra; Mills, John R.; Theis, Jason D.; Zimmermann, Michael T.; Fonseca, Rafael; Vrana, Julie A.; Lester, Steven J.; McLaughlin, Brooke M.; Gillespie, Robert; Highsmith, W. Edward; Lee, John J.; Dispenzieri, Angela

2017-01-01

Lysozyme amyloidosis (ALys) is a rare form of hereditary amyloidosis that typically manifests with renal impairment, gastrointestinal (GI) symptoms, and sicca syndrome, whereas cardiac involvement is exceedingly rare and neuropathy has not been reported. Here, we describe a 40-year-old man with renal impairment, cardiac and GI symptoms, and peripheral neuropathy. Renal biopsy specimen analysis revealed amyloidosis with extensive involvement of glomeruli, vessels, and medulla. Amyloid was also detected in the GI tract. Echocardiographic and electrocardiographic findings were consistent with cardiac involvement. Proteomic analysis of Congo red–positive renal and GI amyloid deposits detected abundant lysozyme C protein. DNA sequencing of the lysozyme gene in the patient and his mother detected a heterozygous c.305T>C alteration in exon 3, which causes a leucine to serine substitution at codon 102 (Human Genome Variation Society nomenclature: p.Leu102Ser; legacy designation: L84S). We also detected the mutant peptide in the proband’s renal and GI amyloid deposits. PolyPhen analysis predicted that the mutation damages the encoded protein. Molecular dynamics simulations suggested that the pathogenesis of ALys p.Leu102Ser is mediated by shifting the position of the central β-hairpin coordinated with an antiparallel motion of the C-terminal helix, which may alter the native-state structural ensemble of the molecule, leading to aggregation-prone intermediates. PMID:28049649

Protein-lipid interactions at the air/water interface.

PubMed

Lad, Mitaben D; Birembaut, Fabrice; Frazier, Richard A; Green, Rebecca J

2005-10-07

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.

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.

Mucous lysozyme levels in hatchery coho salmon (Oncorhynchus kisutch) and spring chinook salmon (O. tshawytscha) early in the parr-smolt transformation

USGS Publications Warehouse

Schrock, R.M.; Smith, S.D.; Maule, A.G.; Doulos, S.K.; Rockowski, J.J.

2001-01-01

Mucous lysozyme concentrations were determined in juvenile coho salmon (Oncorhynchus kisutch) and spring chinook salmon (O. tshawytscha) to establish reference levels during the time associated with the parr-smolt transformation. The first reported naris and vent mucous lysozyme levels are provided for spring chinook salmon and coho salmon. Naris mucous lysozyme levels ranged between 300 and 700 ??g ml-1, vent mucous lysozyme from 100 to 300 ??g ml-1, and skin mucous lysozyme levels were below 130 ??g ml-1. Lysozyme levels in the two species showed the same relationship with the highest levels in naris mucous, and the lowest in skin mucous. A seasonal decrease occurred in both species with a significant decrease in naris mucous lysozyme between February and March. Gill ATPase levels used to monitor smolt development during the same period did not reach ranges reported for smolts for either species during emigration. Identification of seasonal levels of lysozyme activity in mucous provides an alternative determination of developmental status prior to release of fish from the hatchery when salmonids are still undergoing the parr-smolt transformation. ?? 2001 Elsevier Science B.V.

Protein crystals as scanned probes for recognition atomic force microscopy.

PubMed

Wickremasinghe, Nissanka S; Hafner, Jason H

2005-12-01

Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

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.

Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response.

PubMed

Derbise, Anne; Pierre, François; Merchez, Maud; Pradel, Elizabeth; Laouami, Sabrina; Ricard, Isabelle; Sirard, Jean-Claude; Fritz, Jill; Lemaître, Nadine; Akinbi, Henry; Boneca, Ivo G; Sebbane, Florent

2013-05-15

Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

Control of Bacteriophage T4 Tail Lysozyme Activity During the Infection Process

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

Kanamaru, Shuji; Ishiwata, Yasutaka; Suzuki, Toshiharu

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,more » 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.« less

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function.

PubMed

Umasuthan, Navaneethaiyer; Bathige, S D N K; Kasthuri, Saranya Revathy; Wan, Qiang; Whang, Ilson; Lee, Jehee

2013-08-01

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nucleation and convection effects in protein crystal growth

NASA Technical Reports Server (NTRS)

Rosenberger, Franz (Principal Investigator)

1996-01-01

The following activities are reported on: repartitioning of NaCl and protein impurities in lysozyme crystallization; dependence of lysozyme growth kinetics on step sources and impurities; facet morphology response to nonuniformities in nutrient and impurity supply; interactions in undersaturated and supersaturated lysozyme solutions; heterogeneity determination and purification of commercial hen egg white lysozyme; nonlinear response of layer growth dynamics in the mixed kinetics-bulk transport regime; development of a simultaneous multiangle light scattering technique; and x-ray topography of tetragonal lysozyme grown by the temperature-control technique.

Technology Optimization of Lysozyme's Fresh Maintaining Effect on Apple.

PubMed

Jun-Hong, Liu; Kun-Yu, Wang

2016-10-03

Lysozyme is a kind of alkaline globin, which functions well in the degradation of the cell wall of microbe. Currently, lysozyme is widely used in various fields, such as medicine, fruit, and vegetable industry, dairy industry, and so on. Therefore, the exploitation and utilization of lysozyme is of significant economic benefit. Taking apple as material, weight loss ratio and reducing sugar content as objectives, this paper studied the fresh-keeping effect of lysozyme. Influential factors, lysozyme concentration, soaking time, modified temperature, and reaction time were discussed in detail. The results showed that reducing sugar content was 2.043% and the weight loss ratio was the minimum in the presence of soaking time of 2 min, modified temperature of 65 °C, reaction time of 4 d, and lysozyme concentration of 0.5 g/L. © 2016 Institute of Food Technologists®.

Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.

PubMed

Anema, Skelte G; de Kruif, C G Kees

2013-07-24

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ∼50 to 100 nm. The κ-casein content decreased markedly and the αS-casein/β-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/κ-casein ratio was constant for all micelle sizes, whereas the lactoferrin/αS-casein and lactoferrin/β-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/αS-casein and lysozyme/β-casein ratios were nearly constant, whereas the lysozyme/κ-casein ratio increased with increasing micelle size, indicating that lysozyme bound to αS-casein and β-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged αS-casein and β-casein.

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

PubMed

Ji, Shuying; Thulstrup, Peter Waaben; Mu, Huiling; Hansen, Steen Honoré; van de Weert, Marco; Rantanen, Jukka; Yang, Mingshi

2016-11-20

In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme did not compromise the conformation of the protein after drying, while it improved the inhaled aerosol performance. Copyright © 2016 Elsevier B.V. All rights reserved.

A population-based study of salivary lysozyme concentrations and candidal counts.

PubMed

Yeh, C K; Dodds, M W; Zuo, P; Johnson, D A

1997-01-01

The relationship between salivary lysozyme concentration and oral candida load was examined in 595 adults. Unstimulated whole saliva, and citrate-stimulated parotid and submandibular/sublingual saliva were collected from each participant. Candida colony-forming units (c.f.u.) in unstimulated whole saliva were determined. An enzyme-linked immunosorbent assay for lysozyme using commercially available antibodies was developed. This assay showed a linear relation of salivary lysozyme concentrations from 0.5 to 4.0 ng/ml. Significant negative relations were observed between lysozyme concentration and flow rate: r = -0.16 (p < 0.001) for stimulated parotid and r = -0.22 (p < 0.0001) for stimulated submandibular/sublingual saliva. The lysozyme concentration in stimulated submandibular/sublingual saliva was higher in males than in female, but no sex difference was observed for stimulated parotid saliva. The lysozyme concentration of stimulated parotid saliva was positively correlated with candida counts (r = 0.18: p < 0.005). Further study of groups according to their levels of candida in whole saliva revealed that lysozyme concentrations were higher in the high candida (> or = 1000 c.f.u./ml) group than in the zero and moderate candida categories in stimulated parotid saliva (p < 0.001): there were no concentration differences in stimulated submandibular/sublingual saliva. These results suggest that parotid lysozyme concentration increases as candida load increases.

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

PubMed

Buijs; Hlady

1997-06-01

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

EndoU is a novel regulator of AICD during peripheral B cell selection

PubMed Central

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.

2014-01-01

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22−/−[B6]) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag–specific B cells in IgTg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. PMID:24344237

EndoU is a novel regulator of AICD during peripheral B cell selection.

PubMed

Poe, Jonathan C; Kountikov, Evgueni I; Lykken, Jacquelyn M; Natarajan, Abirami; Marchuk, Douglas A; Tedder, Thomas F

2014-01-13

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

Lysozyme pattern formation in evaporating droplets

NASA Astrophysics Data System (ADS)

Gorr, Heather Meloy

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

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

PubMed

Panja, Sudipta; Halder, Mintu

2016-08-01

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

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

PubMed

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

2016-09-16

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

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

PubMed

Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

2015-11-21

Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis).

PubMed

Ko, Jiyeon; Wan, Qiang; Bathige, S D N K; Lee, Jehee

2016-11-01

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu 71 , Asp 84 , and Asp 95 ) and seven N-acetyl-d-glucosamine binding sites (Glu 71 , Asp 95 , Tyr 98 , His 99 , Ile 117 , Tyr 145 , and Asn 146 ). Homology analysis demonstrated that the aa sequence of ShLysG shared 60.7-67.4% identity and 72.6-79.3% similarity with the orthologs of other teleosts. Phylogenetic analysis of ShLysG indicated a closest relationship with the ortholog from Gadus morhua. In healthy seahorse, ShLysG mRNA showed a constitutive expression in all the tissues examined, with the highest expression in kidney and the least expression in liver. The ShLysG mRNA levels were also shown significant elevation upon the bacterial and pathogen-associated molecular pattern (PAMPs) challenges. Furthermore, lytic activities of ShLysG recombinant protein were detected against several Gram-negative and Gram-positive bacterial species. Taken together, these results suggest that ShLysG might possess a potential immune defensive role against invading microbial pathogens in seahorse. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): innate immune responses and expression profile of some immune related genes.

PubMed

Dash, Pujarini; Kar, Banya; Mishra, Arpita; Sahoo, P K

2014-03-01

The monogenean ectoparasite, Dactylogyrus sp. is a major pathogen in freshwater aquaculture. The immune responses in parasitized fish were analyzed by quantitation of innate immune factors (natural agglutinin level, haemolysin titre, antiprotease, lysozyme and myeloperoxidase activities) in serum and immune-relevant gene expression in gill and anterior kidney. The antiprotease activity and natural agglutinin level were found to be significantly higher and lysozyme activity was significantly lower in parasitized fish. Most of the genes viz., beta2-microglobulin (beta2M), major histocompatibility complex I (MHCI), MHCII, tumor necrosis factor alpha (TNFalpha) and toll-like receptor 22 (TLR22) in gill samples were significantly down-regulated in the experimental group. In the anterior kidney, the expression of superoxide dismutase and interleukin 1beta (IL1beta) were significantly up-regulated whereas a significant down regulation of MHCII and TNFalpha was also observed. The down-regulation of most of the genes viz, MHCI, beta2M, MHCII, TLR22 and TNFalpha in infected gills indicated a well evolved mechanism in this parasite to escape the host immune response. The modulation of innate and adaptive immunity by this parasite can be further explored to understand host susceptibility.

Three newly identified galectin homologues from triangle sail mussel (Hyriopsis cumingii) function as potential pattern-recognition receptors.

PubMed

Zhao, Ling-Ling; Hui, Kaimin; Wang, Yu-Qing; Wang, Yue; Ren, Qian; Li, Xin-Cang

2018-05-01

Galactoside-binding lectins, also known as galectins, play crucial roles in innate immune response in invertebrates. In this study, three cDNA sequences from Hyriopsis cumingii were identified and collectively called HcGalec genes. Each of the three deduced HcGalec proteins contained a galactose-binding lectin domain or a GLECT domain. All the three HcGalec genes are mainly present in the hepatopancreas and gills, and their expression is induced at 24 h after bacterial challenge. Three recombinant HcGalec proteins can bind and agglutinate (Ca 2+ -dependent) various microorganisms, including Gram-positive and Gram-negative bacteria. These proteins can attach to mannan and peptidoglycan. Meanwhile, the expression of the three HcGalec genes in the gills were significantly down-regulated after dsRNA interference (HcGalec1-RNAi, HcGalec2-RNAi, and HcGalec3-RNAi) and Vibrio parahaemolyticus injection. The expression levels of some antimicrobial peptides, including lysozyme 1 and lysozyme 2, were also markedly decreased after dsRNA interference. Overall, these results suggested that these three HcGalec proteins may function as potential receptors participating in the innate immune responses of H. cumingii against bacterial infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inhibition of intracellular bacterial replication in fibroblasts is dependent on the perforin-like protein (Perforin-2) encoded by macrophage expressed gene 1

PubMed Central

McCormack, Ryan; de Armas, Lesley R.; Shiratsuchi, Motoaki; Ramos, Jay; Podack, Eckhard R.

2013-01-01

Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage expressed gene 1 (mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knock-down of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with an RFP-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria including Gram positive, Gram negative and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEFs damage cell walls of intracellular bacteria by insertion, polymerization and pore-formation of the perforin-like protein, analogous to pore-formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2. PMID:23257510

Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

PubMed Central

Dvořák, Jiří; Mančíková, Veronika; Pižl, Václav; Elhottová, Dana; Šilerová, Marcela; Roubalová, Radka; Škanta, František; Procházková, Petra; Bilej, Martin

2013-01-01

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins. PMID:24223917

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

PubMed

Lavine, M D; Chen, G; Strand, M R

2005-12-01

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

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

PubMed

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

2011-08-15

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

Molecular Dynamics Analysis of Lysozyme Protein in Ethanol- Water Mixed Solvent

DTIC Science & Technology

2012-01-01

molecular dynamics simulations of solvent effect on lysozyme protein, using water, ethanol, and different concentrations of water-ethanol mixtures as...understood. This work focuses on detailed molecular dynamics simulations of solvent effect on lysozyme protein, using water, ethanol, and different...using GROMACS molecular dynamics simulation (MD) code. Compared to water environment, the lysozyme structure showed remarkable changes in water

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.

Study on the interaction between cinnamic acid and lysozyme

NASA Astrophysics Data System (ADS)

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

2011-02-01

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

Histological and immunohistochemical features of histiocytic sarcoma in four domestic ferrets (Mustela putorius furo).

PubMed

Thongtharb, Atigan; Uchida, Kazuyuki; Chambers, James K; Miwa, Yasutsugu; Murata, Yousuke; Nakayama, Hiroyuki

2016-03-01

Four cases of histiocytic sarcoma in domestic ferrets (Mustela putorius furo) are described in the present study. Tumor samples obtained from the abdominal viscera, including the spleen, were submitted for histologic examination. Microscopically, poorly demarcated masses contained numerous round- to pleomorphic-shaped cells with coarsely vacuolated and eosinophilic cytoplasm. Bizarre, binucleated tumor cells and multinucleated giant tumor cells with low phagocytic activity were commonly observed. Immunohistochemically, tumor cells in all of the cases were positive for vimentin, human leukocyte antigen-DR, ionized calcium-binding adapter molecule-1, and lysozyme, but some of them lacked cluster of differentiation (CD)163 or CD208 expression. The survival time after surgical resection was 9 days to 5 months. Histiocytic sarcoma in the ferret is a rare, but highly aggressive, tumor commonly found in the spleen. © 2016 The Author(s).

COLLABORATIVE RESEARCH AND DEVELOPMENT CONTRACT. Delivery Order 0038: Microbial Biotechnology and Biocatalysis

DTIC Science & Technology

2010-04-01

encapsulated lysozyme. Bacteriolytic activity was inves- tigated with Micrococcus lysodeikticus cells and by hydroly- sis of a synthetic membrane mimic...immobilized lysozyme in silica or tita- nia nanoparticles. Lysozyme activity determined by lytic assay with Micrococcus lysodeikticus cells (&) or with...voltage of 200 V and according to the manufacturer’s instruc- tions. Lysozyme activity assays were performed with Micrococcus lysodeikticus cells

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

NASA Astrophysics Data System (ADS)

Bernard, C.; Leduc, A.; Barbeau, J.; Saoudi, B.; Yahia, L'H.; DeCrescenzo, G.

2006-08-01

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen-oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integrity of Proteins in Human Saliva after Sterilization by Gamma Irradiation▿

PubMed Central

Ruhl, Stefan; Berlenbach, Pereshia; Langenfelder, Sabine; Hörl, Dagmar; Lehn, Norbert; Hiller, Karl-Anton; Schmalz, Gottfried; Durchschlag, Helmut

2011-01-01

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10−6 was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form. PMID:21148692

Magnetic Fluids Have Ability to Decrease Amyloid Aggregation Associated with Amyloid-Related Diseases

NASA Astrophysics Data System (ADS)

Antosova, Andrea; Koneracka, Martina; Siposova, Katarina; Zavisova, Vlasta; Daxnerova, Zuzana; Vavra, Ivo; Fabian, Martin; Kopcansky, Peter; Gazova, Zuzana

2010-12-01

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several amyloid-related diseases. We have investigated the effect of four magnetic fluids (MFs)—electrostatically stabilized Fe3O4 magnetic nanoparticles (MF1) and sterically stabilized Fe3O4 magnetic nanoparticles by sodium oleate (MF2, MF3 and MF4) with adsorbed BSA (MF2) or dextran (MF4)—on amyloid aggregation of two proteins, human insulin and chicken egg lysozyme. The morphology, particle size and size distribution of the prepared magnetic fluids were characterized. We have found that MFs are able to decrease amyloid aggregation of both studied proteins and the extent of depolymerization depended on the MF properties. The most effective reduction was observed for MF4 as 90% decrease of amyloids was detected for insulin and lysozyme amyloid aggregates. Our findings indicate that MFs have potential to be used for treatment of amyloid diseases.

Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme.

PubMed

Huang, Peng; Shi, Jinlei; Sun, Qingwen; Dong, Xianping; Zhang, Ning

2018-04-13

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na + . In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

Crawford, Lisa; Karr, Laurel; Pusey, Marc

1998-01-01

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

Crowding of white shrimp Litopenaeus vananmei depresses their immunity to and resistance against Vibrio alginolyticus and white spot syndrome virus.

PubMed

Lin, Yong-Chin; Chen, Jiann-Chu; Chen, Yu-Yuan; Yeh, Su-Tuen; Chen, Li-Li; Huang, Chien-Lun; Hsieh, Jen-Fang; Li, Chang-Che

2015-07-01

Immunity parameters and the expression levels of several immune-related proteins, including lipopolysaccharide and β-glucan binding protein (LGBP), peroxinectin (PX), intergin β (IB), prophenoloxidase (proPO) I, proPO II, α2-macroglobulin (α2-M), cytosolic mangangese superoxide dismutase (cytMnSOD), mitochondria manganese superoxide dismutase (mtMnSOD), catalase, glutathione peroxidase (GPx), lysozyme, and penaeidin 3a were examined in white shrimp Litopenaeus vannamei reared at stocking densities of 2, 10, 20, 30, and 40 shrimp L(-1) after 3, 6, and 12 h. All immune parameters including haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, lysozyme activity, and haemolymph protein were negatively related to density and time. The PO activity, SOD activity, and lysozyme activity of shrimp reared at 10 shrimp L(-1) after 12 h significantly decreased. The transcript levels of these immune-related proteins were down-regulated in shrimp reared at 20, 30, and 40 shrimp L(-1) after 12 h. Phagocytic activity and clearance efficiency to Vibrio alginolyticus were significantly lower in shrimp reared at 30 and 40 shrimp L(-1) after 12 h. The mortality rates of shrimp reared at 20 and 40 shrimp L(-1) were significantly higher than shrimp reared at 2 shrimp L(-1) over 12-144 h and 12-48 h, respectively. Shrimp reared at high densities (>10 shrimp L(-1)) exhibited decreased resistance against pathogens as evidenced by reductions in immune parameters together with decreased expression levels of immune-related proteins, indicating perturbations of the immune system. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metal-chelating active packaging film enhances lysozyme inhibition of Listeria monocytogenes.

PubMed

Roman, Maxine J; Decker, Eric A; Goddard, Julie M

2014-07-01

Several studies have demonstrated that metal chelators enhance the antimicrobial activity of lysozyme. This study examined the effect of metal-chelating active packaging film on the antimicrobial activity of lysozyme against Listeria monocytogenes. Polypropylene films were surface modified by photoinitiated graft polymerization of acrylic acid (PP-g-PAA) from the food contact surface of the films to impart chelating activity based on electrostatic interactions. PP-g-PAA exhibited a carboxylic acid density of 113 ± 5.4 nmol cm(-2) and an iron chelating activity of 53.7 ± 9.8 nmol cm(-2). The antimicrobial interaction of lysozyme and PP-g-PAA depended on growth media composition. PP-g-PAA hindered lysozyme activity at low ionic strength (2.48-log increase at 64.4 mM total ionic strength) and enhanced lysozyme activity at moderate ionic strength (5.22-log reduction at 120 mM total ionic strength). These data support the hypothesis that at neutral pH, synergy between carboxylate metal-chelating films (pKa(bulk) 6.45) and lysozyme (pI 11.35) is optimal in solutions of moderate to high ionic strength to minimize undesirable charge interactions, such as lysozyme absorption onto film. These findings suggest that active packaging, which chelates metal ions based on ligand-specific interactions, in contrast to electrostatic interactions, may improve antimicrobial synergy. This work demonstrates the potential application of metal-chelating active packaging films to enhance the antimicrobial activity of membrane-disrupting antimicrobials, such as lysozyme.

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.

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

PubMed

Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

2015-07-01

To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Coal dust contiguity-induced changes in the concentration of TNF- and NF- B p65 on the ocular surface

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

Sun, Z.Y.; Hong, J.; Liu, Z.Y.

2009-07-01

To observe the influence of coal dust on ocular surface of coal miners and rabbits with coal dust contiguity on expression TNF- and NF- Bp65 and dry eye occurrence. Expression TNF- and NF- Bp65 in ocular surface were determined. Results showed tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Coal dust exposure was linked to development of xerophthalmia, and induced a higher expression of NF- B p65 and TNF- perhaps as a mechanism to resist coal dust ocular surface injury.

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.

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

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

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

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

Salivary lysozyme in smoking alcohol dependent persons.

PubMed

Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Zalewska, Anna; Waszkiewicz, Magdalena; Szajda, Slawomir Dariusz; Repka, Bernadeta; Szulc, Agata; Kepka, Alina; Minarowska, Alina; Ladny, Jerzy Robert; Zwierz, Krzysztof

2012-01-01

The purpose of this study was to evaluate the effect of chronic alcohol intoxication and smoking on the concentration and output of salivary lysozyme. Thirty seven men participated in the study, including 17 male smoking alcohol-dependent patients after chronic alcohol intoxication (AS), and 20 control non-smoking male social drinkers (CNS) with no history of alcohol abuse or smoking. The level of lysozyme was assessed by the radial immunodiffusion method. Significantly lower lysozyme output in the AS group compared to the CNS group was found. Moreover, gingival index was significantly higher in AS than in the CNS group. It appeared that the reduced salivary lysozyme output was more likely the result of ethanol action than smoking. In conclusion, persons addicted to alcohol and nicotine have a poorer periodontal status than non-smoking social drinkers, which may partially be due to the diminished protective effects of lysozyme present in the saliva.

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

PubMed

Altamar-Ríos, J

1990-01-01

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

Determination of monomer concentrations in crystallizing lysozyme solutions

NASA Technical Reports Server (NTRS)

Wilson, L. J.; Pusey, Marc L.

1992-01-01

We have developed a non-optical technique for the study of aggregation in lysozyme and other protein solutions. By monitoring the rate at which lysozyme traverses a semipermeable membrane it was possible to quantitate the degree of aggregation in supersaturated solutions. Using this technique, we have measured the concentration of monomers and larger aggregates in under- and oversaturated lysozyme solutions, and in the presence of crystals, at pH 4.0 and 3 percent NaCl (0.1M NaAc). Comparison of these concentration profiles with (110) face growth rate data supports the theory that tetragonal lysozyme crystals grow by addition of preformed aggregates and not by monomer addition. The data suggest that a considerable population of aggregates larger than dimers are present at lysozyme concentrations above 22 mg/ml. Determination of dimer concentrations, and equilibrium constants for subsequent aggregation levels, are currently underway.

Transciptomic study of mucosal immune, antioxidant and growth related genes and non-specific immune response of common carp (Cyprinus carpio) fed dietary Ferula (Ferula assafoetida).

PubMed

Safari, Roghieh; Hoseinifar, Seyed Hossein; Nejadmoghadam, Shabnam; Jafar, Ali

2016-08-01

A 8-weeks feeding trial was conducted to examine the effects of different levels (0, 0.5, 1 and 2%) of dietary Ferula (Ferula assafoetida) on expression of antioxidant enzymes (GSR, GPX and GSTA), immune (TNF-alpha, IL1B, IL- 8 and LYZ) and growth (GH, IGF1 and Ghrl) genes as well as cutaneous mucus and serum non-specific immune response in common carp. The results revealed Ferula significantly increased antioxidant gene expression (GSR and GSTA) in a dose dependent manner (P < 0.05). The expression of immune growth related genes were significantly higher in Ferula fed fish compared control group (P < 0.05). The effects of Ferula on expression of genes was more pronounced in higher doses. Feeding on Ferula supplemented diet remarkably increased skin mucus lysozyme activity (P < 0.05). However, evaluation of mucus total Ig and protease activity revealed no significant difference between control and treated groups (P > 0.05). Regarding non-specific humoral response, serum total Ig, lysozyme and ACH50 showed no remarkable variation between Ferula fed carps and control group (P > 0.05). These results indicated up-regulation of growth and health related genes in Ferula fed common carp. Further studies using pathogen or stress challenge is required to conclude that transcriptional modulation is beneficial in common carp. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative proteomics and expression analysis of five genes in Epicauta chinensis larvae from the first to fifth instar.

PubMed

Li, Qiurong; Wang, Dun; Lv, Shumin; Zhang, Yalin

2014-01-01

Blister beetle is an important insect model for both medicinal and pure research. Previous research has mainly focused on its biology and biochemistry, but very little data is yet available in the molecular biology. This study uses differential proteomics technology to analyze the soluble proteins extracted from each of the 5 instars larvae of Epicauta chinensis. 42 of the differentially-expressed proteins were identified successfully by MALDI-TOF/TOF-MS. Some of these proteins' function and their expression profiles are analyzed. Our analysis revealed dynamics regulation of the following proteins: Axin-like protein pry-1 (APR-1), dihydrolipoyl dehydrogenase (DLD), vitellogenin (Vg) and lysozyme C (Lmz-S). APR-1 negatively regulates the Wnt signaling pathway. Its overexpression could result in embryo, leg, eye and ovary ectopica or malformation. DLD catalyzes the pyruvate into acetyl-CoA, the latter is the starting material of juvenile hormone (JH) and ipsdienol biosynthesis through the MVA pathway in insects. While Vg synthesis can be regulated by JH and stimulated by food factors. So DLD may affect the synthesis of JH, ipsdienol and Vg indirectly. The activity of lysozyme is an indicator of the immunity. Nutrition/food should be taken into account for its potential role during the development of larva in the future. Among the five genes and their corresponding proteins' expression, only hsc70 gene showed a good correspondence with the protein level. This reflects the fluctuating relationship between mRNA and protein levels.

The Preventive Effect of L-Lysine on Lysozyme Glycation in Type 2 Diabetes.

PubMed

Mirmiranpour, Hossein; Khaghani, Shahnaz; Bathaie, S Zahra; Nakhjavani, Manouchehr; Kebriaeezadeh, Abbas; Ebadi, Maryam; Gerayesh-Nejad, Siavash; Zangooei, Mohammad

2016-01-01

Lysozyme is a bactericidal enzyme whose structure and functions change in diabetes. Chemical chaperones are small molecules including polyamines (e.g. spermine), amino acids (e.g. L-lysine) and polyols (e.g. glycerol). They can improve protein conformation in several stressful conditions such as glycation. In this study, the authors aimed to observe the effect of L-lysine as a chemical chaperone on structure and function of glycated lysozyme. In this study, in vitro and in vivo effects of L-lysine on lysozyme glycation were investigated. Lysozyme was incubated with glucose and/or L-lysine, followed by an investigation of its structure by electrophoresis, fluorescence spectroscopy, and circular dichroism spectroscopy and also assessment of its bactericidal activity against M. lysodeikticus. In the clinical trial, patients with type 2 diabetes mellitus (T2DM) were randomly divided into two groups of 25 (test and control). All patients received metformin and glibenclamide for a three months period. The test group was supplemented with 3 g/day of L-lysine. The quantity and activity of lysozyme and other parameters were then measured. Among the test group, L-lysine was found to reduce the advanced glycation end products (AGEs) in the sera of patients with T2DM and in vitro condition. This chemical chaperone reversed the alteration in lysozyme structure and function due to glycation and resulted in increased lysozyme activity. Structure and function of glycated lysozyme are significantly improved by l-lysine; therefore it can be considered an effective therapeutic supplementation in T2DM, decreasing the risk of infection in these patients.

B-Lymphocytes Expressing an Ig Specificity Recognizing the Pancreatic β-Cell Autoantigen Peripherin Are Potent Contributors to Type 1 Diabetes Development in NOD Mice

PubMed Central

Leeth, Caroline M.; Racine, Jeremy; Chapman, Harold D.; Arpa, Berta; Carrillo, Jorge; Carrascal, Jorge; Wang, Qiming; Ratiu, Jeremy; Egia-Mendikute, Leire; Rosell-Mases, Estela; Stratmann, Thomas

2016-01-01

Although the autoimmune destruction of pancreatic β-cells underlying type 1 diabetes (T1D) development is ultimately mediated by T cells in NOD mice and also likely in humans, B cells play an additional key pathogenic role. It appears that the expression of plasma membrane–bound Ig molecules that efficiently capture β-cell antigens allows autoreactive B cells that bypass normal tolerance induction processes to be the subset of antigen-presenting cells most efficiently activating diabetogenic T cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or are not (hen egg lysozyme [HEL]) expressed by β-cells have proven useful in dissecting the developmental basis of diabetogenic B cells. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B cells in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin-autoreactive B cells infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach. PMID:26961115

Low-frequency vibrational properties of lysozyme in sugar aqueous solutions: A Raman scattering and molecular dynamics simulation study

NASA Astrophysics Data System (ADS)

Lerbret, A.; Affouard, F.; Bordat, P.; Hédoux, A.; Guinet, Y.; Descamps, M.

2009-12-01

The low-frequency (ω <400 cm-1) vibrational properties of lysozyme in aqueous solutions of three well-known protecting sugars, namely, trehalose, maltose, and sucrose, have been investigated by means of complementary Raman scattering experiments and molecular dynamics simulations. The comparison of the Raman susceptibility χ″(ω) of lysozyme/water and lysozyme/sugar/water solutions at a concentration of 40 wt % with the χ″ of dry lysozyme suggests that the protein dynamics mostly appears in the broad peak around 60-80 cm-1 that reflects the vibrations experienced by atoms within the cage formed by their neighbors, whereas the broad shoulder around 170 cm-1 mainly stems from the intermolecular O-H⋯O stretching vibrations of water. The addition of sugars essentially induces a significant high frequency shift and intensity reduction of this band that reveal a slowing down of water dynamics and a distortion of the tetrahedral hydrogen bond network of water, respectively. Furthermore, the lysozyme vibrational densities of states (VDOS) have been determined from simulations of lysozyme in 37-60 wt % disaccharide aqueous solutions. They exhibit an additional broad peak around 290 cm-1, in line with the VDOS of globular proteins obtained in neutron scattering experiments. The influence of sugars on the computed VDOS mostly appears on the first peak as a slight high-frequency shift and intensity reduction in the low-frequency range (ω <50 cm-1), which increase with the sugar concentration and with the exposition of protein residues to the solvent. These results suggest that sugars stiffen the environment experienced by lysozyme atoms, thereby counteracting the softening of protein vibrational modes upon denaturation, observed at high temperature in the Raman susceptibility of the lysozyme/water solution and in the computed VDOS of unfolded lysozyme in water. Finally, the Raman susceptibility of sugar/water solutions and the calculated VDOS of water in the different lysozyme solutions confirm that sugars induce a significant strengthening of the hydrogen bond network of water that may stabilize proteins at high temperatures.

Binding study of lysozyme with Al(III) using chemiluminescence analysis.

PubMed

Liu, Jiangman; Luo, Kai; Song, Zhenghua

2014-09-01

The binding behavior of lysozyme with Al(III) is described using luminol as a luminescence probe by flow injection-chemiluminescence (FI-CL) analysis. It was found that the CL intensity of the luminol-lysozyme reaction could be markedly enhanced by Al(III), and the increase in CL intensity was linear with the Al(III) concentration over the range 0.3-30.0  pg  mL(-1) , with a detection limit of 0.1 pg  mL(-1) (3σ). Based on the interaction model of lysozyme with Al(III), lg[(I - I0 )/(2I0  - I)] = lgK + nlg[M], the binding constant K = 6.84 × 10(6)  L mol(-1) and the number of binding sites (n) = 0.76. The relative standard deviations were 3.2, 2.4 and 2.0% for 10.0, 20.0 and 30.0  pg  mL(-1) Al(III) (n = 7), respectively. This new method was successfully applied to continuous, quantitative monitoring of picogram level Al(III) in human saliva following oral intake of compound aluminum hydroxide tablets. It was found that Al(III) in saliva reached a maximum of 101.2  ng  mL(-1) at 3.0 h. The absorption rate constant ka , elimination rate constant k and half-life time t1/2 of Al(III) were 1.378  h(-1) , 0.264  h(-1) and 2.624  h, respectively. Copyright © 2013 John Wiley & Sons, Ltd.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

PubMed Central

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A.; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P.

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches. PMID:29423380

Proteflazid® and local immunity in diseases caused by human papillomavirus, herpesvirus and mixed urogenital infections.

PubMed

Kaminsky, Vjacheslav; Chernyshov, Viktor; Grynevych, Oleksandr; Benyuk, Vasil; Kornatskaya, Alla; Shalko, Miroslava; Usevich, Igor; Revenko, Oleg; Shepetko, Maxim; Solomakha, Ludmila

2017-03-21

Reporting of clinical trials results for Proteflazid® in the drug formulation suppositories and vaginal swabs soaked in the solution of the drug to the local immunity of the female reproductive tract. The aim of study was to examine the state of local immunity in the reproductive tract of women with sexually transmitted diseases caused by human papillomavirus, herpes viruses (Type 1, 2) and mixed infection (herpes viruses + chlamydia). The trials involved 216 women with viral sexually transmitted diseases: Cervical Dysplasia associated with papillomavirus infection (HPV) (Group 1); Herpes genitalis type 1 (HSV- 1) and type 2 (HSV-1) (Group 2); mixed infection - HSV-1, HSV-2 and chlamydia (Group 3). Treatment results have confirmed that Proteflazid® contributes to sustainable performance improvement of basic factors of local immunity - sIgA, lysozyme and complement component C3 in the cervical mucus for all three groups of women. Proteflazid® enhances level of local immunity markers (sIgA, lysozyme, C3 complement component) and improves their ratios. Also it intensifies anticontagious activity of mucosal protection and female reproductive system as whole, during treatment diseases caused by human papillomavirus, herpesvirus and mixed urogenital infections (herpesvirus and chlamydia).

The Effect of Lysozyme on Reducing Biofilms by Staphylococcus aureus, Pseudomonas aeruginosa, and Gardnerella vaginalis: An In Vitro Examination.

PubMed

Hukić, Mirsada; Seljmo, Dzenita; Ramovic, Amra; Ibrišimović, Monia Avdić; Dogan, Serkan; Hukic, Jasna; Bojic, Elma Feric

2018-05-01

Two basic questions about lysozyme activities on the selected microorganisms were investigated, namely whether lysozyme inhibits biofilm production and which concentrations of the enzyme have the ability to change the natural biofilm producing capacity of different strains of Staphylococcus aureus (methicillin sensitive and resistant), Streptococcus pyogenes, Pseudomonas aeruginosa, and Gardnerella vaginalis. The effect of lysozyme on biofilm formation capacities of 16 strains of selected microorganisms was investigated, whereby four testing replicates have been performed in vitro using the Test Tube method, and the potential of lysozyme to change biofilm forming capacities depending on its concentration, species, and strains of microorganisms is demonstrated. A lysozyme concentration of 30 μg/ml indicated to have the highest inhibiting effect on all tested microorganisms. Furthermore, G. vaginalis was the most sensitive of them all, as its biofilm formation was inhibited in the presence of as low as 2.5 μg/ml of lysozyme. At enzyme concentrations of 7.5-50 μg/ml (with the exception of 30 μg/ml) the biofilm forming capacities of P. aeruginosa were enhanced. Depending on the strain of P. aeruginosa, the total biofilm quantity was either reduced or unaffected at lysozyme concentrations of 2.5, 5, 7.5, and 30 μg/ml. In contrast, lysozyme concentrations below 15 or 20 μg/ml did not affect or increase the volume of biofilm formation, while higher concentrations (15, 20, 25 μg/ml) reduced biofilm formation by 50% (3/6) and 30 μg/ml of biofilm reduced biofilm forming capacity of S. aureus by 100% (6/6). The results of this study are a strong foundation for further research on lysozyme as a modulator of the biofilm forming capacity of different species with the potential to aid in the development of new drugs for the treatment of oral and vaginal infections.

Theoretical analysis of bimetallic nanorod dimer biosensors for label-free molecule detection

NASA Astrophysics Data System (ADS)

Das, Avijit; Talukder, Muhammad Anisuzzaman

2018-02-01

In this work, we theoretically analyze a gold (Au) core within silver (Ag) shell (Au@Ag) nanorod dimer biosensor for label-free molecule detection. The incident light on an Au@Ag nanorod strongly couples to localized surface plasmon modes, especially around the tip region. The field enhancement around the tip of a nanorod or between the tips of two longitudinally aligned nanorods as in a dimer can be exploited for sensitive detection of biomolecules. We derive analytical expressions for the interactions of an Au@Ag nanorod dimer with the incident light. We also study the detail dynamics of an Au@Ag nanorod dimer with the incident light computationally using finite difference time domain (FDTD) technique when core-shell ratio, relative position of the nanorods, and angle of incidence of light change. We find that the results obtained using the developed analytical model match well with that obtained using FDTD simulations. Additionally, we investigate the sensitivity of the Au@Ag nanorod dimer, i.e., shift in the resonance wavelength, when a target biomolecule such as lysozyme (Lys), human serum albumin (HSA), anti-biotin (Abn), human catalase (CAT), and human fibrinogen (Fb) protein molecules are attached to the tips of the nanorods.

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

PubMed Central

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

2015-01-01

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

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

Regenerated cellulose fiber and film immobilized with lysozyme

USDA-ARS?s Scientific Manuscript database

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...

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

PubMed

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

2013-01-01

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

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions.

PubMed

Cheng, Yu-Chia; Lobo, Raul F; Sandler, Stanley I; Lenhoff, Abraham M

2006-05-05

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Interaction of Lysozyme with Rhodamine B: A combined analysis of spectroscopic & molecular docking.

PubMed

Millan, Sabera; Satish, Lakkoji; Kesh, Sandeep; Chaudhary, Yatendra S; Sahoo, Harekrushna

2016-09-01

The interaction of Rhodamine B (RB) with Lysozyme (Lys) was investigated by different optical spectroscopic techniques such as absorption, fluorescence, and circular-dichroism (CD), along with molecular docking studies. The fluorescence results (including steady-state and time-resolved mode) revealed that the addition of RB effectively causes strong quenching of intrinsic fluorescence in Lysozyme and mostly, by the static quenching mechanism. Different binding and thermodynamic parameters were calculated at different temperatures and the binding constant value was found to be 2963.54Lmol(-1) at 25°C. The average distance (r0) was found to be 3.31nm according to Förster's theory of non-radiative energy transfer between Lysozyme and RB. The conformational change in Lysozyme during interaction with RB was confirmed from absorbance, synchronous fluorescence, and circular dichroism measurements. Finally, molecular docking studies were done to confirm that the dye binds with Lysozyme. Copyright © 2016 Elsevier B.V. All rights reserved.

Laboratory multiple-crystal X-ray topography and reciprocal-space mapping of protein crystals: influence of impurities on crystal perfection

NASA Technical Reports Server (NTRS)

Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

2001-01-01

Double-axis multiple-crystal X-ray topography, rocking-curve measurements and triple-axis reciprocal-space mapping have been combined to characterize protein crystals using a laboratory source. Crystals of lysozyme and lysozyme crystals doped with acetylated lysozyme impurities were examined. It was shown that the incorporation of acetylated lysozyme into crystals of lysozyme induces mosaic domains that are responsible for the broadening and/or splitting of rocking curves and diffraction-space maps along the direction normal to the reciprocal-lattice vector, while the overall elastic lattice strain of the impurity-doped crystals does not appear to be appreciable in high angular resolution reciprocal-space maps. Multiple-crystal monochromatic X-ray topography, which is highly sensitive to lattice distortions, was used to reveal the spatial distribution of mosaic domains in crystals which correlates with the diffraction features in reciprocal space. Discussions of the influence of acetylated lysozyme on crystal perfection are given in terms of our observations.

Laboratory multiple-crystal X-ray topography and reciprocal-space mapping of protein crystals: influence of impurities on crystal perfection.

PubMed

Hu, Z W; Thomas, B R; Chernov, A A

2001-06-01

Double-axis multiple-crystal X-ray topography, rocking-curve measurements and triple-axis reciprocal-space mapping have been combined to characterize protein crystals using a laboratory source. Crystals of lysozyme and lysozyme crystals doped with acetylated lysozyme impurities were examined. It was shown that the incorporation of acetylated lysozyme into crystals of lysozyme induces mosaic domains that are responsible for the broadening and/or splitting of rocking curves and diffraction-space maps along the direction normal to the reciprocal-lattice vector, while the overall elastic lattice strain of the impurity-doped crystals does not appear to be appreciable in high angular resolution reciprocal-space maps. Multiple-crystal monochromatic X-ray topography, which is highly sensitive to lattice distortions, was used to reveal the spatial distribution of mosaic domains in crystals which correlates with the diffraction features in reciprocal space. Discussions of the influence of acetylated lysozyme on crystal perfection are given in terms of our observations.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protein deposition on a lathe-cut silicone hydrogel contact lens material.

PubMed

Subbaraman, Lakshman N; Woods, Jill; Teichroeb, Jonathan H; Jones, Lyndon

2009-03-01

To determine the quantity of total protein, total lysozyme, and the conformational state of lysozyme deposited on a novel, lathe-cut silicone hydrogel (SiHy) contact lens material (sifilcon A) after 3 months of wear. Twenty-four subjects completed a prospective, bilateral, daily-wear, 9-month clinical evaluation in which the subjects were fitted with a novel, custom-made, lathe-cut SiHy lens material. The lenses were worn for three consecutive 3-month periods, with lenses being replaced after each period of wear. After 3 months of wear, the lenses from the left eye were collected and assessed for protein analysis. The total protein deposited on the lenses was determined by a modified Bradford assay, total lysozyme using Western blotting and the lysozyme activity was determined using a modified micrococcal assay. The total protein recovered from the custom-made lenses was 5.3 +/- 2.3 microg/lens and the total lysozyme was 2.4 +/- 1.2 microg/lens. The denatured lysozyme found on the lenses was 1.9 +/- 1.0 microg/lens and the percentage of lysozyme denatured was 80 +/- 10%. Even after 3 months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lens materials was very similar to that found on similar surface-coated SiHy lenses after 2 to 4 weeks of wear. These results indicate that extended use of the sifilcon A material is not deleterious in terms of the quantity and quality of protein deposited on the lens.

An electron microscopy study of the diversity of Streptococcus sanguinis cells induced by lysozyme in vitro.

PubMed

Hao, Yuqing; Li, Li; Li, Wei; Zhou, Xuedong; Lu, Junjun

2010-01-01

Bacterial virulence could be altered by the antimicrobial agents of the host. Our aim was to identify the damage and survival of Streptococcus sanguinis induced by lysozymes in vitro and to analyse the potential of oral microorganisms to shirk host defences, which cause infective endocarditis. S. sanguinis ATCC 10556 received lysozyme at concentrations of 12.5, 25, 50 and 100 microg/ml. Cells were examined by electron microscopy. The survival was assessed by colony counting and construction of a growth curve. Challenged by lysozymes, cells mainly exhibited cell wall damage, which seemed to increase with increasing lysozyme concentration and longer incubation period in the presence of ions. Cells with little as well as apparent lesion were observed under the same treatment set, and anomalous stick and huge rotund bodies were occasionally observed. After the removal of the lysozyme, some damaged cells could be reverted to its original form with brain heart infusion (BHI), and their growth curve was similar to the control cells. After further incubation in BHI containing lysozyme, S. sanguinis cell damage stopped progressing, and their growth curve was also similar to the control cells. The results suggested that the S. sanguinis lesions caused by the lysozyme in the oral cavity may be nonhomogeneous and that some damaged cells could self-repair and survive. It also indicated that S. sanguinis with damaged cell walls may survive and be transmitted in the bloodstream.

Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

PubMed Central

Mechaly, Ariel E.; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M.; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general. PMID:24823716

Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

PubMed

Correa, Agustín; Pacheco, Sabino; Mechaly, Ariel E; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

The VraSR regulatory system contributes to virulence in Streptococcus suis via resistance to innate immune defenses

PubMed Central

Chang, Peixi; Li, Weitian; Shi, Guolin; Li, Huan; Yang, Xiaoqing; Xia, Zechen; Ren, Yuan; Li, Zhiwei; Chen, Huanchun; Bei, Weicheng

2018-01-01

ABSTRACT Streptococcus suis is a highly invasive pathogen that can cause sepsis and meningitis in pigs and humans. However, we have limited understanding of the mechanisms S. suis uses to evade innate immunity. To investigate the involvement of the two-component signal transduction system of S. suis in host immune defense, we examined the expression of 15 response regulators of S. suis following stimulation with polymorphonuclear leukocytes (PMNs). We found that several response regulators were significantly up-regulated including vraR. Thus, we constructed an isogenic deletion mutant of vraSR genes in S. suis and demonstrated VraSR promotes both bacterial survival in human blood and resistance to human PMN-mediated killing. The VraSR mutant was more susceptible to phagocytosis by human PMNs and had greater sensitivity to oxidant and lysozyme than wild-type S. suis. Furthermore, in vitro findings and in vivo evidence from a mouse infection model together strongly demonstrate that ΔvraSR had greatly attenuated virulence compared with wild-type S. suis. Collectively, our data reveal that VraSR is a critical regulatory system that contributes to the survival of S. suis and its ability to defend against host innate immunity. PMID:29471718

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

NASA Astrophysics Data System (ADS)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

PubMed

Shao, Cui-Ying; Secombes, Chris J; Porter, Andrew J

2007-01-01

The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus

PubMed Central

Kobayashi, Kazuya; Matsuura, Kenji

2017-01-01

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole. PMID:28410430

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus.

PubMed

Mitaka, Yuki; Kobayashi, Kazuya; Matsuura, Kenji

2017-01-01

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

Small angle neutron scattering study on the structural variation of lysozyme in bioprotectants

NASA Astrophysics Data System (ADS)

Koda, Shota; Takayama, Haruki; Shibata, Tomohiko; Mori, Tatsuya; Kojima, Seiji; Park, In-Sung; Shin, Tae-Gyu

2015-05-01

The thermal denaturation and subsequent structural variation of lysozyme in various bioprotectant candidate solutions such as trehalose and choline acetate have been investigated by using small angle neutron scattering and differential scanning calorimetry. The gyration radius shows little change with the addition of additives in a native state at room temperature. On heating the lysozyme solution, a remarkable increase in the gyration radius is observed at temperatures above the denaturation temperature without any bioprotectants. Such an increase is suppressed by the additives owing to the intermolecular interactions between the lysozyme molecules and the bioprotectants of trehalose and choline acetate. The fractal dimension of lysozyme varies slightly with the addition of the bioprotectant solutions, and shows a remarkable drop in the vicinity of the denaturation temperature for all the solutions.

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

NASA Astrophysics Data System (ADS)

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

2014-11-01

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

Resolving Single Molecule Lysozyme Dynamics with a Carbon Nanotube Electronic Circuit

NASA Astrophysics Data System (ADS)

Choi, Yongki; Moody, Issa S.; Perez, Israel; Sheps, Tatyana; Weiss, Gregory A.; Collins, Philip G.

2011-03-01

High resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing.

Isolation and characterization of a c-type lysozyme from the nurse shark.

PubMed

Hinds Vaughan, Nichole; Smith, Sylvia L

2013-12-01

Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-06-01

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

Biological and Clinical Implications of Lysozyme Deposition on Soft Contact Lenses

PubMed Central

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

2015-01-01

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

Convective flow effects on protein crystal growth

NASA Technical Reports Server (NTRS)

Rosenberger, Franz; Monaco, Lisa A.

1995-01-01

During the fourth semi-annual period under this grant we have pursued the following activities: (1) crystal growth morphology and kinetics studies with tetragonal lysozyme. These clearly revealed the influence of higher molecular weight protein impurities on interface shape; (2) characterization of the purity and further purification of lysozyme solutions. These efforts have, for the first time, resulted in lysozyme free of higher molecular weight components; (3) continuation of the salt repartitioning studies with Seikagaku lysozyme, which has a lower protein impurity content that Sigma stock. These efforts confirmed our earlier findings of higher salt contents in smaller crystals. However, less salt is in corporated into the crystals grown from Seikagaku stock. This strongly suggests a dependence of salt repartitioning on the concentration of protein impurities in lysozyme. To test this hypothesis, repartitioning studies with the high purity lysozyme prepared in-house will be begun shortly; (4) numerical modelling of the interaction between bulk transport and interface kinetics. These simulations have produced interface shapes which are in good agreement with out experimental observations; and (5) light scattering studies on under- and supersaturated lysozyme solutions. A consistent interpretation of the static and dynamic data leaves little doubt that pre-nucleation clusters, claimed to exist even in undersaturated solutions, are not present. The article: 'Growth morphology response to nutrient and impurity nonuniformities' is attached.

Human Milk-Treatment and Quality of Banked Human Milk.

PubMed

Picaud, Jean-Charles; Buffin, Rachel

2017-03-01

The aim of human milk banks is to deliver safe and high quality donor human milk. Treatment of human milk has to destroy most microorganisms while preserving immunological and nutrient components, which is obtained when using low time low temperature pasteurization. However it destroys bile-simulated lipase, reduces lactoferrin, lysozyme, immunoglobulins, and bactericidal capacity of human milk. New methods are under investigation such as high temperature short time pasteurization, high pressure processing, or ultraviolet irradiation. They have been tested in experimental conditions and there are promising results, but they have to be tested in real conditions in human milk bank. Copyright © 2016 Elsevier Inc. All rights reserved.

Dietary sodium propionate affects mucosal immune parameters, growth and appetite related genes expression: Insights from zebrafish model.

PubMed

Hoseinifar, Seyed Hossein; Safari, Roghieh; Dadar, Maryam

2017-03-01

Propionate is a short-chain fatty acid (SCFA) that improves physiological and pathophysiological properties. However, there is limited information available about the effects of SCFAs on mucosal immune parameters as well as growth and appetite related genes expression. The aim of the present study was to evaluate the effect of sodium propionate (SP) intake on the mucosal immune parameters, growth and appetite related genes expression using zebrafish (Danio rerio) as model organism. Zebrafish fed control or diet supplemented with different levels (0.5, 1 and 2%) of SP for 8weeks. At the end of feeding trial, the expression of the key genes related to growth and appetite (GH, IGF1, MYSTN and Ghrl) was evaluated. Also, mucosal immune parameters (Total Ig, lysozyme and protease activity) were studied in skin mucus of zebrafish. The results showed that dietary administration of SP significantly (P<0.05) up-regulated the expression of GH, IGF1 and down-regulated MYSTN gene. Also, feeding zebrafish with SP supplemented diet significantly increased appetite related gene expression (P<0.05) with a more pronounced effect in higher inclusion levels. Compared with control group, the expression of appetite related gene (Ghrl) was remarkably (P<0.05) higher in SP fed zebrafish. Also, elevated mucosal immune parameters was observed in zebrafish fed SP supplemented diet. The present results revealed beneficial effects of dietary SP on mucosal immune response and growth and appetite related genes expression. These results also highlighted the potential use of SP as additive in human diets. Copyright © 2016 Elsevier Inc. All rights reserved.

Innate immune parameters and haemolymph protein expression profile to evaluate the immunotoxicity of tributyltin on abalone (Haliotis diversicolor supertexta).

PubMed

Zhou, Jin; Cai, Zhong-Hua; Zhu, Xiao-Shan; Li, Lei; Gao, Yun-Feng

2010-10-01

The immunotoxicity of tributyltin (TBT) on marine gastropods has been comparatively little studied although risks to wildlife associated with this compound are well known. In this study, a 30-day trial was conducted to evaluate the immunotoxic effects on abalone (Haliotis diversicolor supertexta) by exposing a range of doses of TBT (0, 2, 10, and 50 ng/L). Innate immune parameters, including phagocytic ability (PA), lysozyme activity, phenoloxidase (PO) level and superoxide dismutase (SOD) activity were monitored at intervals of 5, 15 and 30 days. Haemolymph protein expression profile was also examined at the end of the experiment. The results showed that PA value, lysozyme activity and PO level significantly decreased compared with the controls (P < 0.05), which indicated that TBT exposure markedly suppressed non-specific immune competence. Exposure to TBT also caused variation in protein expression patterns of haemolymph. Among the protein spots of differential expressions, seven proteins from the haemolymph of TBT-treated abalone were successfully identified by MALDI-TOF-MS analysis. Three protein spots increased and were identified as carrier-like peptide, peroxidase 21 precursor and creatine phosphokinase. These proteins are believed to up-regulate in expression as a response to detoxification and antioxidative stress mechanisms. The other four protein spots that down-regulated in TBT-treated groups were identified as aromatase-like protein, protein kinase C, ceruloplasmin and microtubule-actin crosslinking factor 1, and these proteins play an important role in endocrine regulation and immune defense. Taken together, the results demonstrate that TBT impair abalone immunological ability and is a potential immune disruptor. 2010 Elsevier Ltd. All rights reserved.

Structure and Interaction in the pH-Dependent Phase Behavior of Nanoparticle-Protein Systems.

PubMed

Yadav, Indresh; Kumar, Sugam; Aswal, Vinod K; Kohlbrecher, Joachim

2017-02-07

The pH-dependent structure and interaction of anionic silica nanoparticles (diameter 18 nm) with two globular model proteins, lysozyme and bovine serum albumin (BSA), have been studied. Cationic lysozyme adsorbs strongly on the nanoparticles, and the adsorption follows exponential growth as a function of lysozyme concentration, where the saturation value increases as pH approaches the isoelectric point (IEP) of lysozyme. By contrast, irrespective of pH, anionic BSA does not show any adsorption. Despite having a different nature of interactions, both proteins render a similar phase behavior where nanoparticle-protein systems transform from being one-phase (clear) to two-phase (turbid) above a critical protein concentration (CPC). The measurements have been carried out for a fixed concentration of silica nanoparticles (1 wt %) with varying protein concentrations (0-5 wt %). The CPC is found to be much higher for BSA than for lysozyme and increases for lysozyme but decreases for BSA as pH approaches their respective IEPs. The structure and interaction in these systems have been examined using dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The effective hydrodynamic size of the nanoparticles measured using DLS increases with protein concentration and is related to the aggregation of the nanoparticles above the CPC. The propensity of the nanoparticles to aggregate is suppressed for lysozyme and enhanced for BSA as pH approached their respective IEPs. This behavior is understood from SANS data through the interaction potential determined by the interplay of electrostatic repulsion with a short-range attraction for lysozyme and long-range attraction for BSA. The nanoparticle aggregation is caused by charge neutralization by the oppositely charged lysozyme and through depletion for similarly charged BSA. Lysozyme-mediated attractive interaction decreases as pH approaches the IEP because of a decrease in the charge on the protein. In the case of BSA, a decrease in the BSA-BSA repulsion enhances the depletion attraction between the nanoparticles as pH is shifted toward the IEP. The morphology of the nanoparticle aggregates is found to be mass fractal.

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

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

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

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

PubMed

Primo, Emiliano D; Otero, Lisandro H; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

Self perceived work related stress and the relation with salivary IgA and lysozyme among emergency department nurses

PubMed Central

Yang, Y; Koh, D; Ng, V; Lee, C; Chan, G; Dong, F; Goh, S; Anantharaman, V; Chia, S

2002-01-01

Aims: To assess and compare the self perceived work related stress among emergency department (ED) and general ward (GW) nurses, and to investigate its relation with salivary IgA and lysozyme. Methods: One hundred and thirty two of 208 (63.5%) registered female ED and GW nurses participated in the study. A modified mental health professional stress scale (PSS) was used to measure self perceived stress. ELISA methods were used to determine the salivary IgA and lysozyme levels. Results: On PSS, ED nurses had higher scores (mean 1.51) than GW nurses (1.30). The scores of PSS subscales such as organisational structure and processes (OS), lack of resources (RES), and conflict with other professionals (COF) were higher in ED than in GW nurses. ED nurses had lower secretion rates of IgA (geometric mean (GM) 49.1 µg/min) and lysozyme (GM 20.0 µg/min) than GW nurses (68.2 µg/min, 30.5 µg/min). Significant correlations were observed between PSS and log IgA and lysozyme secretion rates. OS, RES, and COF were correlated with log IgA and lysozyme levels. Conclusion: ED nurses, who reported a higher level of professional stress, showed significantly lower secretion rates of salivary IgA and lysozyme compared to GW nurses. Salivary IgA and lysozyme were inversely correlated with self perceived work related stress. As these salivary biomarkers are reflective of the mucosal immunity, results support the inverse relation between stress and mucosal immunity. PMID:12468751

Complex coacervate core micelles with a lysozyme-modified corona.

PubMed

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

2007-07-17

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

CdTe/CdSe quantum dot-based fluorescent aptasensor with hemin/G-quadruplex DNzyme for sensitive detection of lysozyme using rolling circle amplification and strand hybridization.

PubMed

Qiu, Zhenli; Shu, Jian; He, Yu; Lin, Zhenzhen; Zhang, Kangyao; Lv, Shuzhen; Tang, Dianping

2017-01-15

Lysozyme with a small monomeric globular enzymatic protein is part of the innate immune system, and its deficiency can cause the increased incidence of disease. Herein, we devise a new signal-enhanced fluorescence aptasensing platform for quantitative screening of lysozyme by coupling with rolling circle amplification (RCA) and strand hybridization reaction, accompanying the assembly of CdTe/CdSe quantum dots (QDs) and hemin/G-quadruplex DNzyme. Initially, target-triggered release of the primer was carried out from DNA duplex via the reaction of the aptamer with the analyte, and the released primer could be then utilized as the template to produce numerous repeated oligonucleotide sequences by the RCA reaction. Following that, the formed long-stranded DNA simultaneously hybridized with the CdTe/CdSe QD-labeled probe and hemin/G-quadruplex DNzyme strand in the system, thereby resulting in the quenching of QD fluorescent signal through the proximity hemin/G-quadruplex DNzyme on the basis of transferring photoexcited conduction band electrons of quantum dots to Fe(III)/Fe(II)-protoporphyrin IX (hemin) complex. Under optimal conditions, the fluorescent signal decreased with the increasing target lysozyme within the dynamic range from 5.0 to 500nM with a detection limit (LOD) of 2.6nM at the 3s blank criterion. Intra-assay and interassay coefficients of variation (CVs) were below 8.5% and 11.5%, respectively. Finally, the system was applied to analyze spiked human serum samples, and the recoveries in all cases were 85-111.9%. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of lysozyme or antibiotics on fecal zoonotic pathogens in nursery pigs

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on zoonotic pathogen shedding in feces in nursery pigs housed without and with an indirect disease challenge. Two replicates of 600 pigs eac...

Formation of hydrogels based on chitosan/alginate for the delivery of lysozyme and their antibacterial activity.

PubMed

Wu, Tiantian; Huang, Jiaqi; Jiang, Yangyang; Hu, Yaqin; Ye, Xingqian; Liu, Donghong; Chen, Jianchu

2018-02-01

Novel hydrogels based on chitosan/sodium alginate (CS-ALG) were prepared to deliver and protect lysozyme while eliminating food-borne microorganisms. These hydrogels were characterized according to the zeta potential, optical microscopy, scanning electron microscopy (SEM), UV-visible spectroscopy (UV-vis), fourier transform infrared (FT-IR), and small-angle X-ray scattering (SAXS). The results demonstrated that the resultant hydrogels were negatively charged and spherical in shape. In addition, the maximum swelling ratio was 45.66±7.62 for CS-ALG hydrogels loaded with lysozyme. The relative activity of the released lysozyme was 87.72±3.96%, indicating that CS-ALG hydrogels are promising matrices for enzyme loading and adsorption. Furthermore, a 100% bacterial clearance rate of CS/ALG loaded with lysozyme was observed to correspond to the superposition effect stimulated by CS and lysozyme, which improved the antibacterial activity against E. coli and S. aureus compared to CS/ALG, suggesting its potential use in the food industry as well as other applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of cevimeline on salivary components in patients with Sjögren syndrome.

PubMed

Suzuki, Kimihiro; Matsumoto, Mitsuyo; Nakashima, Masahiro; Takada, Kunio; Nakanishi, Takashi; Okada, Makoto; Ohsuzu, Fumitaka

2005-05-01

The aim of this study is to clarify the effects of cevimeline on various components in human saliva, such as immunoglobulin A (IgA), lysozyme, alpha-amylase and squamous cell carcinoma (SCC) antigen. Twelve female patients with Sjögren syndrome (SS) and 14 healthy women were enrolled. After the first saliva collection, one capsule (30 mg) of cevimeline was administered to each subject. Saliva was collected again after 90 min. The salivary flow rate and concentration of each component were measured. In both groups the salivary flow rate and amylase concentration were significantly increased by cevimeline. The lysozyme and IgA concentrations did not change significantly in both groups. The SCC antigen concentration did not change significantly in the SS group, but it decreased significantly in the control group. The secretion rates of amylase and IgA showed significant increases in both groups. The secretion rate of lysozyme significantly increased only in the control group, while the secretion rate of SCC significantly increased only in the SS group. Cevimeline augments not only the salivary flow rate but also the secretion rate of some digestive and/or defense factors from infections. It may be beneficial for SS patients to continue taking cevimeline to prevent oral infections, and other serious sequelae. Copyright (c) 2005 S. Karger AG, Basel.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

Binding patterns and structure-affinity relationships of food azo dyes with lysozyme: a multitechnique approach.

PubMed

Peng, Wei; Ding, Fei; Peng, Yu-Kui; Jiang, Yu-Ting; Zhang, Li

2013-12-18

Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the toxicity of food dyes. Results from this work testify that model protein, though an indirect method, provides a more comprehensive profile of the essence of toxicity evaluation of food dyes.

Designer milk.

PubMed

Sabikhi, Latha

2007-01-01

Dairy biotechnology is fast gaining ground in the area of altering milk composition for processing and/or animal and human health by employing nutritional and genetic approaches. Modification of the primary structure of casein, alteration in the lipid profile, increased protein recovery, milk containing nutraceuticals, and replacement for infant formula offer several advantages in the area of processing. Less fat in milk, altered fatty acid profiles to include more healthy fatty acids such as CLA and omega-fats, improved amino acid profiles, more protein, less lactose, and absence of beta-lactoglobulin (beta-LG) are some opportunities of "designing" milk for human health benefits. Transgenic technology has also produced farm animals that secrete in their milk, human lactoferrin, lysozyme, and lipase so as to simulate human milk in terms of quality and quantity of these elements that are protective to infants. Cow milk allergenicity in children could be reduced by eliminating the beta-LG gene from bovines. Animals that produce milk containing therapeutic agents such as insulin, plasma proteins, drugs, and vaccines for human health have been genetically engineered. In order to cater to animal health, transgenic animals that express in their mammary glands, various components that work against mastitis have been generated. The ultimate acceptability of the "designer" products will depend on ethical issues such as animal welfare and safety, besides better health benefits and increased profitability of products manufactured by the novel techniques.

A longitudinal study of human milk composition in the second year postpartum: implications for human milk banking.

PubMed

Perrin, Maryanne T; Fogleman, April D; Newburg, David S; Allen, Jonathan C

2017-01-01

While the composition of human milk has been studied extensively in the first year of lactation, there is a paucity of data regarding human milk composition beyond one year postpartum. Policies vary at milk banks around the world regarding how long lactating women are eligible to donate their milk. The primary purpose of this study is to describe longitudinal changes in human milk composition in the second year postpartum to support the development of evidence based guidelines regarding how long lactating women can donate human milk to a milk bank. Nineteen lactating women in North Carolina provided monthly milk samples from 11 months to 17 months postpartum (N = 131), and two non-profit milk banks provided (N = 33) pooled, unpasteurized milk samples from 51 approved donors less than one year postpartum. There was a significant increase (P < 0.05) in the concentration of total protein, lactoferrin, lysozyme, Immunoglobulin A, oligosaccharides and sodium in longitudinal samples of mother's milk between 11 and 17 months postpartum, while zinc and calcium concentrations declined, and no changes were observed in lactose, fat, iron and potassium. Human milk in the second year postpartum contained significantly higher concentrations of total protein, lactoferrin, lysozyme and Immunoglobulin A, than milk bank samples, and significantly lower concentrations of zinc, calcium, iron and oligosaccharides. Accepting milk bank donations beyond one year postpartum is a potential strategy for increasing the supply of donor milk, but may require mineral fortification. © 2016 John Wiley & Sons Ltd.

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

PubMed

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

2011-02-28

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

An electrochemical aptasensor based on a TiO2/three-dimensional reduced graphene oxide/PPy nanocomposite for the sensitive detection of lysozyme.

PubMed

Wang, Minghua; Zhai, Shuyong; Ye, Zihan; He, Linghao; Peng, Donglai; Feng, Xiaozhong; Yang, Yanqin; Fang, Shaoming; Zhang, Hongzhong; Zhang, Zhihong

2015-04-14

A sensitive aptasensor based on a nanocomposite of hollow titanium dioxide nanoball, three-dimensional reduced graphene oxide, and polypyrrole (TiO2/3D-rGO/PPy) was developed for lysozyme detection. A lysozyme aptamer was easily immobilized onto the TiO2/3D-rGO/PPy nanocomposite matrix by assembling the aptamer onto graphene through simple π-stacking interactions and electrostatic interactions between PPy molecular chains and aptamer strands. In the presence of lysozyme, the aptamer on the adsorbent layer catches the target on the electrode interface, which generates a barrier for electrons and inhibits electron transfer, subsequently resulting in decreased electrochemically differential pulse voltammetric signals of a gold electrode modified with TiO2/3D-rGO/PPy. Using this strategy, a low limit of detection of 0.085 ng mL(-1) (5.5 pM) for detecting lysozyme was observed within the detection range of 0.1-50 ng mL(-1) (0.007-3.5 nM). The aptasensor also presents high specificity for lysozyme, which is unaffected by the coexistence of other proteins. Such an aptasensor opens a rapid, selective, and sensitive route to lysozyme detection. This finding indicates that the TiO2/3D-rGO/PPy nanocomposite could be used as an electrochemical biosensor for detecting proteins in the biomedical field.

High-temperature short-time pasteurisation of human breastmilk is efficient in retaining protein and reducing the bacterial count.

PubMed

Klotz, Daniel; Joellenbeck, Mirjam; Winkler, Karl; Kunze, Mirjam; Huzly, Daniela; Hentschel, Roland

2017-05-01

Milk banks are advised to use Holder pasteurisation to inactivate the cytomegalovirus, but the process adversely affects the bioactive properties of human breastmilk. This study explored the antibacterial efficacy of an alternative high-temperature short-time (HTST) treatment of human breastmilk and its effect on marker proteins, compared with the Holder method. Breastmilk samples were obtained from 27 mothers with infants in a German neonatal intensive care unit. The samples were either heated to 62°C for five seconds using HTST or processed using Holder pasteurisation, at 63 ± 0.5°C for 30 minutes. Immunoglobulin A, lactoferrin, lysozyme, alkaline phosphatase and bile salt-stimulated lipase concentrations and bacterial colony-forming units/mL were measured before and after heating. HTST-treated samples retained higher rates of immunoglobulin A (95% versus 83%), alkaline phosphatase (6% versus 0%) and bile salt-stimulated lipase (0.8% versus 0.4%) than Holder pasteurisation samples (all p < 0.01), but not lactoferrin (32% versus 20%, p = 0.18) and lysozyme (72% versus 65%, p = 1). No difference in antibacterial efficacy was noted between the two groups (p = 0.29). Using the HTST treatment protocol retained some of the bioactive properties of human breastmilk and appeared to have similar antibacterial efficacy to Holder pasteurisation. ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Isothermal Titration Calorimetry and Macromolecular Visualization for the Interaction of Lysozyme and Its Inhibitors

ERIC Educational Resources Information Center

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

2015-01-01

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

Lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine if lysozyme in nursery diets improved growth performance and gastrointestinal health of pigs weaned from the sow at 24 d of age. Two replicates of 96 pigs (192 total 96 males,...

Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old pigs

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine the effect of a purified granulated lysozyme, compared to antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-...

Granulated lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology of 10-day-old pigs

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine the efficacy of granulated lysozyme, compared to antibiotics, on growth performance, small intestinal morphology, and Campylobacter shedding in 10-d-old pigs. Forty-eight pigs ...

Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect immune challenge. Two replicates of 600 pigs each were weaned from the sow at 2...

Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment

ERIC Educational Resources Information Center

Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.

2010-01-01

We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

Modulatory effects of levamisole and garlic oil on the immune response of Wistar rats: Biochemical, immunohistochemical, molecular and immunological study.

PubMed

Mohamed, Essam Hassan; Baiomy, Ahmed Abdel-Aziz; Ibrahim, Zein Shaban; Soliman, Mohamed Mohamed

2016-09-01

Levamisole (LEVA) and garlic are prevalent immunomodulators in humans and animals. Therefore, the present study aimed to examine the immunomodulatory effects of LEVA and garlic oil (GO) alone or in combination on the immune response of Wistar rats. A total of 24 male Wistar rats were allocated into four equal groups: Control group, which was given ad libitum access to food and water; and groups 2‑4, which were orally administered LEVA [2.5 mg/kg body weight (BW) every 2 days], GO, (5 ml/kg BW daily), or LEVA plus GO, respectively for 4 consecutive weeks. Serum immunoglobulin (Ig)G and IgM levels were measured using a radial immunodiffusion assay. Serum cytokine levels, including interferon (IFN)-γ, interleukin (IL)-5 and tumor necrosis factor (TNF)-α, were measured using enzyme‑linked immunosorbent assay kits. Total blood counts were measured automatically using a cell counter. Serum lysozyme enzymatic activity was determined by measuring the diameters of the zones of clearance relative to lysozyme. Immunohistochemical detection of CD4 and CD8 was carried out using the streptavidin-biotin-peroxidase method. Furthermore, the mRNA expression levels of IL‑4, IL‑5 and IL‑12 were measured in the leukocytes and thymus gland by semi-quantitative polymerase chain reaction. The results revealed that LEVA increased serum levels of IFN‑γ, IL‑5 and TNF‑α cytokines, whereas co‑administration of LEVA and GO decreased the stimulatory action of LEVA alone. LEVA and GO alone increased the serum levels of IgG, IgM and total blood cell counts, and co‑administration of GO and LEVA inhibited the effects of LEVA. At the cellular level, in the spleen, LEVA increased immunoreactivity of CD4 and CD8, whereas co‑administration of GO with LEVA decreased this strong expression. At the molecular level, in leukocytes, LEVA upregulated the mRNA expression levels of IL‑2, IL‑4 and IL‑5, whereas GO alone downregulated mRNA expression. Co‑administration of GO with LEVA inhibited the LEVA‑induced upregulation of IL‑2, IL‑4 and IL‑5 mRNA expression. In the thymus, both LEVA and GO upregulated the mRNA expression levels of IL‑4 and IL‑5, whereas LEVA alone did not affect IL‑12 mRNA expression. Co‑administration of GO with LEVA inhibited LEVA‑induced upregulation of IL‑4 and GO‑induced upregulation of IL‑12 expression, and had an additive upregulatory effect on IL‑5 expression. In conclusion, LEVA stimulated T‑helper (Th)1 cytokines, whereas GO stimulated a Th2 response, and co‑administration of GO with LEVA inhibited the stimulatory effects of LEVA and balanced the Th1/Th2 response.

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

PubMed

Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

2017-05-13

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

PubMed Central

Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

2017-01-01

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

Phage lytic enzymes: a history.

PubMed

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

A role for antimicrobial peptides in intestinal microsporidiosis

PubMed Central

Leitch, Gordon J.; Ceballos, Carolina

2009-01-01

SUMMARY Clinical isolates from three microsporidia species, Encephalitozoon intestinalis and Encephalitozoon hellem, and the insect parasite Anncaliia (Brachiola, Nosema) algerae, were used in spore germination and enterocyte-like (C2Bbe1) cell infection assays to determine the effect of a panel of antimicrobial peptides. Spores were incubated with lactoferrin (Lf), lysozyme (Lz), and human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1), alone and in combination with Lz, prior to germination. Of the Encephalitozoon species only E. hellem spore germination was inhibited by HNP1, while A. algerae spore germination was inhibited by Lf, HBD2, HD5 and HNP1, although HBD2 and HD5 inhibition required the presence of Lz. The effects of HBD2 and HD5 on microsporidia enterocyte infection paralleled their effects on spore germination. Lysozyme alone only inhibited infection with A. algerae, while Lf inhibited infection by E. intestinalis and A. algerae. HNP1 significantly reduced enterocyte infection by all three parasite species and a combination of Lf, Lz and HNP1 caused a further reduced infection with A. algerae. These data suggest that intestinal antimicrobial peptides contribute to the defense of the intestine against infection by luminal microsporidia spores and may partially determine which parasite species infects the intestine. PMID:19079820

Persistent oral human papillomavirus infection is associated with smoking and elevated salivary immunoglobulin G concentration.

PubMed

Haukioja, Anna; Asunta, Maribel; Söderling, Eva; Syrjänen, Stina

2014-09-01

Prevalence and risk factors for human papillomavirus (HPV) persistence in oral mucosa are largely unknown. Furthermore, the antiviral effects of saliva in the outcome of oral HPV infections are unexplored. To compare the levels of selected salivary defence proteins in women with a persistent oral HPV infection and in those without any signs of oral HPV. Lifestyle factors including the use of oral contraceptives, oral sex, smoking and alcohol drinking habits were also assessed. This nested case-control study of the Finnish Family HPV Study included 60 women with a persistent oral HPV infection and 117 women who remained HPV DNA negative throughout a 6-year follow-up. Whole saliva samples and oral scrapings for HPV testing were collected at the same visit. The oral HPV status was related to salivary concentrations of lactoferrin, lysozyme, IgA, IgG, total protein and sodium as well as to the use of oral contraceptives, oral sex, smoking and alcohol drinking habits. Women with a persistent oral HPV infection had higher salivary levels of IgG (p=0.007) and lysozyme (p=0.002, when adjusted to the total protein concentration), than those without an HPV infection. Lactoferrin and IgA concentrations were not related to the HPV-status. Smoking increased the risk of a persistent oral HPV infection (p=0.020), but the oral HPV status was not related to other life-style factors studied. Smoking is a risk factor for a persistent oral HPV infection. Oral HPV infection may be associated with increased concentrations of salivary IgG and lysozyme. Copyright © 2014 Elsevier B.V. All rights reserved.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

NASA Astrophysics Data System (ADS)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces.

PubMed

Samaranayake, Y H; Cheung, B P K; Parahitiyawa, N; Seneviratne, C J; Yau, J Y Y; Yeung, K W S; Samaranayake, L P

2009-02-01

Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated. (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes. The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay. The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p<0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 microg/ml) significantly (p<0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 microg/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 microg/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 microg/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends. Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections.

Lysis of grouped and ungrouped streptococci by lysozyme.

PubMed

Coleman, S E; van de Rijn, I; Bleiweis, A S

1970-11-01

Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 m tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 mug/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 mug/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 mug of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.

Coassembly of Lysozyme and Amphiphilic Biomolecules Driven by Unimer-Aggregate Equilibrium.

PubMed

Tao, Yuanyuan; Ma, Xiaoteng; Cai, Yaqian; Liu, Li; Zhao, Hanying

2018-04-12

Synthesis and self-assembly of bioconjugates composed of proteins and synthetic molecules have been widely studied because of the potential applications in medicine, biotechnology, and nanotechnology. One of the challenging research studies in this area is to develop organic solvent-free approaches to the synthesis and self-assembly of amphiphilic bioconjugates. In this research, dialysis-assisted approach, a method based on unimer-aggregate equilibrium, was applied in the coassembly of lysozyme and conjugate of cholesterol and glutathione (Ch-GSH). In phosphate buffer solution, amphiphilic Ch-GSH conjugate self-assembles into vesicles, and the vesicle solution is dialyzed against lysozyme solution. Negatively charged Ch-GSH unimers produced in the unimer-vesicle exchange equilibrium, diffuse across the dialysis membrane and have electrostatic interaction with positively charged lysozyme, resulting in the formation of Ch-GSH-lysozyme bioconjugate. Above a critical concentration, the three-component bioconjugate molecules self-assemble into bioactive vesicles.

Stability of Lysozyme in Aqueous Extremolyte Solutions during Heat Shock and Accelerated Thermal Conditions

PubMed Central

van Streun, Erwin L. P.; Frijlink, Henderik W.; Hinrichs, Wouter L. J.

2014-01-01

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account. PMID:24465983

Stability of lysozyme in aqueous extremolyte solutions during heat shock and accelerated thermal conditions.

PubMed

Avanti, Christina; Saluja, Vinay; van Streun, Erwin L P; Frijlink, Henderik W; Hinrichs, Wouter L J

2014-01-01

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.

Sexual Signaling and Immune Function in the Black Field Cricket Teleogryllus commodus

PubMed Central

Drayton, Jean M.; Hall, Matthew D.; Hunt, John; Jennions, Michael D.

2012-01-01

The immunocompetence handicap hypothesis predicts that male sexual trait expression should be positively correlated with immunocompetence. Here we investigate if immune function in the cricket, Teleogryllus commodus, is related to specific individual components of male sexual signals, as well as to certain multivariate combinations of these components that females most strongly prefer. Male T. commodus produce both advertisement and courtship calls prior to mating. We measured fine-scale structural parameters of both call types and also recorded nightly advertisement calling effort. We then measured two standard indices of immune function: lysozyme-like activity of the haemolymph and haemocyte counts. We found a weak, positive relationship between advertisement calling effort and lysozyme-like activity. There was, however, little evidence that individual structural call components or the net multivariate attractiveness of either call type signalled immune function. The relationships between immunity and sexual signaling did not differ between inbred and outbred males. Our data suggest that it is unlikely that females assess overall male immune function using male calls. PMID:22808047

Lysozyme as an alternative to antibiotics improves growth performance and tumor necrosis factor-a levels during an indirect immune challenge

USDA-ARS?s Scientific Manuscript database

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect disease challenge. Two replicates of 720 pigs each were weaned from the sow at ...

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

PubMed

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

2006-03-01

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

The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.

PubMed

Arabski, Michał; Konieczna, Iwona; Tusińska, Ewa; Wąsik, Sławomir; Relich, Inga; Zając, Krzysztof; Kamiński, Zbigniew J; Kaca, Wiesław

2015-01-01

Lysozyme (1,4-β-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur. Copyright © 2014 Elsevier GmbH. All rights reserved.

Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-02-16

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

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.

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

PubMed

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

2016-04-19

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

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

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

PubMed

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

2016-10-01

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

Sulfur Mustard (SM) Lesions in Organ-Cultured Human Skin: Markers of Injury and Inflammatory Mediators

DTIC Science & Technology

1990-04-16

18. SUB3ECT TERMS (oont’d) epidermal injury organ culture •ranuaear vacuoles C-leucine incorpora’tion by full-thickness human akin explants hi stamine ...mast- cell degranulation prostaglandin E2 lysobomal enzymes: acid phosphatase, B-glucuronidase, 0-galactcsidase, lysozyme and lactic dehydrogenase...that histamline (from local mast cells ), and PA and POgk (probably from mast cells and epidermal cells ) are s3e of the early mediators of the inflmma

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.

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

DTIC Science & Technology

1984-06-25

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

Lysozyme encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics.

PubMed

Russell, B A; Jachimska, B; Komorek, P; Mulheran, P A; Chen, Y

2017-03-08

The study of gold nanoclusters (AuNCs) has seen much interest in recent history due to their unique fluorescence properties and environmentally friendly synthesis method using proteins as a growth scaffold. The differences in the physicochemical properties of lysozyme encapsulated AuNCs in comparison to natural lysozyme are characterised in order to determine the effects AuNCs have on natural protein behaviour. The hydrodynamic radius (dynamic light scattering), light absorbance (UV-Vis), electrophoretic mobility, relative density, dynamic viscosity, adsorption (quartz crystal microbalance) and circular dichroism (CD) characteristics of the molecules were studied. It was found that lysozyme forms small dimer/trimer aggregates upon the synthesis of AuNCs within the protein. The diameter of Ly-AuNCs was found to be 8.0 nm across a pH range of 2-11 indicating dimer formation, but larger aggregates with diameters >20 nm were formed between pH 3 and 6. The formation of larger aggregates limits the use of Ly-AuNCs as a fluorescent probe in this pH range. A large shift in the protein's isoelectric point was also observed, shifting from 11.0 to 4.0 upon AuNC synthesis. This resulted in major changes to the adsorption characteristics of lysozyme, observed using a QCM. A monolayer of 8 nm was seen for Ly-AuNCs at pH 4, offering further evidence that the proteins form small aggregates, unlike the natural monomer form of lysozyme. The adsorption of Ly-AuNCs was seen to decrease as pH was increased; this is in major contrast to the lysozyme adsorption behaviour. A decrease in the α-helix content was observed from 25% in natural lysozyme to 1% in Ly-AuNCs. This coincided with an increase in the β-sheet content after AuNC synthesis indicating that the natural structure of lysozyme was lost. The formation of protein dimers, the change in the protein surface charge from positive to negative, and secondary structure alteration caused by the AuNC synthesis must be considered before attempting to utilise Ly-AuNCs as in vivo probes.

Erythrocyte Saturation with IgG Is Required for Inducing Antibody-Mediated Immune Suppression and Impacts Both Erythrocyte Clearance and Antigen-Modulation Mechanisms.

PubMed

Cruz-Leal, Yoelys; Marjoram, Danielle; Lazarus, Alan H

2018-02-15

Anti-D prevents hemolytic disease of the fetus and newborn, and this mechanism has been referred to as Ab-mediated immune suppression (AMIS). Anti-D, as well as other polyclonal AMIS-inducing Abs, most often induce both epitope masking and erythrocyte clearance mechanisms. We have previously observed that some Abs that successfully induce AMIS effects could be split into those that mediate epitope masking versus those that induce erythrocyte clearance, allowing the ability to analyze these mechanisms separately. In addition, AMIS-inducing activity has recently been shown to induce Ag modulation (Ag loss from the erythrocyte surface). To assess these mechanisms, we immunized mice with transgenic murine RBCs expressing a single Ag protein comprising a recombinant Ag composed of hen egg lysozyme, OVA sequences comprising aa 251-349, and the human Duffy transmembrane protein (HOD-Ag) with serial doses of polyclonal anti-OVA IgG as the AMIS-inducing Ab. The anti-OVA Ab induced AMIS in the absence of apparent epitope masking. AMIS occurred only when the erythrocytes appeared saturated with IgG. This Ab was capable of inducing HOD-RBC clearance, as well as loss of the OVA epitope at doses of Ab that caused AMIS effects. HOD-RBCs also lost reactivity with Abs specific for the hen egg lysozyme and Duffy portions of the Ag consistent with the initiation of Ag modulation and/or trogocytosis mechanisms. These data support the concept that an AMIS-inducing Ab that does not cause epitope masking can induce AMIS effects in a manner consistent with RBC clearance and/or Ag modulation. Copyright © 2018 by The American Association of Immunologists, Inc.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome.

PubMed

Massie, Isobel; Spaniol, Kristina; Barbian, Andreas; Poschmann, Gereon; Stühler, Kai; Geerling, Gerd; Metzger, Marco; Mertsch, Sonja; Schrader, Stefan

2017-10-01

Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Lysozyme activity and nitroblue-tetrazolium reduction in leukaemic cells

PubMed Central

Catovsky, D.; Galton, D. A. G.

1973-01-01

The cytochemical methods for lysozyme and nitroblue-tetrazolium reduction have been used to study the blast cells of acute myeloid leukaemia. Both proved useful in characterizing the cases with predominant monocytic differentiation. The demonstration of lysozyme activity helped to define two main groups: (a) with predominantly lysozyme-negative cells (myeloblastic-promyelocytic), and (b) with considerable numbers of positive cells (monoblastic-monocytic). In addition this test was also of value in the differentiation of other leukaemic disorders. Reduction of nitroblue-tetrazolium was also a feature of monocytic differentiation. The combination of these two methods with those for myeloperoxidase and non-specific esterase activity contributes to the cytological characterization of acute myeloid leukaemia. Images PMID:4511938

Osmotic pressures and second virial coefficients for aqueous saline solutions of lysozyme

DOE PAGES

Moon, Y. U.; Anderson, C. O.; Blanch, H. W.; ...

2000-03-27

Experimental data at 25 °C are reported for osmotic pressures of aqueous solutions containing lysozyme and any one of the following salts: ammonium sulfate, ammonium oxalate and ammonium phosphate at ionic strength 1 or 3M. Data were obtained using a Wescor Colloid Membrane Osmometer at lysozyme concentrations from about 4 to 20 grams per liter at pH 4, 7 or 8. Osmotic second virial coefficients for lysozyme were calculated from the osmotic-pressure data. All coefficients were negative, increasing in magnitude with ionic strength. Furthermore, tesults are insensitive to the nature of the anion, but rise slightly in magnitude as themore » size of the anion increases.« less

Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

NASA Astrophysics Data System (ADS)

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

2016-05-01

The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. The magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.

Location of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

Locations of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system.

PubMed

Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu

2015-05-15

The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

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

Yadav, Indresh, E-mail: iykumarindresh288@gmail.com; Aswal, V. K.; Kohlbrecher, J.

The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. Themore » magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.« less

Molecular dynamics simulations of lysozyme-lipid systems: probing the early steps of protein aggregation.

PubMed

Trusova, Valeriya M; Gorbenko, Galyna P

2017-07-10

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of β-structures. Along with this, the formation of protein-lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.

Macromolecular powder diffraction : structure solution via molecular.

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

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

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

Fluorinated ionic liquids for protein drug delivery systems: Investigating their impact on the structure and function of lysozyme.

PubMed

Alves, Márcia; Vieira, Nicole S M; Rebelo, Luís Paulo N; Araújo, João M M; Pereiro, Ana B; Archer, Margarida

2017-06-30

Since the approval of recombinant human insulin by FDA in 1982, more than 200 proteins are currently available for pharmaceutical use to treat a wide range of diseases. However, innovation is still required to develop effective approaches for drug delivery. Our aim is to investigate the potential use of fluorinated ionic liquids (FILs) as drug delivery systems (DDS) for therapeutic proteins. Some initial parameters need to be assessed before further studies can proceed. This work evaluates the impact of FILs on the stability, function, structure and aggregation state of lysozyme. Different techniques were used for this purpose, which included differential scanning fluorimetry (DSF), spectrophotometric assays, circular dichroism (CD), dynamic light scattering (DLS), and scanning and transmission electron microscopy (SEM/TEM). Ionic liquids composed of cholinium-, imidazolium- or pyridinium- derivatives were combined with different anions and analysed at different concentrations in aqueous solutions (below and above the critical aggregation concentration, CAC). The results herein presented show that the addition of ionic liquids had no significant effect on the stability and hydrolytic activity of lysozyme. Moreover, a distinct behaviour was observed in DLS experiments for non-surfactant and surfactant ionic liquids, with the latter encapsulating the protein at concentrations above the CAC. These results encourage us to further study ionic liquids as promising tools for DDS of protein drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Vertically Aligned Nitrogen-Doped Carbon Nanotube Carpet Electrodes: Highly Sensitive Interfaces for the Analysis of Serum from Patients with Inflammatory Bowel Disease.

PubMed

Wang, Qian; Subramanian, Palaniappan; Schechter, Alex; Teblum, Eti; Yemini, Reut; Nessim, Gilbert Daniel; Vasilescu, Alina; Li, Musen; Boukherroub, Rabah; Szunerits, Sabine

2016-04-20

The number of patients suffering from inflammatory bowel disease (IBD) is increasing worldwide. The development of noninvasive tests that are rapid, sensitive, specific, and simple would allow preventing patient discomfort, delay in diagnosis, and the follow-up of the status of the disease. Herein, we show the interest of vertically aligned nitrogen-doped carbon nanotube (VA-NCNT) electrodes for the required sensitive electrochemical detection of lysozyme in serum, a protein that is up-regulated in IBD. To achieve selective lysozyme detection, biotinylated lysozyme aptamers were covalently immobilized onto the VA-NCNTs. Detection of lysozyme in serum was achieved by measuring the decrease in the peak current of the Fe(CN)6(3-/4-) redox couple by differential pulse voltammetry upon addition of the analyte. We achieved a detection limit as low as 100 fM with a linear range up to 7 pM, in line with the required demands for the determination of lysozyme level in patients suffering from IBD. We attained the sensitive detection of biomarkers in clinical samples of healthy patients and individuals suffering from IBD and compared the results to a classical turbidimetric assay. The results clearly indicate that the newly developed sensor allows for a reliable and efficient analysis of lysozyme in serum.

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.

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

NASA Technical Reports Server (NTRS)

Burke, Michael; Judge, Russell; Pusey, Marc

2000-01-01

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

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

NASA Technical Reports Server (NTRS)

Wilson, Lori June

1990-01-01

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

In Vitro Effect of Lysozyme on Albumin Deposition to Hydrogel Contact Lens Materials.

PubMed

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

2017-11-01

Albumin deposition on contact lenses could be detrimental to contact lens (CL) wear because this may increase the risk of bacterial binding and reduce comfort. Lysozyme deposition on selected lens materials would reduce albumin deposition on lenses. This study aims to determine if lysozyme deposition on CLs could act as a barrier against subsequent albumin adsorption, using an in vitro model. Six hydrogel CL materials (etafilcon A, polymacon, nelfilcon A, omafilcon A, ocufilcon B, and nesofilcon A) were evaluated. Four CLs of each type were soaked in lysozyme solution for 16 hours at 37°C. Lysozyme-coated lenses were then placed in vials with 1.5 mL of artificial tear solution containing I-labeled albumin for 16 hours at 37°C with shaking. Four uncoated lenses of each type were used as controls. Lenses soaked in radiolabeled albumin were rinsed in a phosphate-buffered saline solution, and radioactive counts were measured directly on lenses using a gamma counter. Albumin uptake on lenses was measured using a calibration curve by plotting radioactive counts versus protein concentration. Results are reported as mean ± SD. Lysozyme-coated etafilcon A lenses exhibited lower levels of deposited albumin than uncoated etafilcon A lenses (58 ± 12 vs. 84 ± 5 ng/lens; P < .05). There were no differences in albumin adsorption between control (uncoated) and lysozyme-coated polymacon (105 ± 10 vs. 110 ± 34 ng/lens), nelfilcon A (51 ± 7 vs. 42 ± 20 ng/lens), omafilcon A (90 ± 20 vs. 80 ± 38 ng/lens), ocufilcon B (87 ± 20 vs. 115 ± 50 ng/lens), and nesofilcon A (170 ± 29 vs. 161 ± 10 ng/lens) lens materials (P > .05). Uncoated nesofilcon A lenses deposited the highest amount of albumin when compared with other uncoated lenses (P < .05). This study demonstrates that lysozyme deposited onto etafilcon A resists the deposition of albumin, which may potentially be beneficial to CL wearers.

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

PubMed

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

2016-09-21

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

Solubility of lysozyme in polyethylene glycol-electrolyte mixtures: the depletion interaction and ion-specific effects.

PubMed

Boncina, Matjaz; Rescic, Jurij; Vlachy, Vojko

2008-08-01

The solubility of aqueous solutions of lysozyme in the presence of polyethylene glycol and various alkaline salts was studied experimentally. The protein-electrolyte mixture was titrated with polyethylene glycol, and when precipitation of the protein occurred, a strong increase of the absorbance at 340 nm was observed. The solubility data were obtained as a function of experimental variables such as protein and electrolyte concentrations, electrolyte type, degree of polymerization of polyethylene glycol, and pH of the solution; the last defines the net charge of the lysozyme. The results indicate that the solubility of lysozyme decreases with the addition of polyethylene glycol; the solubility is lower for a polyethylene glycol with a higher degree of polymerization. Further, the logarithm of the protein solubility is a linear function of the polyethylene glycol concentration. The process is reversible and the protein remains in its native form. An increase of the electrolyte (NaCl) concentration decreases the solubility of lysozyme in the presence and absence of polyethylene glycol. The effect can be explained by the screening of the charged amino residues of the protein. The solubility experiments were performed at two different pH values (pH = 4.0 and 6.0), where the lysozyme net charge was +11 and +8, respectively. Ion-specific effects were systematically investigated. Anions such as Br(-), Cl(-), F(-), and H(2)PO(4)(-) (all in combination with Na(+)), when acting as counterions to a protein with positive net charge, exhibit a strong effect on the lysozyme solubility. The differences in protein solubility for chloride solutions with different cations Cs(+), K(+), and Na(+) (coions) were much smaller. The results at pH = 4.0 show that anions decrease the lysozyme solubility in the order F(-) < H(2)PO(4)(-) < Cl(-) < Br(-) (the inverse Hofmeister series), whereas cations follow the direct Hofmeister series (Cs(+) < K(+) < Na(+)) in this situation.

The nutritive and immunoprotective quality of human milk beyond 1 year postpartum: are lactation-duration-based donor exclusions justified?

PubMed

Perrin, Maryanne Tigchelaar; Fogleman, April; Allen, Jonathan C

2013-08-01

Donor human milk is critical for the fragile preterm infant who does not have access to his or her mother's milk, improving survival rates and quality of survival and decreasing hospital stay. Despite the opening of donor milk banks around the world, shortages continue as demand for donor milk exceeds supply. One potential means of increasing supply is by reducing exclusion criteria that prohibit mothers from donating milk based on duration of lactation. Minimal research has been done on the composition of human milk during the second year of lactation, with most research focusing on the nutritive compounds and not the immunoprotective compounds. Several immunoprotective compounds, including lysozyme, lactoferrin, secretory immunoglobulin A, and oligosaccharides, are abundant in human milk compared to bovine-based infant formula and are partially or fully retained during Holder pasteurization, making them an important differentiating feature of donor milk. A PubMed search was conducted to review studies in human milk composition during the second year of lactation. Limitations of existing research include sample collection protocols, small study sizes, and use of populations that may have been at risk for nutritional deficiencies. Stable concentrations of several components were reported including protein, lactose, iron, copper, lactoferrin, and secretory immunoglobulin A. Lysozyme concentration increased during extended lactation, while zinc and calcium concentrations declined into the second year. Conflicting findings were reported on fat content, and no information was available regarding oligosaccharide content. More research is needed to create evidence-based guidelines regarding the nutritive and immunoprotective value of donor milk throughout the course of lactation.

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

PubMed

Shbailat, Seba Jamal; Aslan, Ibtisam Omar

2018-01-01

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

The trenbolone acetate affects the immune system in rainbow trout, Oncorhynchus mykiss.

PubMed

Massart, Sophie; Redivo, Baptiste; Flamion, Enora; Mandiki, S N M; Falisse, Elodie; Milla, Sylvain; Kestemont, Patrick

2015-06-01

In aquatic systems, the presence of endocrine-disrupting chemicals (EDC) can disrupt the reproductive function but also the immune system of wildlife. Some studies have investigated the effects of androgens on the fish immune parameters but the mechanisms by which the xenoandrogens alter the immunity are not well characterized. In order to test the effects of trenbolone acetate (TbA) on fish immune system, we exposed rainbow trout male juveniles during three weeks to TbA levels at 0.1 and 1μg/L. The present results suggest that TbA impacts, in a tissue-dependent manner, the rainbow trout immunity by affecting primarily the humoral immunity. Indeed, TbA inhibited lysozyme activity in plasma and liver and enhanced the alternative complement pathway activity (ACH50) in kidney. In plasma, the modulation of the complement system was time-dependent. The mRNA expression of genes encoding some cytokines such as renal TGF-β1, TNF-α in skin and hepatic IL-1β was also altered in fish exposed to TbA. Regarding the cellular immunity, no effect was observed on the leucocyte population. However, the expression of genes involved in the development and maturation of lymphoid cells (RAG-1 and RAG-2) was decreased in TbA-treated fish. Among those effects, we suggest that the modulation of RAG-1 and mucus apolipoprotein-A1 gene expression as well as plasma and hepatic lysozyme activities are mediated through the action of the androgen receptor. All combined, we conclude that trenbolone affects the rainbow trout immunity. Copyright © 2015 Elsevier B.V. All rights reserved.

Immune Modulation by Coxiella burnetii: Characterization of a Phase 1 Immunosuppressive Complex Differentially Expressed among Strains

DTIC Science & Technology

1988-01-01

components may bear compositional similarities to a Mycobacterium leprae glycolipid, which is capable of inducing suppression of mitogenic responses of...of the phenolic glycolipid of M. leprae (20). Suppressor cell- inducing epitopes have also been demonstrated in the lysozyme 256 WAAG AND WILLIAMS...V., Brennan. P. J., Rada, E., Convit, J., and Bloom, B. R. Lymphocyte suppression in leprosy induced by a unique M. leprae glycolipid. Nature 308:194

[Markers of dental children`s health in the application of therapeutic orthodontic equipment].

PubMed

Пачевська, Аліса В; Білошицька, Аліна В

Treatment of teeth anomalies using removable and non-removable orthodontic devices in children leads to complications such as caries, gingivitis, periodontitis, oral mucosa hyperplasia. Etiopathogenetical of these diseases can be associated with biochemical changes in the composition of saliva. To determine the activity of lysozyme and amylase in oral fluid in children when using a fixed and removable orthodontic devices. Amylase and lysozyme were studied in oral fluid. Analyzed the biochemical composition of the freshly samples of oral fluid that was obtained in the control, experimental group 1 and 2 (children ages 7-18 years, which were used medical non-removable and removable orthodontic devices). Saliva was collected at the beginning of the therapeutic use of orthodontic devices (the first day of treatment), on 3 and 6 months of treatment. Assessment of lysozyme activity was carried nephelometric method on the ability of lysozyme to dissolve indicator organism Micrococcus lysodeicticus. To construct a calibration graph using dry lysozyme company Sigma. Salivary amylase activity was determined by hydrolysis of starch. The results were subjected to statistical analysis by standard methods. Data processed using software packages applied statistical analysis Statistica 6.0, Microsoft Excel, 2003. The use of a fixed and removable orthodontic equipment led to a decrease in saliva amylase, major changes are observed on the 6th month of treatment. The activity of lysozyme in saliva decreased the mostin patients with a permanent equipment. Major changes were also recorded on the 6th month of treatment. Complications of orthodontic treatment teeth anomalies in children (caries, gingivitis, periodontitis) caused by changes in the biochemical composition of saliva. For the prevention of the emergence and development of these complications is necessary to control the level of amylase and lysozyme in the mouth.

Adsorption of lysozyme to phospholipid and meibomian lipid monolayer films.

PubMed

Mudgil, Poonam; Torres, Margaux; Millar, Thomas J

2006-03-15

It is believed that a lipid layer forms the outer layer of the pre-ocular tear film and this layer helps maintain tear film stability by lowering its surface tension. Proteins of the aqueous layer of the tear film (beneath the lipid layer) may also contribute to reducing surface tension by adsorbing to, or penetrating the lipid layer. The purpose of this study was to compare the penetration of lysozyme, a tear protein, into films of meibomian lipids and phospholipids held at different surface pressures to determine if lysozyme were part of the surface layer of the tear film. Films of meibomian lipids or phospholipids were spread onto the surface of a buffered aqueous subphase. Films were compressed to particular pressures and lysozyme was injected into the subphase. Changes in surface pressure were monitored to determine adsorption or penetration of lysozyme into the surface film. Lysozyme penetrated a meibomian lipid film at all pressures tested (max=20 mN/m). It also penetrated phosphatidylglycerol, phosphatidylserine or phosphatidylethanolamine lipid films up to a pressure of 20 mN/m. It was not able to penetrate a phosphatidylcholine film at pressures >or=10 mN/m irrespective of the temperature being at 20 or 37 degrees C. However, it was able to penetrate it at very low pressures (<10 mN/m). Epifluorescence microscopy showed that the protein either adsorbs to or penetrates the lipid layer and the pattern of mixing depended upon the lipid at the surface. These results indicate that lysozyme is present at the surface of the tear film where it contributes to decreasing the surface tension by adsorbing and penetrating the meibomian lipids. Thus it helps to stabilize the tear film.

The effects of hyaluronic acid incorporated as a wetting agent on lysozyme denaturation in model contact lens materials.

PubMed

Weeks, Andrea; Boone, Adrienne; Luensmann, Doerte; Jones, Lyndon; Sheardown, Heather

2013-09-01

Conventional and silicone hydrogels as models for contact lenses were prepared to determine the effect of the presence of hyaluronic acid on lysozyme sorption and denaturation. Hyaluronic acid was loaded into poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) hydrogels, which served as models for conventional and silicone hydrogel contact lens materials. The hyaluronic acid was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of dendrimers. Active lysozyme was quantified using a Micrococcus lysodeikticus assay while total lysozyme was determined using 125-I radiolabeled protein. To examine the location of hyaluronic acid in the gels, 6-aminofluorescein labeled hyaluronic acid was incorporated into the gels using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry and the gels were examined using confocal laser scanning microscopy. Hyaluronic acid incorporation significantly reduced lysozyme sorption in poly(2-hydroxyethyl methacrylate) (p < 0.00001) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) (p < 0.001) hydrogels, with the modified materials sorbing only 20% and 16% that of the control, respectively. More importantly, hyaluronic acid also decreased lysozyme denaturation in poly(2-hydroxyethyl methacrylate) (p < 0.005) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) (p < 0.02) hydrogels. The confocal laser scanning microscopy results showed that the hyaluronic acid distribution was dependent on both the material type and the molecular weight of hyaluronic acid. This study demonstrates that hyaluronic acid incorporated as a wetting agent has the potential to reduce lysozyme sorption and denaturation in contact lens applications. The distribution of hyaluronic acid within hydrogels appears to affect denaturation, with more surface mobile, lower molecular weight hyaluronic acid being more effective in preventing denaturation.

Allergenicity of milk of different animal species in relation to human milk.

PubMed

Pastuszka, Robert; Barłowska, Joanna; Litwińczuk, Zygmunt

2016-12-31

Protein content in cow milk (with over 20 proteins, and peptides may also occur as a result of enzymatic hydrolysis) ranges from 2.5% to 4.2% and is about 1.5-2 times higher than in human milk. Its most important allergens are considered to be β-lactoglobulin (absent in human milk) and αs1-casein. The most similar in composition to human milk is horse and donkey milk. It contains considerably more whey proteins (35-50%) than cow milk (about 20%), and the concentration of the most allergenic casein fraction αs1 is 1.5-2.5 g/l. In comparison, the content of αs1-casein in cow milk is about 10 g/l. β-lactoglobulin present in donkey milk is a monomer, while in milk of ruminants it is a dimer. Like human milk, it contains a substantial amount of lactose (about 7%), which determines its flavour and facilitates calcium absorption. The high lysozyme content (about 1 g/l) gives it antibacterial properties (compared to trace amounts in ruminants). Camel milk is also more digestible and induces fewer allergic reactions, because it lacks β-lactoglobulin, and its β-casein has a different structure. It also contains (compared to cow milk) more antibacterial substances such as lysozyme, lactoferrin and immunoglobulins, and furthermore the number of immunoglobulins is compatible with human ones. Goat milk components have a higher degree of assimilability as compared to cow milk. Its main protein is β-casein, with total protein content depending on the αs1-casein genetic variant. Goats with the '0' variant do not synthesize this allergenic protein. Clinical and immunochemical studies indicate, however, that it cannot be a substitute for cow milk without the risk of an anaphylactic reaction.

Efficacy of Spirulina platensis diet supplements on disease resistance and immune-related gene expression in Cyprinus carpio L. exposed to herbicide atrazine.

PubMed

Khalil, Samah R; Reda, Rasha M; Awad, Ashraf

2017-08-01

The present study evaluated the immunotoxicological effects of the herbicide atrazine (ATZ) at sub-lethal concentrations and the potential ameliorative influence of Spirulina platensis (SP) over a sub-chronic exposure period on Cyprinus carpio L., also known as common carp. Common carp was sampled after a 40-days exposure to ATZ (428 μg/L) and SP (1%), individually or in combination to assess the non-specific immune response, changes in mRNA expression of immune-related genes [lysozyme (LYZ), immunoglobulin M (IgM), and complement component 3 (C3)] in the spleen, and inflammatory cytokines (interleukins IL-1ß and IL-10) in the head kidney using real-time PCR. Additionally, disease resistance to Aeromonas sobria was evaluated. The results revealed that ATZ exposure caused a significant decline in most of the hematological variables, lymphocyte viability, and lysozyme and bactericidal activity. Moreover, ATZ increased the susceptibility to disease, reflected by a significantly lower post-challenge survival rate of the carp. ATZ may induce dysregulated expression of immune-related genes leading to downregulation of mRNA levels of IgM and LYZ in the spleen. However, expression of C3 remained unaffected. Of the cytokine-related genes examined, IL-1B was up-regulated in the head kidney. In contrast, the expression of IL-10 gene was down-regulated in the ATZ-exposed group. The SP supplementation resulted in a significant improvement in most indices; however, these values did not match with that of the controls. These results may conclude that ATZ affects both innate and adaptive immune responses through the negative transcriptional effect on genes involved in immunity and also due to the inflammation of the immune organs. In addition, dietary supplements with SP could be useful for modulation of the immunity in response to ATZ exposure, thereby presenting a promising feed additive for carps in aquaculture. Copyright © 2017 Elsevier Ltd. All rights reserved.

The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

PubMed

Luo, Jian-Bo; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

2014-09-01

This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) (P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression (P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P < 0.05), and improved Claudin 15 expression in the fish intestine (P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quinopeptide formation associated with the disruptive effect of epigallocatechin-gallate on lysozyme fibrils.

PubMed

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

2015-01-01

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

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

Marana, S. R.; Cançado, F. C.; Valério, A. A.

The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases. Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1more » and 2 crystals belong to the monoclinic space group P2{sub 1} (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, β = 102.97°) and the orthorhombic space group P2{sub 1}2{sub 1}2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.« less

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-10-20

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-11-03

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

1988-01-01

Non-immune SJL (H-2s) spleen cells were fused with (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B- cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones were able to present both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Zouhair Atassi, M. Z.

1989-01-01

Non-immune SJL (H-2s) spleen cells were fused with non-secreting, non-antigen presenting (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B-cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones presented both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

Mechanical Insight into Resistance of Betaine to Urea-Induced Protein Denaturation.

PubMed

Chen, Jiantao; Gong, Xiangjun; Zeng, Chaoxi; Wang, Yonghua; Zhang, Guangzhao

2016-12-08

It is known that urea can induce protein denaturation that can be inhibited by osmolytes. Yet, experimental explorations on this mechanism at the molecular level are still lacking. We have investigated the resistance of betaine to the urea-induced denaturation of lysozyme in aqueous solutions using low-field NMR. Our study demonstrates that urea molecules directly interact with lysozyme, leading to denaturation. However, betaine molecules interacting with urea more strongly than lysozyme can pull the bound urea molecules from lysozyme so that the protein is protected from denaturation. The number of urea molecules bound to a betaine molecule is given under different conditions. Proton NMR spectroscopy ( 1 H-NMR) and Fourier transform infrared spectroscopy reveal that the interaction between betaine and urea is through hydrogen bonding.

The effects of biological buffers TRIS, TAPS, TES on the stability of lysozyme.

PubMed

Pannuru, Pavani; Rani, Anjeeta; Venkatesu, Pannuru; Lee, Ming-Jer

2018-06-01

To explore the mechanism of lysozyme stabilization in buffer system, we have investigated the interactions between lysozyme and the biological buffers (TRIS, TAPS, and TES) using spectroscopic techniques, including ultraviolet-visible (UV-Vis), fluorescence, thermal fluorescence, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. From the series of spectroscopic studies, it is found that the native structure of the protein remains intact in the different concentrations (0.05, 0.1, 0.25, 0.5, and 1.0M) of the biological buffer aqueous solutions at pH7.0. Moreover, all these three investigated buffers are able to protect lysozyme against thermal denaturation, particularly in high concentration (1.0M) of the buffer aqueous solutions. Copyright © 2018 Elsevier B.V. All rights reserved.

Combined pulmonary involvement in hereditary lysozyme amyloidosis with associated pulmonary sarcoidosis: a case report.

PubMed

McCarthy, Cormac; Deegan, Alexander P; Garvey, John F; McDonnell, Timothy J

2013-12-17

Sarcoidosis is a multisystem inflammatory disorder of unknown cause which can affect any organ system. Autosomal dominant lysozyme amyloidosis is a very rare form of hereditary amyloidosis. The Arg64 variant is extraordinarily rare with each family showing a particular pattern of organ involvement, however while Sicca syndrome, gastrointestinal involvement and renal failure are common, lymph node involvement is very rare. In this case report we describe the first reported case of sarcoidosis in association with hereditary lysozyme amyloidosis.

Polysaccharides from marine macroalga, Padina gymnospora improve the nonspecific and specific immune responses of Cyprinus carpio and protect it from different pathogens.

PubMed

Rajendran, Priyatharsini; Subramani, Parasuraman Aiya; Michael, Dinakaran

2016-11-01

Immunostimulation by plant-derived compounds presents a fascinating alternative to vaccines and antibiotics in aquaculture. Fish farmers are longing for immunostimulants that activate both specific and nonspecific immune responses of fish and protect fishes from all possible infections. In this study, we observed that polysaccharide fraction from marine macroalga, Padina gymnospora stimulated the immune response of common carp Cyprinus carpio (Filed for patent, Indian patent no. 201641027311 dated:10-Aug-2016). Our results indicate that fish fed with polysaccharides as feed supplement improved all the immune parameters tested which include serum lysozyme, myeloperoxidase activities and antibody response. Further, polysaccharide fraction protected the fish from its common bacterial pathogens namely Aeromonas hydrophila and Edwardsiella tarda with relative percent survival (RPS) values of 80 and 60 respectively. Gene expression studies, indicate that the immunostimulation by P. gymnospora might be at least in part due to the upregulation of the cytokine interleukin-1β (IL-1β) and antimicrobial peptide lysozyme-C. Copyright © 2016 Elsevier Ltd. All rights reserved.

Innate Immune Responses of Drosophila melanogaster Are Altered by Spaceflight

PubMed Central

Marcu, Oana; Lera, Matthew P.; Sanchez, Max E.; Levic, Edina; Higgins, Laura A.; Shmygelska, Alena; Fahlen, Thomas F.; Nichol, Helen; Bhattacharya, Sharmila

2011-01-01

Alterations and impairment of immune responses in humans present a health risk for space exploration missions. The molecular mechanisms underpinning innate immune defense can be confounded by the complexity of the acquired immune system of humans. Drosophila (fruit fly) innate immunity is simpler, and shares many similarities with human innate immunity at the level of molecular and genetic pathways. The goals of this study were to elucidate fundamental immune processes in Drosophila affected by spaceflight and to measure host-pathogen responses post-flight. Five containers, each containing ten female and five male fruit flies, were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours. A new generation of flies was reared in microgravity. In larvae, the immune system was examined by analyzing plasmatocyte number and activity in culture. In adults, the induced immune responses were analyzed by bacterial clearance and quantitative real-time polymerase chain reaction (qPCR) of selected genes following infection with E. coli. The RNA levels of relevant immune pathway genes were determined in both larvae and adults by microarray analysis. The ability of larval plasmatocytes to phagocytose E. coli in culture was attenuated following spaceflight, and in parallel, the expression of genes involved in cell maturation was downregulated. In addition, the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria, and of lysozymes, antimicrobial peptide (AMP) pathway and immune stress genes, hallmarks of humoral immunity, were also reduced in larvae. In adults, the efficiency of bacterial clearance measured in vivo following a systemic infection with E. coli post-flight, remained robust. We show that spaceflight altered both cellular and humoral immune responses in Drosophila and that the disruption occurs at multiple interacting pathways. PMID:21264297

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

PubMed

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-05-17

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes*

PubMed Central

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-01-01

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane. PMID:23580643

Causes for massive bacterial colonization on mucosal membranes during infectious mononucleosis: implications for acute otitis media.

PubMed

Stenfors, Lars-Eric; Bye, Helga-Marie; Räisänen, Simo

2002-09-24

A common complication of virus-induced upper respiratory tract infections is acute otitis media caused by bacterial pathogens. Simultaneously, increased bacterial colonization in the nasopharynx occurs. Our intention in this study was to identify the causes of this increased colonization of bacteria by evaluating their coating with the antibacterial substances lysozyme, lactoferrin and immunoglobulins IgG, S-IgA and IgM and their ability to penetrate epithelial cells during infectious mononucleosis (IM) caused by Epstein-Barr virus. Cellular samples were collected from the oropharynx of 21 patients (16 males, five females; age range 10-21 years) with current IM. An immunocytochemical assay using gold-labelled antiserum to human lysozyme, lactoferrin, IgG, S-IgA and IgM followed by gold particle and epithelial cell tracing in the transmission electron microscope. A significant reduction in bacterial coating with IgG (P<0.05) and S-IgA (P<0.01) was noted, whereas there was a significant increase in coating with lactoferrin (P<0.01) and IgM (P<0.01). No significant change in lysozyme coating of the bacteria was noted, compared with healthy controls. Bacterial penetration into epithelial cells was seen particularly in patients culture-positive for beta-haemolytic streptococci. Reduced bacterial coating with IgG and S-IgA immunoglobulins, combined with bacterial penetration into epithelial cells, may exacerbate the bacterial colonization on oropharyngeal mucosal membranes observed during IM.

Metalloporphyrin-based porous polymers prepared via click chemistry for size-selective adsorption of protein.

PubMed

Zhu, Dailian; Qin, Cunqi; Ao, Shanshi; Su, Qiuping; Sun, Xiying; Jiang, Tengfei; Pei, Kemei; Ni, Huagang; Ye, Peng

2018-08-01

Zinc porphyrin-based porous polymers (PPs-Zn) with different pore sizes were prepared by controlling the reaction condition of click chemistry, and the protein adsorption in PPs-Zn and the catalytic activity of immobilized enzyme were investigated. PPs-Zn-1 with 18 nm and PPS-Zn-2 with 90 nm of pore size were characterized by FTIR, NMR and nitrogen absorption experiments. The amount of adsorbed protein in PPs-Zn-1 was more than that in PPs-Zn-2 for small size proteins, such as lysozyme, lipase and bovine serum albumin (BSA). And for large size proteins including myosin and human fibrinogen (HFg), the amount of adsorbed protein in PPs-Zn-1 was less than that in PPs-Zn-2. The result indicates that the protein adsorption is size-selective in PPs-Zn. Both the protein size and the pore size have a significant effect on the amount of adsorbed protein in the PPs-Zn. Lipase and lysozyme immobilized in PPs-Zn exhibited excellent reuse stability.

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

PubMed

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

2015-01-01

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

Study on the conformation changes of Lysozyme induced by Hypocrellin A: The mechanism investigation

NASA Astrophysics Data System (ADS)

Ma, Fei; Huang, He-Yong; Zhou, Lin; Yang, Chao; Zhou, Jia-Hong; Liu, Zheng-Ming

2012-11-01

The interactions between Lysozyme and Hypocrellin A are investigated in details using time-resolved fluorescence, fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), three-dimensional fluorescence spectra, and thermal gravimetric analysis (TGA) techniques. The results of time-resolved fluorescence suggest that the quenching mechanism is static quenching. FTIR and CD spectroscopy provide evidences of the reducing of α-helix after interaction. Hypocrellin A could change the micro-environmental of Lysozyme according to hydrophobic interaction between the aromatic ring and the hydrophobic amino acid residues, and the altered polypeptide backbone structures induce the reduction of α-helical structures. Moreover, TGA study further demonstrates the structure changes of Lysozyme on the effect of Hypocrellin A. This study could provide some important information for the derivatives of HA in pharmacy, pharmacology and biochemistry.

Enzyme activities in parotid saliva of patients with the restrictive type of anorexia nervosa.

PubMed

Paszynska, Elzbieta; Slopien, Agnieszka; Dmitrzak-Weglarz, Monika; Hannig, Christian

2017-04-01

In patients with anorexia nervosa (AN) specific signs may occur in the oral cavity, but there are conflicting reports about their significance, especially concerning changes in salivary composition. The aim of this clinical study was to evaluate the resting parotid flow rate (PFR) and the activity of the following enzymes in parotid saliva: amylase, aspartate amino transferase (AST), lysozyme, peroxidase, serine and acidic proteases in the acute phase of the restrictive type of AN and to compare the findings with those in healthy controls. Forty-one subjects participated (20 patients with AN, 21 matched healthy controls), parotid saliva was collected using a modified Lashley cap at rest. Enzyme activities were measured with fluorimetric and photometric assays. The unstimulated PFR was significantly lower than in the controls, lysozyme and AST activity was significantly lower, and amylase showed a high inter-individual variability. A positive correlation for amylase and lysozyme and negative ones for lysozyme and BMI, lysozyme and IBW%, serine protease and salivary flow were observed. The reduced PFR and enzyme activities levels suggest that AN does not only affect the quantity of the saliva but also its quality and, its biological functions. The results obtained should help to provide a better understanding of the effect of AN disease on the pathogenesis of at least some oral diseases. Further research is needed on any possible role of reduced lysozyme and transaminase activity in maintaining oral protection against external toxic agents and bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Relationship between salivary immunoglobulin a, lactoferrin and lysozyme flow rates and lifestyle factors in Japanese children: a cross-sectional study.

PubMed

Ide, Momo; Saruta, Juri; To, Masahiro; Yamamoto, Yuko; Sugimoto, Masahiro; Fuchida, Shinya; Yokoyama, Mina; Kimoto, Shigenari; Tsukinoki, Keiichi

2016-10-01

The antimicrobial substances in saliva contribute to the maintenance of both oral health and overall health of the body. Therefore, the associations among immunoglobulin A (IgA), lactoferrin and lysozyme flow rates in the saliva of children, and their relationships with the physical attributes and lifestyle factors of children, were examined. Saliva was collected from 90 children who visited the Kanagawa Dental University Hospital Pediatric Dentistry, and questionnaires were completed by guardians. IgA, lactoferrin and lysozyme concentrations were measured in the saliva samples using enzyme-linked immunosorbent assays (ELISAs). The IgA flow rate in saliva increased as age, height and weight increased. A correlation was found between lactoferrin and lysozyme flow rates. When the antimicrobial substance flow rates in the saliva were divided into two groups of 22 children each based on the highest and lowest quartiles, children with either a low or high IgA flow rate also had a high or low lactoferrin flow rate, respectively. The same pattern was observed for lactoferrin and lysozyme flow rates. There is a high probability that the IgA flow rate in the saliva of children reflects and corresponds to the developmental status of immune function as the child ages and increases in height and weight. The flow rates of lactoferrin and lysozyme were correlated in children. In addition, regarding lifestyle factors, the duration of sleep and lactoferrin flow rate were also related.

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

PubMed

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

2018-02-01

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

Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles.

PubMed

Ergul Yilmaz, Zeynep; Cordonnier, Thomas; Debuigne, Antoine; Calvignac, Brice; Jerome, Christine; Boury, Frank

2016-11-20

Calcium carbonate particles are promising candidates as proteins carriers for their controlled delivery in the body. The present paper aims at investigating the protein encapsulation by in situ precipitation of calcium carbonate particles prepared by a process based on supercritical CO 2 and using a new type of degradable well-defined double hydrophilic block copolymers composed of poly(ethylene oxide) and polyphosphoester blocks acting as templating agent for the calcium carbonate. For this study, lysozyme was chosen as a model for therapeutic protein for its availability and ease of detection. It was found that by this green process, loading into the CaCO 3 microparticles with a diameter about 2μm can be obtained as determined by scanning electron microscopy. A protein loading up to 6.5% active lysozyme was measured by a specific bioassay (Micrococcus lysodeikticus). By encapsulating fluorescent-labelled lysozyme (lysozyme-FITC), the confocal microscopy images confirmed its encapsulation and suggested a core-shell distribution of lysozyme into CaCO 3 , leading to a release profile reaching a steady state at 59% of release after 90min. Copyright © 2016. Published by Elsevier B.V.

Functional Micrococcus lysodeikticus layers deposited by laser technique for the optical sensing of lysozyme.

PubMed

Dinca, Valentina; Zaharie-Butucel, Diana; Stanica, Luciana; Brajnicov, Simona; Marascu, Valentina; Bonciu, Anca; Cristocea, Andra; Gaman, Laura; Gheorghiu, Mihaela; Astilean, Simion; Vasilescu, Alina

2018-02-01

Whole cell optical biosensors, made by immobilizing whole algal, bacterial or mammalian cells on various supports have found applications in several fields, from ecology and ecotoxicity testing to biopharmaceutical production or medical diagnostics. We hereby report the deposition of functional bacterial layers of Micrococcus lysodeikticus (ML) via Matrix-Assisted Pulsed Laser Evaporation (MAPLE) on poly(diallyldimethylamonium) (PDDA)-coated-glass slides and their application as an optical biosensor for the detection of lysozyme in serum. Lysozyme is an enzyme upregulated in inflammatory diseases and ML is an enzymatic substrate for this enzyme. The MAPLE-deposited bacterial interfaces were characterised by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Fourier-Transformed Infrared Spectroscopy (FTIR), Raman and optical microscopy and were compared with control interfaces deposited via layer-by-layer on the same substrate. After MAPLE deposition and coating with graphene oxide (GO), ML-modified interfaces retained their functionality and sensitivity to lysozyme's lytic action. The optical biosensor detected lysozyme in undiluted serum in the clinically relevant range up to 10μgmL -1 , in a fast and simple manner. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress

NASA Astrophysics Data System (ADS)

Choe, Jong Ryeol; Shin, Yoon Sub; Choi, Ji Yong; Kim, Tae Hwan; Jung, Min-Min; Choi, Cheol Young

2017-12-01

We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m2 intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H2O2), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H2O2 levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H2O2. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.

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 more attractive for larger sized nanoparticles. The nanoparticle aggregates are characterized by mass fractal.

Natural stimulus responsive scaffolds/cells for bone tissue engineering: influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings.

PubMed

Martins, Ana M; Pham, Quynh P; Malafaya, Patrícia B; Raphael, Robert M; Kasper, F Kurtis; Reis, Rui L; Mikos, Antonios G

2009-08-01

This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g/L) showed the formation of pores, indicating an increase of porosity ( approximately 5-55% up to 21 days) resulting in porous three-dimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.

A comparative study for adsorption of lysozyme from aqueous samples onto Fe3O4 magnetic nanoparticles using different ionic liquids as modifier.

PubMed

Kamran, Sedigheh; Absalan, Ghodratollah; Asadi, Mozaffar

2015-12-01

In this paper, nanoparticles of Fe3O4 as well as their modified forms with different ionic liquids (IL-Fe3O4) were prepared and used for adsorption of lysozyme. The mean size and the surface morphology of the nanoparticles were characterized by TEM, XRD and FTIR techniques. Adsorption studies of lysozyme were performed under different experimental conditions in batch system on different modified magnetic nanoparticles such as, lysozyme concentration, pH of the solution, and contact time. Experimental results were obtained under the optimum operational conditions of pH 9.0 and a contact time of 10 min when initial protein concentrations of 0.05-2.0 mg mL(-1) were used. The isotherm evaluations revealed that the Langmuir model attained better fits to the equilibrium data than the Freundlich model. The maximum obtained adsorption capacities were 370.4, 400.0 500.0 and 526.3 mg of lysozyme for adsorption onto Fe3O4 and modified magnetic nanoparticles by [C4MIM][Br], [C6MIM][Br] and [C8MIM][Br] per gram of adsorbent, respectively. The Langmuir adsorption constants were 0.004, 0.019, 0.024 and 0.012 L mg(-1) for adsorptions of lysozyme onto Fe3O4 and modified magnetic nanoparticles by [C4MIM][Br], [C6MIM][Br] and [C8MIM][Br], respectively. The adsorption capacity of lysozyme was found to be dependent on its chemical structure, pH of the solution, temperature and type of ionic liquid as modifier. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated. Furthermore, the thermodynamic parameters were calculated. Protein could desorb from IL-Fe3O4 nanoparticles by using NaCl solution at pH 9.5 and was reused.

An ultrasensitive and selective electrochemical aptasensor based on rGO-MWCNTs/Chitosan/carbon quantum dot for the detection of lysozyme.

PubMed

Rezaei, Behzad; Jamei, Hamid Reza; Ensafi, Ali Asghar

2018-05-09

An aptamer-based method is described for the electrochemical determination of lysozyme. A glassy carbon electrode was modified with a nanocomposite composed of reduced graphene oxide (rGO), multi-walled carbon nanotubes (MWCNTs), chitosan (CS), and a synthesized carbon quantum dot (CQD) from CS. The composition of the nanocomposite (rGO-MWCNT/CS/CQD) warrants a high surface-to-volume ratio, high conductivity, high stability, and great electrocatalytic activity. This nanocomposite provides a suitable site for better immobilization of aptamers due to the existence of many amino and carboxyl functional groups, and remaining oxygen-related defects properties in rGO. In addition, this nanocomposite allows considerable enhancement of the electrochemical signal and contributes to improving sensitivity. The amino-linked lysozyme aptamers were immobilized on the nanocomposite through covalent coupling between the amino groups of the aptamer and the amino groups of the nanocomposite using glutaraldehyde (GLA) linker. The modified electrode was characterized by electrochemical methods including differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). In the presence of lysozyme, the immobilized aptamer selectively caught the target lysozyme on the electrode interface that leads to a decrease in the DPV peak current and an increase in Charge Transfer Resistance (R ct ) in EIS as an analytical signal. Using the obtained data from DPV and EIS techniques, two calibration curves were drawn. The anti-lysozyme aptasensor proposed has two very low LODs. These measures are 3.7 and 1.9 fmol L -1 within the wide detection ranges of 20 fmol L -1 to 10 nmol L -1 , and 10 fmol L -1 to 100 nmol L -1 for DPV and EIS calibration curves, respectively. The GCE/rGO-MWCNT/CS/CQD showed sensitivity, high reproducibility, specificity and rapid response for lysozyme which can be used in biomedical fields. Copyright © 2018 Elsevier B.V. All rights reserved.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

PubMed

Moringo, Nicholas A; Shen, Hao; Tauzin, Lawrence J; Wang, Wenxiao; Bishop, Logan D C; Landes, Christy F

2017-10-17

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content. Single-molecule tracking indicates lysozyme loading capacities, and surface mobility at the polymer interface is hindered as a result of all functionalization techniques. Adsorption dynamics of lysozyme depend on the extent and the specificity of the oxygen functionalities introduced to the polystyrene surface. Hindered adsorption and mobility are dominated by hydrophobic effects attributed to water hydration layer formation at the functionalized polystyrene surfaces.

Salt induced reduction of lysozyme adsorption at charged interfaces

NASA Astrophysics Data System (ADS)

Göhring, Holger; Paulus, Michael; Salmen, Paul; Wirkert, Florian; Kruse, Theresa; Degen, Patrick; Stuhr, Susan; Rehage, Heinz; Tolan, Metin

2015-06-01

A study of lysozyme adsorption below a behenic acid membrane and at the solid-liquid interface between aqueous lysozyme solution and a silicon wafer in the presence of sodium chloride is presented. The salt concentration was varied between 1 mmol L-1 and 1000 mmol L-1. X-ray reflectivity data show a clear dependence of the protein adsorption on the salt concentration. Increasing salt concentrations result in a decreased protein adsorption at the interface until a complete suppression at high concentrations is reached. This effect can be attributed to a reduced attractive electrostatic interaction between the positively charged proteins and negatively charged surfaces by charge screening. The measurements at the solid-liquid interfaces show a transition from unoriented order of lysozyme in the adsorbed film to an oriented order with the short protein axis perpendicular to the solid-liquid interface with rising salt concentration.

Effect of glycerol and dimethyl sulfoxide on the phase behavior of lysozyme: Theory and experiments

NASA Astrophysics Data System (ADS)

Gögelein, Christoph; Wagner, Dana; Cardinaux, Frédéric; Nägele, Gerhard; Egelhaaf, Stefan U.

2012-01-01

Salt, glycerol, and dimethyl sulfoxide (DMSO) are used to modify the properties of protein solutions. We experimentally determined the effect of these additives on the phase behavior of lysozyme solutions. Upon the addition of glycerol and DMSO, the fluid-solid transition and the gas-liquid coexistence curve (binodal) shift to lower temperatures and the gap between them increases. The experimentally observed trends are consistent with our theoretical predictions based on the thermodynamic perturbation theory and the Derjaguin-Landau-Verwey-Overbeek model for the lysozyme-lysozyme pair interactions. The values of the parameters describing the interactions, namely the refractive indices, dielectric constants, Hamaker constant and cut-off length, are extracted from literature or are experimentally determined by independent experiments, including static light scattering, to determine the second virial coefficient. We observe that both, glycerol and DMSO, render the potential more repulsive, while sodium chloride reduces the repulsion.

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.

Effects of melatonin injection or green-wavelength LED light on the antioxidant system in goldfish (Carassius auratus) during thermal stress.

PubMed

Jung, Seo Jin; Choi, Young Jae; Kim, Na Na; Choi, Ji Yong; Kim, Bong-Seok; Choi, Cheol Young

2016-05-01

We tested the mitigating effects of melatonin injections or irradiation from green-wavelength light-emitting diodes (LEDs) on goldfish (Carassius auratus) exposed to thermal stress (high water temperature, 30 °C). The effects of the two treatments were assessed by measuring the expression and activity levels of the antioxidant enzymes, superoxide dismutase and catalase, plasma hydrogen peroxide, lipid hydroperoxide, and lysozyme. In addition, a comet assay was conducted to confirm that high water temperature damaged nuclear DNA. The expression and activity of the antioxidant enzymes, plasma hydrogen peroxide, and lipid hydroperoxide were significantly higher after exposure to high temperature and were significantly lower in fish that received melatonin or LED light than in those that received no mitigating treatment. Plasma lysozyme was significantly lower after exposure to high temperature and was significantly higher after exposure to melatonin or LED light. The comet assay revealed that thermal stress caused a great deal of damage to nuclear DNA; however, treatment with melatonin or green-wavelength LED light prevented a significant portion of this damage from occurring. These results indicate that, although high temperatures induce oxidative stress and reduce immune system strength in goldfish, both melatonin and green-wavelength LED light inhibit oxidative stress and boost the immune system. LED treatment increased the antioxidant and immune system activity more significantly than did melatonin treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Electron Microscopy of Staphylococcus aureus Cell Wall Lysis

PubMed Central

Virgilio, R.; González, C.; Muñoz, Nubia; Mendoza, Silvia

1966-01-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018–2024. 1966.—A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents. Images PMID:5939482

Electron microscopy of Staphylococcus aureus cell wall lysis.

PubMed

Virgilio, R; González, C; Muñoz, N; Mendoza, S

1966-05-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function.

PubMed

Yu, Jinyun; Chen, Tingjin; Xie, Zhizhi; Liang, Pei; Qu, Honglin; Shang, Mei; Mao, Qiang; Ning, Dan; Tang, Zeli; Shi, Mengchen; Zhou, Lina; Huang, Yan; Yu, Xinbing

2015-07-01

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Plant flavones enhance antimicrobial activity of respiratory epithelial cell secretions against Pseudomonas aeruginosa

PubMed Central

Hariri, Benjamin M.; McMahon, Derek B.; Chen, Bei; Adappa, Nithin D.; Palmer, James N.; Kennedy, David W.

2017-01-01

Flavones are a class of natural plant secondary metabolites that have anti-inflammatory and anti-bacterial effects. Some flavones also activate the T2R14 bitter taste receptor, which is expressed in motile cilia of the sinonasal epithelium and activates innate immune nitric oxide (NO) production. Flavones may thus be potential therapeutics for respiratory infections. Our objective was to examine the anti-microbial effects of flavones on the common sinonasal pathogens Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, evaluating both planktonic and biofilm growth. Flavones had only very low-level antibacterial activity alone. They did not reduce biofilm formation, but did reduce production of the important P. aeruginosa inflammatory mediator and ciliotoxin pyocyanin. However, flavones exhibited synergy against P. aeruginosa in the presence of antibiotics or recombinant human lysozyme. They also enhanced the efficacy of antimicrobials secreted by cultured and primary human airway cells grown at air-liquid interface. This suggests that flavones may have anti-gram-negative potential as topical therapeutics when combined with antibiotics or in the context of innate antimicrobials secreted by the respiratory or other epithelia. This may have an additive effect when combined with T2R14-activated NO production. Additional studies are necessary to understand which flavone compounds or mixtures are the most efficacious. PMID:28931063

An investigation of konjac glucomannan-keratin hydrogel scaffold loaded with Avena sativa extracts for diabetic wound healing.

PubMed

Veerasubramanian, Praveen Krishna; Thangavel, Ponrasu; Kannan, Ramya; Chakraborty, Sudip; Ramachandran, Balaji; Suguna, Lonchin; Muthuvijayan, Vignesh

2018-05-01

We have developed a novel hydrogel composed of konjac glucomannan (KGM), human hair proteins (KER), and an ethanolic extract of Avena sativa (OAT) and evaluated its potential as a dressing material for diabetic wounds. KGM is an excellent biocompatible gelling agent that stimulates fibroblast proliferation and immunomodulation. Human hair proteins (KER) are biocompatible, biodegradable, and possess abundant cell adhesion sites. KER also promotes fibroblast attachment and proliferation, keratinocyte migration, and collagen expression, which can accelerate wound healing. OAT consists of oat β-glucans and several anti-inflammatory and antioxidant moieties that can reduce prolonged inflammation in chronic wounds. SEM images confirm the highly porous architecture of the scaffolds. When immersed in PBS, KGM+KER+OAT hydrogels absorb 7.5 times their dry weight. These hydrogels display a measured rate of degradation in lysozyme. KGM+KER+OAT hydrogels showed no significant cytotoxicity against NIH/3T3 fibroblasts. DAPI and SEM images obtained after 48h of cell culture illustrate the attachment and infiltration of fibroblasts. In vivo studies performed using a diabetic rat excision wound model showed that KGM+KER+OAT hydrogels significantly accelerated wound healing compared to the control and the KGM+KER hydrogels. Copyright © 2018 Elsevier B.V. All rights reserved.

Apple cider vinegar boosted immunomodulatory and health promoting effects of Lactobacillus casei in common carp (Cyprinus carpio).

PubMed

Safari, Roghieh; Hoseinifar, Seyed Hossein; Nejadmoghadam, Shabnam; Khalili, Mohsen

2017-08-01

The present study was performed to investigate the immunomodulatory and health promoting effects of combined or singular administration of apple cider vinegar (ACV) and Lactobacillus casei in common carp (Cyprinus carpio) diet. An 8-week feeding trial was designed with following treatments: Control (basal diet), Pro (contains 10 7  CFU g -1 L. casei), LACV (contains 1% ACV), HACV (contains 2% ACV), Pro + LACV (contains 10 7  CFU g -1 L. casei plus 1% ACV) and Pro + HACV (contains 10 7  CFU g -1 L. casei plus 2% ACV). Evaluation of skin mucus revealed notable increase of total Ig level and lysozyme activity in Pro + LACV and Pro + HACV treatments compared other groups (P < 0.05). Similarly, serum total Ig and lysozyme activity in HACV, Pro + LACV and Pro + HACV fed carps was remarkably higher than other groups (P < 0.05). However, regarding serum alternative complement (ACH50) activity significant difference was observed just between Pro + HACV and control treatment (P < 0.05). The highest expression of immune related (LYZ, TNF-alpha, IL1b, IL8) and antioxidant enzymes genes (GSR, GST) were observed in carps fed Pro + HACV and Pro + LACV. The expression of GH gene expression in Pro, LACV and HACV treatments was significantly higher than those in control group (P < 0.05). The highest expression level of GH and IGF1 was observed in fish fed combined Pro and ACV (P < 0.05). These results indicated that co-administration of ACV boosted immunomodulatory and health promoting effects of L. casei and can be considered as a promising immunostimulants in early stage of common carp culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of brevetoxin exposure on the immune system of loggerhead sea turtles.

PubMed

Walsh, Catherine J; Leggett, Stephanie R; Carter, Barbara J; Colle, Clarence

2010-05-10

Blooms of the toxic dinoflagellate, Karenia brevis, occur almost annually off the Florida coast. These blooms, commonly called "red tides", produce a group of neurotoxins collectively termed brevetoxins. Many species of sealife, including sea turtles, are severely impacted by brevetoxin exposure. Effects of brevetoxins on immune cells were investigated in rescued loggerhead sea turtles, Caretta caretta, as well as through in vitro experiments using peripheral blood leukocytes (PBL) collected from captive sea turtles. In rescued animals, plasma brevetoxin concentrations were measured using a competitive ELISA. Plasma lysozyme activity was measured using a turbidity assay. Lysozyme activity correlated positively with plasma brevetoxin concentrations. Differential expression of genes affected by brevetoxin exposure was determined using two separate suppression subtractive hybridization experiments. In one experiment, genes from PBL collected from sea turtles rescued from red tide toxin exposure were compared to genes from PBL collected from healthy captive loggerhead sea turtles. In the second experiment, PBL from healthy captive loggerhead sea turtles were exposed to brevetoxin (500 ng PbTx-2/ml) in vitro for 18 h and compared to unexposed PBL. Results from the subtraction hybridization experiment conducted with red tide rescued sea turtle PBL indicated that genes involved in oxidative stress or xenobiotic metabolism were up-regulated. Using quantitative real-time PCR, a greater than 2-fold increase in superoxide dismutase and thioredoxin and greater than 10-fold increase in expression of thiopurine S-methyltransferase were observed. Results from the in vitro subtraction hybridization experiment indicated that genes coding for cytochrome c oxidases were the major up-regulated genes. Using quantitative real-time PCR, a greater than 8-fold increase in expression of beta-tubulin and greater than 3-fold increase in expression of ubiquinol were observed. Brevetoxin exposure may have significant implications for immune function in loggerhead sea turtles. 2009 Elsevier B.V. All rights reserved.

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

PubMed Central

Lewis, C E; McCarthy, S P; Lorenzen, J; McGee, J O

1990-01-01

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/11. This enabled the direct effects of LPS, IFN-gamma and TNF alpha on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-gamma or recombinant human TNF alpha. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNF alpha but not by LPS or IFN-gamma. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNF alpha, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZM-stimulating effects of LPS and IFN-gamma. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-gamma, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNF alpha is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity. Images Figure 1 Figure 1 PMID:2107146

A microcrystal selection technique in protein crystallization

NASA Astrophysics Data System (ADS)

Han, Qing; Lin, Sheng-Xiang

1996-10-01

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

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.

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

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Spectrophotometric studies on the interaction between (-)-epigallocatechin gallate and lysozyme

NASA Astrophysics Data System (ADS)

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

2008-02-01

Various reported antibacterial activities of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea prompted us to study its binding with lysozyme. This has been investigated by fluorescence, circular dichroism (CD) and protein-ligand docking. The binding parameters were determined using a modified Stern-Volmer equation. The thermodynamic parameters are indicative of an initial hydrophobic association. The complex is, however, held together predominantly by van der Waals interactions and hydrogen bonding. CD studies do not indicate any significant changes in the secondary structure of lysozyme. Docking studies revealed that specific interactions are observed with residues Trp 62 and Trp 63.

Three-dimensional structure of an antibody-antigen complex.

PubMed

Sheriff, S; Silverton, E W; Padlan, E A; Cohen, G H; Smith-Gill, S J; Finzel, B C; Davies, D R

1987-11-01

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.

Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: An analysis by solvent exchange model

NASA Astrophysics Data System (ADS)

Spinozzi, Francesco; Ortore, Maria Grazia; Sinibaldi, Raffaele; Mariani, Paolo; Esposito, Alessandro; Cinelli, Stefania; Onori, Giuseppe

2008-07-01

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.

Lysozyme adsorption onto mesoporous materials: effect of pore geometry and stability of adsorbents.

PubMed

Vinu, Ajayan; Miyahara, Masahiko; Hossain, Kazi Zakir; Takahashi, Motoi; Balasubramanian, Veerappan Vaithilingam; Mori, Toshiyuki; Ariga, Katsuhiko

2007-03-01

In this paper, adsorption of lysozyme onto two kinds of mesoporous adsorbents (KIT-5 and AISBA-15) has been investigated and the results on the effects of pore geometry and stability of the adsorbents are also discussed. The KIT-5 mesoporous silica materials possess cage-type pore geometry while the AISBA-15 adsorbent has mesopores of cylindrical type with rather large diameter (9.7 nm). Adsorption of lysozyme onto AISBA-15 aluminosilicate obeys a Langmuir isotherm, resulting in pore occupation of 25 to 30%. In contrast, the KIT-5 adsorbents showed very small adsorption capacities for the lysozyme adsorption, typically falling in 6 to 13% of pore occupation. The cage-type KIT-5 adsorbents have narrow channel (4 to 6 nm) where penetration of the lysozyme (3 x 3 x 4.5 nm) might be restricted. The KIT-5 adsorbent tends to collapse after long-time immersion in water, as indicated by XRD patterns, while the AISBA-15 adsorbent retains its regular structure even after immersion in basic water for 4 days. These facts confirm superiority of the AISBA-15 as an adsorbent as compared with the KIT-5 mesoporous silicates. This research strikingly demonstrates the selection of mesoporous materials is crucial to achieve efficient immobilization of biomaterials in aqueous environment.

Adsorption of lysozyme by alginate/graphene oxide composite beads with enhanced stability and mechanical property.

PubMed

Li, Jiwei; Ma, Jianwei; Chen, Shaojuan; Huang, Yudong; He, Jinmei

2018-08-01

The large-scale applications of lysozyme in the pharmaceutical industry and food industry require more efficient and cost-effective techniques for its separation/purification. In the present study, graphene oxide (GO) was encapsulated into environmentally benign sodium alginate (SA) to prepare a Ca 2+ crosslinked alginate/graphene oxide composite gel beads (Ca-SA/GO) which were then used to adsorb lysozyme from aqueous solutions. Compared with pure Ca 2+ crosslinked alginate gel beads (Ca-SA), the as-prepared Ca-SA/GO has a lower swelling degree, an improved gel stability in salt solutions, and a higher mechanical performance. This can be explained by the uniform distribution of GO sheets in the Ca-SA matrix and the existence of hydrogen bonding and high interfacial adhesion between GO filler and SA matrix demonstrated by SEM, FTIR, XRD, and TGA. Batch adsorption experiments found that the lysozyme adsorption capacity of Ca-SA/GO can reach 278.28 mg g -1 and it can be regenerated and reused at least 4 times. Moreover, in column adsorption, the Ca-SA/GO showed excellent dynamic adsorption property. With good stability, adsorption capacity, and regeneration ability, the Ca-SA/GO could be a promising adsorbent for lysozyme from aqueous solutions. Copyright © 2018. Published by Elsevier B.V.

Formation of Silica-Lysozyme Composites Through Co-Precipitation and Adsorption

NASA Astrophysics Data System (ADS)

van den Heuvel, Daniela B.; Stawski, Tomasz M.; Tobler, Dominique J.; Wirth, Richard; Peacock, Caroline L.; Benning, Liane G.

2018-04-01

Interactions between silica and proteins are crucial for the formation of biosilica and the production of novel functional hybrid materials for a range of industrial applications. The proteins control both precipitation pathway and the properties of the resulting silica-organic composites. Here we present data on the formation of silica-lysozyme composites through two different synthesis approaches (co-precipitation vs. adsorption) and show that the chemical and structural properties of these composites, when analyzed using a combination of synchrotron-based scattering (total scattering and SAXS), spectroscopic, electron microscopy and potentiometric methods vary dramatically. We document that while lysozyme was not incorporated into nor did its presence alter the molecular structure of silica, it strongly enhanced the aggregation of silica particles due to electrostatic and potentially hydrophobic interactions, leading to the formation of composites with characteristics differing from pure silica. The differences increased with increasing lysozyme content for both synthesis approaches. Yet, the absolute changes differ substantially between the two sets of composites, as lysozyme did not just affect aggregation during co-precipitation but also particle growth and likely polymerization during co-precipitation. Our results improve the fundamental understanding of how organic macromolecules interact with dissolved and nanoparticulate silica and how these interactions control the formation pathway of silica-organic composites from sodium silicate solutions, a widely available and cheap starting material.

Electrophoretic Approach for the Simultaneous Deposition and Functionalization of Reduced Graphene Oxide Nanosheets with Diazonium Compounds: Application for Lysozyme Sensing in Serum.

PubMed

Wang, Qian; Vasilescu, Alina; Wang, Qi; Coffinier, Yannick; Li, Musen; Boukherroub, Rabah; Szunerits, Sabine

2017-04-12

Electrophoretic deposition (EPD) of reduced graphene oxide nanosheets (rGO) offers several advantages over other surface coating approaches, including process simplicity, uniformity of the deposited films, and good control of the film thickness. The EPD conditions might also be of interest for the reduction of diazonium salts, which upon the release of N 2 molecules and generation of radicals, can form covalent bonds with the sp 2 hybridized carbon lattice atoms of rGO films. In this work, we report on the coating of gold electrodes in one step with rGO/polyethylenimine (PEI) thin films and their simultaneous modification using different phenyl (Ph) diazonium salt precursors bearing various functionalities such as -B(OH) 2 , -COOH, and -C≡CH. We show further the interest of such interfaces for designing highly sensitive sensing platforms. Azide-terminated lysozyme aptamers were clicked onto the rGO/PEI/Ph-alkynyl matrix and used for the sensing of lysozyme levels in patients suffering from inflammatory bowel disease (IBD), where lysozyme levels are up-regulated. The approach attained the required demand for the determination of lysozyme level in patients suffering from IBD with a 200 fM detection limit and a linear range up to 20 pM without signal amplification.

Development of lysozyme-combined antibacterial system to reduce sulfur dioxide and to stabilize Italian Riesling ice wine during aging process

PubMed Central

Chen, Kai; Han, Shun-yu; Zhang, Bo; Li, Min; Sheng, Wen-jun

2015-01-01

For the purpose of SO2 reduction and stabilizing ice wine, a new antibacterial technique was developed and verified in order to reduce the content of sulfur dioxide (SO2) and simultaneously maintain protein stability during ice wine aging process. Hazardous bacterial strain (lactic acid bacteria, LAB) and protein stability of Italian Riesling ice wine were evaluated in terms of different amounts of lysozyme, SO2, polyphenols, and wine pH by single-factor experiments. Subsequently, a quadratic rotation-orthogonal composite design with four variables was conducted to establish the multiple linear regression model that demonstrated the influence of different treatments on synthesis score between LAB inhibition and protein stability of ice wine. The results showed that, synthesis score can be influenced by lysozyme and SO2 concentrations on an extremely significant level (P < 0.01). Furthermore, the lysozyme-combined antibacterial system, which is specially designed for ice wine aging, was optimized step by step by response surface methodology and ridge analysis. As a result, the optimal proportion should be control in ice wine as follows: 179.31 mg L−1 lysozyme, 177.14 mg L−1 SO2, 0.60 g L−1 polyphenols, and 4.01 ice wine pH. Based on this system, the normalized synthesis score between LAB inhibition and protein stability can reach the highest point 0.920. Finally, by the experiments of verification and comparison, it was indicated that lysozyme-combined antibacterial system, which was a practical and prospective method to reduce SO2 concentration and effectively prevent contamination from hazardous LAB, can be used to stabilize ice wine during aging process. PMID:26405531

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

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

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

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

[Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

PubMed

Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

2017-03-01

Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes.

PubMed

Mossberg, Ann-Kristin; Hun Mok, Kenneth; Morozova-Roche, Ludmilla A; Svanborg, Catharina

2010-11-01

Human α-lactalbumin made lethal to tumor cells (HAMLET) and equine lysozyme with oleic acid (ELOA) are complexes consisting of protein and fatty acid that exhibit cytotoxic activities, drastically differing from the activity of their respective proteinaceous compounds. Since the discovery of HAMLET in the 1990s, a wealth of information has been accumulated, illuminating the structural, functional and therapeutic properties of protein complexes with oleic acid, which is summarized in this review. In vitro, both HAMLET and ELOA are produced by using ion-exchange columns preconditioned with oleic acid. However, the complex of human α-lactalbumin with oleic acid with the antitumor activity of HAMLET was found to be naturally present in the acidic fraction of human milk, where it was discovered by serendipity. Structural studies have shown that α-lactalbumin in HAMLET and lysozyme in ELOA are partially unfolded, 'molten-globule'-like, thereby rendering the complexes dynamic and in conformational exchange. HAMLET exists in the monomeric form, whereas ELOA mostly exists as oligomers and the fatty acid stoichiometry varies, with HAMLET holding an average of approximately five oleic acid molecules, whereas ELOA contains a considerably larger number (11- 48). Potent tumoricidal activity is found in both HAMLET and ELOA, and HAMLET has also shown strong potential as an antitumor drug in different in vivo animal models and clinical studies. The gain of new, beneficial function upon partial protein unfolding and fatty acid binding is a remarkable phenomenon, and may reflect a significant generic route of functional diversification of proteins via varying their conformational states and associated ligands. © 2010 The Authors Journal compilation © 2010 FEBS.

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

PubMed Central

Zhang, Zecai; Shen, Peng; Yang, Zhengtao; Zhang, Naisheng

2017-01-01

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. PMID:28184027

Off-resonance R1rho relaxation outside of the fast exchange limit: an experimental study of a cavity mutant of T4 lysozyme.

PubMed

Korzhnev, Dmitry M; Orekhov, Vladislav Yu; Dahlquist, Frederick W; Kay, Lewis E

2003-05-01

An (15)N off-resonance R(1rho) spin relaxation study of an L99A point mutant of T4 lysozyme is presented. Previous CPMG-based relaxation dispersion studies of exchange in this protein have established that the molecule interconverts between a populated ground state and an excited state (3.4%) with an exchange rate constant of 1450 s(-1) at 25 degrees C. It is shown that for the majority of residues in this protein the offset dependence of the R(1rho) relaxation rates cannot be well fit using models which are only valid in the fast exchange regime. In contrast, a recently derived expression by Trott and Palmer (J. Magn. Reson., 154, 157-160, 2002) which is valid over a wider window of exchange than other relations, is shown to fit the data well. Values of (signed) chemical shift differences between exchanging sites have been extracted and are in reasonable agreement with shift differences measured using CPMG methods. A set of simulations is presented which help establish the exchange regimes that are best suited to analysis by off-resonance R(1rho) techniques.

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

PubMed

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

2015-01-20

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

Impact of lysozyme on stability mechanism of nanozirconia aqueous suspension

NASA Astrophysics Data System (ADS)

Szewczuk-Karpisz, Katarzyna; Wiśniewska, Małgorzata

2016-08-01

The effect of lysozyme (LSZ) presence on the zirconium(IV) oxide (ZrO2) aqueous suspension stability was examined. The applied zirconia contains mesopores (with a diameter about 30 nm) and its mean particle size is about 100 nm. To determine the stability mechanism of ZrO2 suspension in the biopolymer presence, the adsorption and electrokinetic (surface charge density and zeta potential) measurements were performed in the pH range 3-10. The lysozyme adsorption on the nanozirconia surface proceeds mainly through electrostatic forces. Under solid-polymer repulsion conditions, there is no adsorption of lysozyme (pH < 6, CNaCl 0.01 mol/dm3). The increase of solution ionic strength to 0.2 mol/dm3 causes screening of unfavourable forces and biopolymer adsorption becomes possible. The LSZ addition to the ZrO2 suspension influences its stability. At pH 3, 4.6 and 7.6, slight improvement of the system stability was obtained. In turn, at pH 9 considerable destabilization of nanozirconia particles covered by polymeric layers occurs.

Microcalorimetric study of the adsorption of PEGylated lysozyme and PEG on a mildly hydrophobic resin: influence of ammonium sulfate.

PubMed

Werner, Albert; Blaschke, Tim; Hasse, Hans

2012-08-07

Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl PPG-600M, a mildly hydrophobic resin is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements in sodium phosphate buffer at pH 7.0 and 25 °C. For PEGylation two different PEG sizes are used (5 and 10 kDa) which leads to six different forms of PEGylated lysozyme all of which are systematically studied. Additionally, the adsorption of five pure PEGs is explored. The ammonium sulfate concentration is varied from 600 to 1200 mM. The molar enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It is found to be endothermic in all experiments. The comparison of the adsorption of different PEGylated forms shows that the adsorption of PEGylated lysozyme is driven by the adsorption of the PEG chain. The results provide insight into the adsorption mechanisms of polymer-modified proteins on hydrophobic chromatographic resins.

Osmotic second virial cross-coefficient measurements for binary combination of lysozyme, ovalbumin, and α-amylase in salt solutions.

PubMed

Mehta, Chirag M; White, Edward T; Litster, James D

2013-01-01

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross-interactions were measured as the osmotic second virial cross-coefficients (B23 ) for the three binary protein systems involving lysozyme, ovalbumin, and α-amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross-interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross-interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B23 value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers.

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

PubMed

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

2018-02-01

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

Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study.

PubMed

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

2017-12-01

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates. Copyright © 2017 Elsevier B.V. All rights reserved.

Improvement of wine aromatic quality using mixtures of lysozyme and dimethyl dicarbonate, with low SO2 concentration.

PubMed

Nieto-Rojo, Rodrigo; Luquin, Asuncion; Ancín-Azpilicueta, Carmen

2015-01-01

The use of sulphur dioxide (SO2) in the treatment of foodstuffs presents some problems as it could lead to pseudo-allergies in some people. The aim of this research work was to study the addition of different preservative mixtures and their influence on the concentration of volatile compounds and sensorial quality in wine. To do so, vinifications were carried out using Garnacha must to which lysozyme, dimethyl dicarbonate (DMDC) and mixtures of these with SO2 were added at different doses (25 and 50 mg l(-1)). The results were compared with a control sample to which only SO2 had been added (50 mg l(-1)). In general, mixtures of SO2 with lysozyme and DMDC favoured the formation of volatile compounds in the wines. Wines obtained from the mixtures of lysozyme and DMDC with 25 mg l(-1) of SO2 had better sensorial quality than the wines obtained with 50 mg l(-1) as the only preservative used.

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

PubMed

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

2014-01-01

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

Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorption methods by kinetics of thermal inactivation

NASA Astrophysics Data System (ADS)

Puentes-Camacho, Daniel; Velázquez, Enrique F.; Rodríguez-Félix, Dora E.; Castillo-Ortega, Mónica; Sotelo-Mundo, Rogerio R.; del Castillo-Castro, Teresa

2017-12-01

Proteins suffer changes in their tertiary structure when they are immobilized, and enzymatic activity is affected due to the low biocompatibility of some supporting materials. In this work immobilization of lysozyme on carbon nanotubes previously functionalized by microwave irradiation was studied. The effectiveness of the microwave-assisted acid treatment of carbon nanotubes was evaluated by XPS, TEM, Raman and FTIR spectroscopy. The carboxylic modification of nanotube surfaces by this fast, simple and feasible method allowed the physical adsorption and covalent linking of active lysozyme onto the carbonaceous material. Thermal inactivation kinetics, thermodynamic parameters and storage stability were studied for adsorbed and covalent enzyme complexes. A major stability was found for lysozyme immobilized by the covalent method, the activation energy for inactivation of the enzyme was higher for the covalent method and it was stable after 50 d of storage at 4 °C. The current study highlights the effect of protein immobilization method on the biotechnological potential of nanostructured biocatalysts.

The influence of size, structure and hydrophilicity of model surfactants on the adsorption of lysozyme to oil-water interface--interfacial shear measurements.

PubMed

Baldursdottir, Stefania G; Jorgensen, Lene

2011-10-01

The flexibility and aggregation of proteins can cause adsorption to oil-water interfaces and thereby create challenges during formulation and processing. Protein adsorption is a complex process and the presence of surfactants further complicates the system, in which additional parameters need to be considered. The purpose of this study is to scrutinize the influence of surfactants on protein adsorption to interfaces, using lysozyme as a model protein and sorbitan monooleate 80 (S80), polysorbate 80 (T80), polyethylene-block-poly(ethylene glycol) (PE-PEG) and polyglycerol polyricinoleate (PG-PR) as model surfactants. Rheological properties, measured using a TA AR-G2 rheometer equipped with a double wall ring (DWR) geometry, were used to compare the efficacy of the surfactant in hindering lysozyme adsorption. The system consists of a ring and a Delrin® trough with a circular channel (interfacial area=1882.6 mm(2)). Oscillatory shear measurements were conducted at a constant frequency of 0.1 Hz, a temperature of 25°C, and with strain set to 1%. The adsorption of lysozyme to the oil-water interface results in the formation of a viscoelastic film. This can be prevented by addition of surfactants, in a manner depending on the concentration and the type of surfactant. The more hydrophilic surfactants are more effective in hindering lysozyme adsorption to oil-water interfaces. Additionally, the larger surfactants are more persistent in preventing film formation, whereas the smaller ones eventually give space for the lysozyme on the interface. The addition of a mixture of two different surfactants was only beneficial when the two hydrophilic surfactants were mixed, in which case a delay in the multilayer formation was detected. The method is able to detect the interfacial adsorption of lysozyme and thus the hindering of film formation by model surfactants. It can therefore aid in processing of any delivery systems for proteins in which the protein is introduced to oil-water interfaces. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ11 dextranase mouthwash

NASA Astrophysics Data System (ADS)

Ren, Wei; Wang, Shujun; Lü, Mingsheng; Wang, Xiaobei; Fang, Yaowei; Jiao, Yuliang; Hu, Jianen

2016-03-01

We adopted the response surface methodology using single factor and orthogonal experiments to optimize four types of antimicrobial agents that could inhibit biofilm formation by Streptococcus mutans, which is commonly found in the human oral cavity and causes tooth decay. The objective was to improve the function of marine Arthrobacter oxydans KQ11 dextranase mouthwash (designed and developed by our laboratory). The experiment was conducted in a three-level, four-variable central composite design to determine the best combination of ZnSO4, lysozyme, citric acid and chitosan. The optimized antibacterial agents were 2.16 g/L ZnSO4, 14 g/L lysozyme, 4.5 g/L citric acid and 5 g/L chitosan. The biofilm formation inhibition reached 84.49%. In addition, microscopic observation of the biofilm was performed using scanning electron microscopy and confocal laser scanning microscopy. The optimized formula was tested in marine dextranase Arthrobacter oxydans KQ11 mouthwash and enhanced the inhibition of S. mutans. This work may be promoted for the design and development of future marine dextranase oral care products.

Crystal Structures of the β2-Adrenergic Receptor

NASA Astrophysics Data System (ADS)

Weis, William I.; Rosenbaum, Daniel M.; Rasmussen, Søren G. F.; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Yao, Xiao-Jie; Day, Peter W.; Parnot, Charles; Fung, Juan J.; Ratnala, Venkata R. P.; Kobilka, Brian K.; Cherezov, Vadim; Hanson, Michael A.; Kuhn, Peter; Stevens, Raymond C.; Edwards, Patricia C.; Schertler, Gebhard F. X.; Burghammer, Manfred; Sanishvili, Ruslan; Fischetti, Robert F.; Masood, Asna; Rohrer, Daniel K.

G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are responsible for the majority of signal transduction events involving hormones and neuro-transmitters across the cell membrane. GPCRs that bind to diffusible ligands have low natural abundance, are relatively unstable in detergents, and display basal G protein activation even in the absence of ligands. To overcome these problems two approaches were taken to obtain crystal structures of the β2-adrenergic receptor (β2AR), a well-characterized GPCR that binds cate-cholamine hormones. The receptor was bound to the partial inverse agonist carazolol and co-crystallized with a Fab made to a three-dimensional epitope formed by the third intracellular loop (ICL3), or by replacement of ICL3 with T4 lysozyme. Small crystals were obtained in lipid bicelles (β2AR-Fab) or lipidic cubic phase (β2AR-T4 lysozyme), and diffraction data were obtained using microfocus technology. The structures provide insights into the basal activity of the receptor, the structural features that enable binding of diffusible ligands, and the coupling between ligand binding and G-protein activation.

Repacking the Core of T4 lysozyme by automated design.

PubMed

Mooers, Blaine H M; Datta, Deepshikha; Baase, Walter A; Zollars, Eric S; Mayo, Stephen L; Matthews, Brian W

2003-09-19

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations

USDA-ARS?s Scientific Manuscript database

Lysozyme was attached through an amide linkage between protein aspartate and glutamate residues to amino-glycine-cellulose (AGC), which was prepared by esterification of glycine to preparations of cotton nanocrystals (CNC). The nanocrystalline preparations were produced through acid hydrolysis and ...

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

PubMed

Pezzini, J; Cabanne, C; Dupuy, J-W; Gantier, R; Santarelli, X

2014-06-01

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigation of structural changes of β-casein and lysozyme at the gas-liquid interface during foam fractionation.

PubMed

Barackov, Ivana; Mause, Anika; Kapoor, Shobhna; Winter, Roland; Schembecker, Gerhard; Burghoff, Bernhard

2012-10-15

Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and β-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH

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

Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose i and ii cotton nanocrystalline preparations

USDA-ARS?s Scientific Manuscript database

Lysozyme was attached through an amide linkage between some of the protein’s aspartate and glutamate residues to amino-glycine-cellulose (AGC), which was prepared by esterification of glycine to preparations of cotton nanocrystals (CNC). The nanocrystalline preparations were produced through acid h...

Lysozyme as an alternative to growth promoting antibiotics in swine production

USDA-ARS?s Scientific Manuscript database

Lysozyme is a naturally occurring enzyme found in bodily secretions such as tears, saliva, and milk. It functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, which leads to cell death. Antibiotics are also antimicrobials and have been fed at subtherape...

Growth-active peptides are produced from alpha-lactalbumin and lysozyme.

PubMed

Kanda, Yoshikazu; Hisayasu, Sanae; Abe, Yasuko; Katsura, Kenichiro; Mashimo, Keico

2007-07-19

We determined the growth-active domains of milk-growth factor (MGF), human alpha-lactalbumin (HMLA) and human lysozyme (HMLZ), and their sequences. Fetal calf serum (FCS) showed inhibitors against proteases. The growth-stimulation of IMR90 cells in CG medium (free-serum) without FCS was induced in a dose-dependent manner up to 400 ng/ml of HMLA, HMLZ or chicken lysozyme (ChLZ), and also in a time-dependent manner until 48 h but was induced gradually until 1000 ng/ml of bovine alpha-lactalbumin (BVLA). The HMLAL6-peptide (HMLAL6), a cleaved product from HMLA by Endpeptidase Lys C, was growth-stimulative. The sequence of HMLAL6 was matched to 35 amino-acid residues (from No. 59 to No. 93 of HMLA), owing to the sequences of HMLAL6R3, HMLAL6R5 and HMLAL6R7 after the reduction of HMLAL6. The sequences of the reduced peptides from MGFL7-peptide (MGFL7: a cleaved product from MGF by Endpeptidase lysine C matched to those of the peptides from HMLAL6, and were similarly identified as the partial sequence of HMLA (59-93, H(2)N-L.W.C.?.K./S.S.Q.V.P.Q.S.R.N.I.?.D.I.S.?.D.K./F.L. D.D.D.I.T.D.D.I.M.?.A.-COOH). The sequence of HMLZ is similar to that of HMLA. HMLZT7-peptide (HMLZT7), a cleaved product of HMLZ by trypsin, was confirmed to have growth-stimulating activity and it's sequence was partially identified as Y. W.?.N.D.G.K.T.P.G.A.V.N.A.?.H.L. -, owing to the results of HMLZT7R1 (reduction of HMLZT7) and HMLZA7R2 (reduction of HMLZA7-peptide (HMLZA7) cleaved product of HMLZ by Endpeptidase Arg C) and is accordingly the sequence from No. 63 to No. 97 of HMLZ. Therefore, the peptides produced from LA and LZ by proteolysis may play a role of growth-stimulation.

Tetragonal Chicken Egg White Lysozyme Solubility in Sodium Chloride Solutions

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth L.; Judge, Russell A.; Pusey, Marc L.

1998-01-01

The solubility of chicken egg white lysozyme, crystallized in the tetragonal form was measured in sodium chloride solutions from 1.6 to 30.7 C, using a miniature column solubility apparatus. Sodium chloride solution concentrations ranged from 1 to 7% (w/v). The solutions were buffered with 0.1 M sodium acetate buffer with the solubility being measured at pH values in 0.2 pH unit increments in the range pH 4.0 to 5.4, with data also included at pH 4.5. Lysozyme solubility was found to increase with increases in temperature and decreasing salt concentration. Solution pH has a varied and unpredictable effect on solubility.

Signals of monocyte activation in patients with SLE.

PubMed Central

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

1983-01-01

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

Immune response of greenback flounder Rhombosolea tapirina after exposure to contaminated marine sediment and diet.

PubMed

Mondon, J A; Duda, S; Nowak, B F

2000-01-01

Non-specific immune response of greenback flounder, Rhombosolea tapirina, exposed to contaminated marine sediments was examined. Reference sediments from Port Sorell and contaminated sediments from Deceitful Cove, Tasmania, Australia were investigated. Hatchery-reared flounder were exposed to reference sediment, contaminated sediment or contaminated sediment and diet for 6 weeks. Phagocytic capacity and lysozyme response in flounder were examined on cessation of exposure trial. Significant differences were found in phagocytic capacity and lysozyme response between treatments. Exposure to contaminated sediment, irrespective of diet or benthic disturbance elicited inhibition of phagocytic efficiency in flounder. Disturbance of contaminated sediment stimulated lysozyme activity. The immune response in flounder indicates potential immunotoxicity of sediment from Deceitful Cove.

Effect of intracrystalline water on longitudinal sound velocity in tetragonal hen-egg-white lysozyme crystals.

PubMed

Tachibana, M; Koizumi, H; Kojima, K

2004-05-01

Longitudinal sound velocity of tetragonal hen-egg-white (HEW) lysozyme crystals was measured during air drying by ultrasonic pulseecho method. The sound velocity increases with exposure to open air and approaches a constant value. The maximum value is approximately 2900 m/s that is about 1.6 times as much as that of original one before drying. In addition, the sound velocity clearly recovers to original one after immersing the dried crystal in solution. Therefore, the sound velocity in tetragonal HEW lysozyme crystals can be reversibly changed due to dehydration and rehydration. These changes in sound velocity are discussed in the light of water-mediated intramolecular and intermolecular interactions in the crystals.

Effect of intracrystalline water on longitudinal sound velocity in tetragonal hen-egg-white lysozyme crystals

NASA Astrophysics Data System (ADS)

Tachibana, M.; Koizumi, H.; Kojima, K.

2004-05-01

Longitudinal sound velocity of tetragonal hen-egg-white (HEW) lysozyme crystals was measured during air drying by ultrasonic pulseecho method. The sound velocity increases with exposure to open air and approaches a constant value. The maximum value is ˜2900 m/s that is about 1.6 times as much as that of original one before drying. In addition, the sound velocity clearly recovers to original one after immersing the dried crystal in solution. Therefore, the sound velocity in tetragonal HEW lysozyme crystals can be reversibly changed due to dehydration and rehydration. These changes in sound velocity are discussed in the light of water-mediated intramolecular and intermolecular interactions in the crystals.

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

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

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

PubMed Central

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

2011-01-01

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

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

PubMed

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

2016-09-01

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

A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

PubMed

Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung

2018-02-27

Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

Lactoferrin and necrotizing enterocolitis.

PubMed

Sherman, Michael P

2013-03-01

Lactoferrin (LF) is a multifunctional protein and a member of the transferrin family. LF and lysozyme in breast milk kill bacteria. In the stomach, pepsin digests and releases a potent peptide antibiotic called lactoferricin from native LF. The antimicrobial characteristics of LF may facilitate a healthy intestinal microbiome. LF is the major whey in human milk; its highest concentration is in colostrum. This fact highlights early feeding of colostrum and also fresh mature milk as a way to prevent necrotizing enterocolitis. Copyright © 2013 Elsevier Inc. All rights reserved.

The Disruptive Effect of Lysozyme on the Bacterial Cell Wall Explored by an "In-Silico" Structural Outlook

ERIC Educational Resources Information Center

Primo, Emiliano D.; Otero, Lisandro H.; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall…

Destruction of single-species biofilms of Escherichia coli or Klebsiella pneumoniae subsp. pneumoniae by dextranase, lactoferrin, and lysozyme

USDA-ARS?s Scientific Manuscript database

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...

Effect of lysozyme or antibiotics on fecal zoonotic pathogens in nursery pigs

USDA-ARS?s Scientific Manuscript database

The objective of this study was to determine the effect of lysozyme and antibiotics on zoonotic pathogen shedding in feces from nursery pigs housed without and with an indirect disease challenge. Two replicates of 600 pigs each were weaned and randomly assigned to one of 24 pens in either a nursery...

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…

Formation of Protoplasts from Resting Spores

PubMed Central

Fitz-James, Philip C.

1971-01-01

Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca2+ but Mn2+ could not replace Mg2+ in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores. Images PMID:4995380

Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

PubMed

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

2016-02-01

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

Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration.

PubMed

Lerbret, Adrien; Affouard, Frédéric

2017-10-12

Water and glycerol are well-known to facilitate the structural relaxation of amorphous protein matrices. However, several studies evidenced that they may also limit fast (∼picosecond-nanosecond, ps-ns) and small-amplitude (∼Å) motions of proteins, which govern their stability in freeze-dried sugar mixtures. To determine how they interact with proteins and sugars in glassy matrices and, thereby, modulate their fast dynamics, we performed molecular dynamics (MD) simulations of lysozyme/trehalose/glycerol (LTG) and trehalose/glycerol (TG) mixtures at low glycerol and water concentrations. Upon addition of glycerol and/or water, the glass transition temperature, T g , of LTG and TG mixtures decreases, the molecular packing of glasses is improved, and the mean-square displacements (MSDs) of lysozyme and trehalose either decrease or increase, depending on the time scale and on the temperature considered. A detailed analysis of the hydrogen bonds (HBs) formed between species reveals that water and glycerol may antiplasticize the fast dynamics of lysozyme and trehalose by increasing the total number and/or the strength of the HBs they form in glassy matrices.

Lysozyme as a recognition element for monitoring of bacterial population.

PubMed

Zheng, Laibao; Wan, Yi; Yu, Liangmin; Zhang, Dun

2016-01-01

Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5) cfu mL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test. Copyright © 2015 Elsevier B.V. All rights reserved.

[Level of selected antibacterial tear proteins in children with diabetes type 1].

PubMed

Moll, Agnieszka; Wyka, Krystyna; Młynarski, Wojciech; Niwald, Anna

2011-01-01

Antibacterial immunity in diabetes is impaired, which increases the risk of general and local infections. The aim of the study was to evaluate non-specific local antibacterial immunity based on lactoferrin and lysozyme concentration in tears in children with diabetes type 1. Children at the age of 10-18 years old were studied. Group 1. consisted of children without diabetes, group 2. included patients with new onset of diabetes and group 3. consisted of children with decade-long diabetes. Among all patients tears were collected from inferior coniunctival fornix with hematocrit glass capillaries in purpose to measure lactoferrin and lysozyme concentration. ELISA method was used in laboratory testing. Level of lactoferrin did not differ significantly among all groups. Concentration of lysozyme was statistically lower in group with decade-long diabetes (group 3.) compared to patients without diabetes. Mild correlation between lactoferrin and lysozyme levels was seen in individual patients in whole group of probands together. Diabetes type 1 in children is associated with significant changes in concentration of tear proteins, which contribute to antibacterial immunity.

Protein nanocrystallography: growth mechanism and atomic structure of crystals induced by nanotemplates.

PubMed

Pechkova, E; Vasile, F; Spera, R; Fiordoro, S; Nicolini, C

2005-11-01

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant alpha-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation.

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

PubMed

Voznesenskaia, F M; Panshinskaia, N M

1976-01-01

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

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.

Co-immobilization of cellulase and lysozyme on amino-functionalized magnetic nanoparticles: An activity-tunable biocatalyst for extraction of lipids from microalgae.

PubMed

Chen, Qingtai; Liu, Dong; Wu, Chongchong; Yao, Kaisheng; Li, Zhiheng; Shi, Nan; Wen, Fushan; Gates, Ian D

2018-05-03

An activity-tunable biocatalyst for Nannochloropsis sp. cell-walls degradation was prepared by co-immobilization of cellulase and lysozyme on the surface of amino-functionalized magnetic nanoparticles (MNPs) employing glutaraldehyde. The competition between cellulase and lysozyme during immobilization was caused by the limited active sites of the MNPs. The maximum recovery of activities (cellulase: 78.9% and lysozyme: 69.6%) were achieved due to synergistic effects during dual-enzyme co-immobilization. The thermal stability in terms of half-life of the co-immobilized enzymes was three times higher than that in free form and had higher catalytic efficiency for hydrolysis of cell walls. Moreover, the co-immobilized enzymes showed greater thermal stability and wider pH tolerance than free enzymes under harsh conditions. Furthermore, the co-immobilized enzymes retained up to 60% of the residual activity after being recycled 6 times. This study provides a feasible approach for the industrialization of enzyme during cell-walls disruption and lipids extraction from Nannochloropsis sp. Copyright © 2018. Published by Elsevier Ltd.

Probabilistic approach to lysozyme crystal nucleation kinetics.

PubMed

Dimitrov, Ivaylo L; Hodzhaoglu, Feyzim V; Koleva, Dobryana P

2015-09-01

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

PubMed

Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P

2013-01-01

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

Relationship between β-relaxation and structural stability of lysozyme: Microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments

NASA Astrophysics Data System (ADS)

Hédoux, Alain; Paccou, Laurent; Guinet, Yannick

2014-06-01

Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein-trehalose interactions in the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.

Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Shi, Ruixin; Hao, Changchun; Chen, Huan; Zhang, Lei; Li, Junhua; Xu, Guoqing; Sun, Runguang

2016-09-01

The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics. The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine (DPPE) by analyzing the data of surface pressure-area (π-A) isotherms and surface pressure-time (π-T) curves. Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 mN/m. However, the changes of DPPE are larger than DPPC, indicating stronger interaction of lysozyme with DPPE than DPPC. The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 mN/m. Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers, which leads to self-aggregation and self-assembly, forming irregular multimers and conical multimeric. Through analysis, we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201603026), and the National University Science and Technology Innovation Project of China (Grant No. 201610718013).

Protein crystal growth in low gravity

NASA Technical Reports Server (NTRS)

Feigelson, Robert S.

1993-01-01

This Final Technical Report for NASA Grant NAG8-774 covers the period from April 27, 1989 through December 31, 1992. It covers five main topics: fluid flow studies, the influence of growth conditions on the morphology of isocitrate lyase crystals, control of nucleation, the growth of lysozyme by the temperature gradient method and graphoepitaxy of protein crystals. The section on fluid flow discusses the limits of detectability in the Schlieren imaging of fluid flows around protein crystals. The isocitrate lyase study compares crystals grown terrestrially under a variety of conditions with those grown in space. The controlling factor governing the morphology of the crystals is the supersaturation. The lack of flow in the interface between the drop and the atmosphere in microgravity causes protein precipitation in the boundary layer and a lowering of the supersaturation in the drop. This lowered supersaturation leads to improved crystal morphology. Preliminary experiments with lysozyme indicated that localized temperature gradients could be used to nucleate crystals in a controlled manner. An apparatus (thermonucleator) was designed to study the controlled nucleation of protein crystals. This apparatus has been used to nucleate crystals of materials with both normal (ice-water, Rochelle salt and lysozyme) and retrograde (horse serum albumin and alpha chymotrypsinogen A) solubility. These studies have lead to the design of an new apparatus that small and more compatible with use in microgravity. Lysozyme crystals were grown by transporting nutrient from a source (lysozyme powder) to the crystal in a temperature gradient. The influence of path length and cross section on the growth rate was demonstrated. This technique can be combined with the thermonucleator to control both nucleation and growth. Graphoepitaxy utilizes a patterned substrate to orient growing crystals. In this study, silicon substrates with 10 micron grooves were used to grow crystals of catalase, lysozyme and canavalin. In all cases, the crystals grew oriented to the substrate. The supersaturation needed for nucleation and growth was lower on the patterned substrates. In some cases, isolated, large crystals were grown.

[Alterations in tears aqueous layer during cytostatics treatment].

PubMed

Wojciechowska, Katarzyna; Wieckowska-Szakiel, Marzena; Rózalska, Barbara; Jurowski, Piotr

2013-01-01

The aim of the study was to evaluate tears secretion, pH and lysozyme activity in tears aqueous layer during chemotherapy in lung, breast and bowel cancer. 36 patients were enrolled to the study. Depending on the type of cancer and type of chemotherapy patients were divided into three groups. Group I (12 patients) diagnosed with non-small-cell lung cancer treated with PE schema (cisplatin, etoposide), Group II (12 patients) with breast cancer treated with FAC schema (fluorouracil, doxorubicin, cyclophosphamide), Group III (12 patients) with bowel cancer treated with FU/LV schema (fluorouracil, leucovorin). In all the patients: Schirmer's I test, pH measurements and lysozyme test were performed. Patients were examined before chemotherapy, after 2nd, 4th, 6th cycle. In group I and II lowering of tears secretion (p < 0.001) was revealed. In group III there was higher tears secretion (p < 0.001). PH was lowered after 2nd chemotherapy course in group I and II. In further treatment pH value were in the same lower level as after the second course. In group III there was higher pH--more alkaline (p < 0.001) after 2nd cycle of treatment and it was on the same level to the end of the examination process. Lowering of lysozyme activity in the tears film in all groups (p < 0.001) was established. The higher alterations of the lysozyme activity were observed in group treated with FAC schema. Cytostatic treatment has major influence on tears aqueous layer causing alterations of tears secretions. PH alterations depending on type of chemotherapy was observed. Lowering of lysozyme activity in tears was observed. All the deteriorations aggravate with duration of chemotherapy. Alterations of tears film parameters during chemotherapy may influence upon eye surface homeostasis and infectious complication. tears aqueous layer, Schirmer's test, lysozyme activity, tears pH.

Does Warming a Lysozyme Solution Cook Ones Data?

NASA Technical Reports Server (NTRS)

Pusey, Marc; Burke, Michael; Judge, Russell

2000-01-01

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

Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

PubMed

Gill, Christina; van de Wijgert, Janneke H H M; Blow, Frances; Darby, Alistair C

2016-01-01

Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies. However, we recommend that the same method is used on all samples within a particular study.

Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough.

PubMed

Dubey, S; Kalia, Y N

2014-06-10

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm(2) (0.7mM; pH6) was surprisingly low (5.37±3.46μg/cm(2) in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13μg/cm(2), respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750μm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750μm dermatomed skin (13.57±12.20 and 5.37±3.46μg/cm(2), respectively), there was a statistically significant increase across tape-stripped skin and heat-separated dermis (36.86±7.48 and 43.42±13.11μg/cm(2), respectively) - although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat-separated dermis - indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skin microenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Anions play an important role in the crystallization of lysozyme, and are known to bind to the crystalline protein. 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. Studies using other approaches have reported more chloride ion binding sites, but their locations were not known. Knowing the precise location of these anions is also useful in determining the correct electrostatic fields surrounding the protein. In the first part of this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from the lysozyme crystals grown in bromide and chloride solutions under identical conditions. The anion locations were then obtained from standard crystallographic methods and five possible anion binding sites were found in this manner. The sole chloride ion binding site found in previous studies was confirmed. The remaining four sites were new ones for tetragonal lysozyme crystals. However, three of these new sites and the previously found one corresponded to the four unique binding sites found for nitrate ions in monoclinic crystals. This suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed. It is unlikely that there are many more anions in the tetragonal lysozyme crystal structure. Assuming osmotic equilibrium it can be shown that there are at most three more anions in the crystal channels. Some of the new anion binding sites found in this study were, as expected, in pockets containing basic residues. However, some of them were near neutral, but polar, residues. Thus, the study also showed the importance of uncharged, but polar groups, on the protein surface in determining its electrostatic field. This was important for the second part of this study where the electrostatic field 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.

Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa

PubMed Central

Evans, David J.; Fleiszig, Suzanne M. J.

2017-01-01

It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen. PMID:28489917

Effects of dietary n-3 highly unsaturated fatty acids (HUFAs) on growth, fatty acid profiles, antioxidant capacity and immunity of sea cucumber Apostichopus japonicus (Selenka).

PubMed

Yu, Haibo; Gao, Qinfeng; Dong, Shuanglin; Zhou, Jishu; Ye, Zhi; Lan, Ying

2016-07-01

The present study was conducted to understand the effects of dietary n-3 highly unsaturated fatty acids (HUFAs) on growth, fatty acid profiles, antioxidant capacity and the immunity of sea cucumber Apostichopus japonicus (Selenka). Five experimental diets were prepared, containing graded levels of n-3 HUFAs (0.46%, 0.85%, 1.25%, 1.61% and 1.95%, respectively), and the 0.46% group was used as control group. The specific growth rates, fatty acid profiles, activities and gene expression of antioxidative enzymes and lysozyme of the sea cucumbers that were fed with the 5 experimental diets were determined. The results showed that the specific growth rate of sea cucumbers in all the treatment groups significantly increased compared to the control group (P < 0.05), indicating the positive effects of n-3 HUFAs on the growth of sea cucumbers. The contents of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in the body wall of the sea cucumbers gradually increased with the increasing levels of n-3 HUFAs in the diets. The suitable supplement of n-3 HUFAs in diets improved the activities of superoxide dismutase (SOD) and catalase (CAT) of sea cucumbers by up-regulating the expression of SOD and CAT mRNA in sea cucumbers. However, excess n-3 HUFAs in diets caused lipid peroxidation, inhibited the expression of lysozyme (LSZ) mRNA and decreased the activities of LSZ in sea cucumbers. In summary, the suitable supplement levels of n-3 HUFAs in diets of sea cucumbers A. japonicus were estimated between 0.85% and 1.25% considering the growth performance, cost and the indicators of antioxidant capacity and immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

ERIC Educational Resources Information Center

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

2008-01-01

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

Inflammasome activation: how macrophages watch what they eat.

PubMed

Vance, Russell E

2010-01-21

Underhill and colleagues (Shimada et al., 2010) provide evidence for an intriguing link between the activity of lysozyme in the phagosome and activation of the Nlrp3 inflammasome, a cytosolic regulator of inflammation and cytokine production. The authors show that resistance to lysozyme allows Staphylococcus aureus to evade Nlrp3 activation. 2010 Elsevier Inc. All rights reserved.

The Effect of Ethylene Glycol, Glycine Betaine, and Urea on Lysozyme Thermal Stability

ERIC Educational Resources Information Center

Schwinefus, Jeffrey J.; Leslie, Elizabeth J.; Nordstrom, Anna R.

2010-01-01

The four-week student project described in this article is an extension of protein thermal denaturation experiments to include effects of added cosolutes ethylene glycol, glycine betaine, and urea on the unfolding of lysozyme. The transition temperatures and van't Hoff enthalpies for unfolding are evaluated for six concentrations of each cosolute,…

Antimicrobial peptide gene expression in periodontitis patients: A pilot study.

PubMed

Jourdain, Marie-Laure; Pierrard, Loïc; Kanagaratnam, Lukshe; Velard, Frédéric; Sergheraert, Johan; Lefèvre, Benoît; Gangloff, Sophie C; Braux, Julien

2018-05-01

Antimicrobial peptides (AMPs) are one of the most active components of innate immunity and have characteristics that could place them at the heart of the pathogenesis of periodontal disease. This study investigated differences in the expression of AMP coding genes obtained using a simple harvesting technique, gingival smear, between two groups of patients: chronic periodontitis subjects versus healthy ones. Twenty-three patients were enrolled in two groups: 12 were diagnosed with moderate or severe generalized chronic periodontitis, and 11 were diagnosed as clinically healthy. Gingival smears were retrieved and studied using reverse transcription-quantitative PCR (RT-qPCR) after mRNA purification. Fifteen gene expressions were obtained using real-time RT-qPCR. Three AMP genes, histatin 3 (HTN3), α-defensin 4 (DEFA4) and lysozyme C (LYZ), presented different expression levels in periodontitis patients compared with healthy subjects. The relative expression level of DEFA4 appeared to be a protective factor against periodontitis. Gingival smears studied by RT-qPCR may be used to assess the expression of AMPs coding genes. A lack of expression of DEFA4 could be a potential indicator of periodontitis status. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

PubMed

Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N

2011-02-21

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.

Preliminary study on the relationship between dexamethasone and pathogen susceptibility on crucian carp (Carassius auratus).

PubMed

Qi, Xiao-Zhou; Li, Dong-Liang; Tu, Xiao; Song, Chen-Guang; Ling, Fei; Wang, Gao-Xue

2016-12-01

Dexamethasone, a known immunosuppressant, can inhibit the immune response and increase the amount of pathogen in body, but the role of dexamethasone affecting susceptibility of crucian carp (Carassius auratus) to pathogen is unclear. The effects of dexamethasone on susceptibility of crucian carp to Aeromonas hydrophila were investigated in this study. The fish were divided into four groups randomly and injected intraperitoneally by dexamethasone for 0 day (group D), 3 days (group C), 6 days (group B), and 9 days (group A), respectively. The serum lysozyme activity was significantly declined in group A, B and C. Relative immune gene expression such as il-1β, cxcl-8, tnfα and crp in kidney were down-regulation compared to group D. After that crucian carp were infected with A. hydrophila, crucian carp treated by dexamethasone had higher mortality (group A 95%, group B 76%, group C 31%) when compared to group D (4% mortality); the amount of pathogen in was significantly increased (P < 0.05) in liver, kidney and spleen of fish in group A-C compared to group D. These results implicated that higher susceptibility caused by dexamethasone may be induced by the decrease of lysozyme activity and the down-regulation of some immune genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmocytomas are unknown.

PubMed

Butler, J E; Wertz, N; Sun, X-Z; Lunney, J K; Muyldermans, S

2013-01-01

The immunoglobulin (Ig) genes of many vertebrates have been characterized but IgG subclasses, IgD and IgE proteins are only available for three species in which plasmacytomas occur. This creates a major problem in the production and specificity verification of diagnostic anti-Ig reagents for the vast majority of mammals. We describe a novel solution using the swine system with its eleven different variants of IgG. It involves the in vitro synthesis of chimeric porcine-camelid heavy chain antibodies (HCAbs) that do not require light chains and therefore only a single transfection vector. The expressed chimeric HCAbs are comprised of the camelid VHH domain encoding specificity for lysozyme and the hinge, CH2 and CH3 domains of the various porcine IgGs. These HCAb retain their antigenic integrity and their ability to recognize lysozyme. The engineered specificity assures that these HCAb can be immobilized in native configuration when used for testing the specificity of anti-swine IgG antibodies. Comparative data to illustrate the importance of this point are provided. These are now available for use in hybridoma selection and as reference standards for evaluating the specificity of currently available anti-swine IgG antibodies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

PubMed

Ben Bacha, Abir; Abid, Islem

2013-03-01

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Diquafosol Tetrasodium Increases the Concentration of Mucin-like Substances in Tears of Healthy Human Subjects.

PubMed

Shigeyasu, Chika; Hirano, Shinichiro; Akune, Yoko; Yamada, Masakazu

2015-09-01

This study was undertaken to determine the effect of topical application of diquafosol tetrasodium on proteins and mucin-like substances from tears of clinically healthy subjects. Tears were collected from both the eyes of 10 healthy volunteers. Diquafosol tetrasodium solution (3%) was applied once to the right eye and 0.9% sodium chloride solution (saline) once to the left eye. Tear samples were collected by Schirmer test strips before application and 5, 15, 30 and 60 min after application. Sialic acid, a marker of mucin-like substances, and major tear proteins including secretory IgA, lactoferrin, lipocalin-1, and lysozyme were measured by high performance liquid chromatography. Levels of total protein, sIgA and lysozyme were transiently decreased in both groups but returned to baseline levels within 15 min after application. The concentration of lactoferrin and lipocalin-1 did not change significantly in both groups. Sialic acid in tears was significantly decreased 5 min after saline application, but significantly increased 5 min after diquafosol application. No significant difference in sialic acid was seen after 15 min in both groups. Topical application of saline and diquafosol resulted in transient decrease of tear proteins possibly due to wash out or dilution effects. In contrast, diquafosol application significantly increased sialic acid, although the effect was transient. This suggests diquafosol stimulates the secretion of mucins from ocular tissues of healthy human subjects.

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

NASA Technical Reports Server (NTRS)

Holmes, Leonard D.

1996-01-01

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

Concentration dependent switch in the kinetic pathway of lysozyme fibrillation: Spectroscopic and microscopic analysis

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

Gold nanoparticles on titanium and interaction with prototype protein.

PubMed

Padmos, J Daniel; Duchesne, Paul; Dunbar, Michael; Zhang, Peng

2010-10-01

Modifying titanium (Ti) implant surfaces with functional proteins can strengthen the interface between prosthesis and bone. A prototype system was developed using gold nanoparticles (AuNPs) to immobilize proteins onto Ti. An electroless (galvanic displacement) deposition method was first used to form AuNPs of controlled size and coverage on commercial Ti foil (giving Ti-AuNPs). Parameters were then modified to create two groups of discs (n = 26) with different average AuNP diameters. Scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the morphology and surface structure of Ti-AuNPs. To study the interaction of Ti-AuNPs with proteins, Ti discs (n = 8) modified with plain AuNPs and discs (n = 8) modified with thiol (HS--R--COOH)-functionalized AuNPs were treated with lysozyme solution. The amount and activity of the lysozyme on the discs were examined with Micro-BCA and enzymatic assays. Lysozyme was immobilized onto the discs, and the assays showed that the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls had average lysozyme adsorptions of 23 x 10(4), 2.3 x 10(4), and 5.7 x 10(4) microg/m2, respectively. The activity assays showed that 21.5, 18.4, and 12.5% of the adsorbed lysozyme was active on the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls, respectively. This technique holds promise for binding functional biomolecules to surgical implants, hence possibly creating implant surfaces that react to their local environment. Copyright 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

The impact of particle preparation methods and polymorphic stability of lipid excipients on protein distribution in microparticles.

PubMed

Liu, Jingying; Christophersen, Philip C; Yang, Mingshi; Nielsen, Hanne M; Mu, Huiling

2017-12-01

The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. Labeled lysozyme was incorporated into SLM prepared with different excipients, i.e. trimyristin (TG14), glyceryl distearate (GDS), and glyceryl monostearate (GMS), by water-oil-water (w/o/w) or solid-oil-water (s/o/w) method. The distribution of lysozyme in SLM and the release of the protein from SLM were evaluated by confocal laser scanning microscopy. The storage stability of SLM was characterized by HPLC, differential scanning calorimetry, X-ray powder diffraction, and scanning electron microscopy. Lysozyme was displayed as small scattered domains inside GDS and GMS SLM, whereas it was incorporated in the core of TG14 SLM formulated by the w/o/w method or evenly distributed in TG14 SLM prepared by the s/o/w method. Stability study at 37 °C revealed that only TG14 SLM made by the w/o/w method was able to maintain the lysozyme amount both on the particle surface and released from the SLM. Elevated storage temperature induced polymorphic transition of lipids in GDS and GMS SLM, which was, however, not remarkable for the TG14 SLM. Lipid excipients and particle preparation methods were found to differently affect the lysozyme distribution in SLM, owning to varied storage stabilities of the lipids. The present study provides updated knowledge for rational development of lipid-based formulations for oral delivery of peptide or protein drugs.

Relationship between β-relaxation and structural stability of lysozyme: Microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments

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

Hédoux, Alain, E-mail: alain.hedoux@univ-lille1.fr; Paccou, Laurent; Guinet, Yannick

Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein–trehalose interactions inmore » the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.« less

An energy landscape approach to protein aggregation

NASA Astrophysics Data System (ADS)

Buell, Alexander; Knowles, Tuomas

2012-02-01

Protein aggregation into ordered fibrillar structures is the hallmark of a class of diseases, the most prominent examples of which are Alzheimer's and Parkinson's disease. Recent results (e.g. Baldwin et al. J. Am. Chem. Soc. 2011) suggest that the aggregated state of a protein is in many cases thermodynamically more stable than the soluble state. Therefore the solubility of proteins in a cellular context appears to be to a large extent under kinetic control. Here, we first present a conceptual framework for the description of protein aggregation ( see AK Buell et al., Phys. Rev. Lett. 2010) that is an extension to the generally accepted energy landscape model for protein folding. Then we apply this model to analyse and interpret a large set of experimental data on the kinetics of protein aggregation, acquired mainly with a novel biosensing approach (see TPJK Knowles et al, Proc. Nat. Acad. Sc. 2007). We show how for example the effect of sequence modifications on the kinetics and thermodynamics of human lysozyme aggregation can be understood and quantified (see AK Buell et al., J. Am. Chem. Soc. 2011). These results have important implications for therapeutic strategies against protein aggregation disorders, in this case lysozyme systemic amyloidosis.

[Potentialization of antibiotics by lytic enzymes].

PubMed

Brisou, J; Babin, P; Babin, R

1975-01-01

Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.

Effect of mobile phone use on salivary concentrations of protein, amylase, lipase, immunoglobulin A, lysozyme, lactoferrin, peroxidase and C-reactive protein of the parotid gland.

PubMed

Hashemipour, M S; Yarbakht, M; Gholamhosseinian, A; Famori, H

2014-05-01

The possibility of side effects associated with the electromagnetic waves emitted from mobile phones is a controversial issue. The present study aimed to evaluate the effect of mobile phone use on parotid gland salivary concentrations of protein, amylase, lipase, immunoglobulin A, lysozyme, lactoferrin, peroxidase and C-reactive protein. Stimulated salivary samples were collected simultaneously from both parotid glands of 86 healthy volunteers. Salivary flow rate and salivary concentrations of proteins, amylase, lipase, lysozyme, lactoferrin, peroxidase, C-reactive protein and immunoglobulin A, were measured. Data were analysed using t-tests and one-way analyses of variance. Salivary flow rate and parotid gland salivary concentrations of protein were significantly higher on the right side compared to the left in those that predominantly held mobile phones on the right side. In addition, there was a decrease in concentrations of amylase, lipase, lysozyme, lactoferrin and peroxidase. The side of dominant mobile phone use was associated with differences in salivary flow rate and parotid gland salivary concentrations, in right-dominant users. Although mobile phone use influenced salivary composition, the relationship was not significant.

Determination of sorption isotherm and rheological properties of lysozyme using a high-resolution humidity scanning QCM-D technique.

PubMed

Graf, Gesche; Kocherbitov, Vitaly

2013-08-29

The high-resolution humidity scanning QCM-D technique enables investigation of hydration of soft matter films using a quartz crystal microbalance with dissipation monitoring (QCM-D) equipped with a humidity module. Based on a continuous increase of relative humidity, properties of soft matter films can be investigated depending on the water content of the surrounding atmosphere. Determination of complete water sorption isotherms is possible via analysis of the overtone dependence of the resonance frequencies. Rheological properties are monitored via measurement of the dissipation. The glass transition can be identified from the change of viscoelastic properties of the film reflected in changes of the dissipation. A high-resolution water sorption isotherm of lysozyme was measured and compared with results from water sorption calorimetry. Analysis of the rheological behavior during hydration of lysozyme films revealed the presence of two separate sharp transitions at the water activities 0.67 and 0.91, which are connected to the glass transition. In previous works, only the existence of a broad glass transition has been reported so far. Combining the QCM-D data with Raman scattering data presented earlier, a new mechanism of isothermal glass transition in lysozyme is proposed.

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method

PubMed Central

Cao, X.M.; Tian, Y.; Wang, Z.Y.; Liu, Y.W.; Wang, C.X.

2016-01-01

ABSTRACT 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. PMID:27459596

Resonance scattering spectra of micrococcus lysodeikticus and its application to assay of lysozyme activity.

PubMed

Jiang, Zhi-Liang; Huang, Guo-Xia

2007-02-01

Several methods, including turbidimetric and colorimetric methods, have been reported for the detection of lysozyme activity. However, there is no report about the resonance scattering spectral (RSS) assay, which is based on the catalytic effect of lysozyme on the hydrolysis of micrococcus lysodeikticus (ML) and its resonance scattering effect. ML has 5 resonance scattering peaks at 360 400, 420, 470, and 520 nm with the strongest one at 470 nm. The concentration of ML in the range of 2.0x10(6)-9.3x10(8) cells/ml is proportional to the RS intensity at 470 nm (I(470 nm)). A new catalytic RSS method has been proposed for 0.24-40.0 U/ml (or 0.012-2.0 mug/ml) lysozyme activity, with a detection limit (3sigma) of 0.014 U/ml (or 0.0007 microg/ml). Saliva samples were assayed by this method, and it is in agreement with the results of turbidimetric method. The slope, intercept and the correlation coefficient of the regression analysis of the 2 assays were 0.9665, -87.50, and 0.9973, respectively. The assay has high sensitivity and simplicity.

In situ study of the growth and degradation processes in tetragonal lysozyme crystals on a silicon substrate by high-resolution X-ray diffractometry

NASA Astrophysics Data System (ADS)

Kovalchuk, M. V.; Prosekov, P. A.; Marchenkova, M. A.; Blagov, A. E.; D'yakova, Yu. A.; Tereshchenko, E. Yu.; Pisarevskii, Yu. V.; Kondratev, O. A.

2014-09-01

The results of an in situ study of the growth of tetragonal lysozyme crystals by high-resolution X-ray diffractometry are considered. The crystals are grown by the sitting-drop method on crystalline silicon substrates of different types: both on smooth substrates and substrates with artificial surface-relief structures using graphoepitaxy. The crystals are grown in a special hermetically closed crystallization cell, which enables one to obtain images with an optical microscope and perform in situ X-ray diffraction studies in the course of crystal growth. Measurements for lysozyme crystals were carried out in different stages of the crystallization process, including crystal nucleation and growth, developed crystals, the degradation of the crystal structure, and complete destruction.

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.

Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum

NASA Astrophysics Data System (ADS)

Sanders, Lori; Slimmer, Scott; Angelini, Thomas; Wong, Gerard C. L.

2003-03-01

Cystic fibrosis sputum is a complex fluid consisting of mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems containing actin, lysozyme, and KCl were created to simulate CF sputum in vitro. These model systems were studied using small angle x-ray scattering and confocal fluorescence microscopy. Preliminary results will be presented. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

Physiological responses of Chinese longsnout catfish to water temperature

NASA Astrophysics Data System (ADS)

Han, Dong; Xie, Shouqi; Zhu, Xiaoming; Yang, Yunxia

2011-05-01

We evaluated the effect of water temperature on the growth and physiology of the Chinese longsnout catfish ( Leiocassis longirostris Günther). The fish were reared at four temperatures (20, 25, 30, and 35°C) and sampled on days 7, 20, and 30. We measured plasma levels of insulin, free thyroxine (FT4), free 3,5,3'-triiodothyronine (FT3), lysozyme and leukocyte phagocytic activity. The optimum water temperature for growth was 27.7°C. The plasma levels of insulin and FT4 declined significantly ( P<0.05) on day 30 at temperatures above 20°C. Lysozyme activity was significantly ( P<0.05) lower at 25°C than at other temperatures. We conclude that final weight, insulin, FT4, and lysozyme were significantly affected by water temperature.

Study of lysozyme mobility and binding free energy during adsorption on a graphene surface

NASA Astrophysics Data System (ADS)

Nakano, C. Masato; Ma, Heng; Wei, Tao

2015-04-01

Understanding protein adsorption is a key to the development of biosensors and anti-biofouling materials. Hydration essentially controls the adsorption process on hydrophobic surfaces, but its effect is complicated by various factors. Here, we present an ideal model system to isolate hydration effects—lysozyme adsorption on a flat hydrophobic graphene surface. Our all-atom molecular dynamics and molecular-mechanics/Poisson-Boltzmann surface area computation study reveal that lysozyme on graphene displays much larger diffusivity than in bulk water. Protein's hydration free energy within the first hydration shell is dominated by the protein-water electrostatic interactions and acts as an energy barrier for protein adsorption. On the other hand, the surface tension, especially that from the hydrophobic graphene, can effectively weaken the barrier to promote adsorption.

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

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

López-Jaramillo, F. J., E-mail: javier@lec.ugr.es; Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, E-18071; Pérez-Banderas, F.

The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purificationmore » glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.« less

Convective flow effects on protein crystal growth

NASA Technical Reports Server (NTRS)

Rosenberger, Franz

1995-01-01

During the fifth semi-annual period under this grant we have pursued the following activities: (1) Characterization of the purity and further purification of lysozyme solutions, these efforts are summarized in Section 2; (2) Crystal growth morphology and kinetics studies with tetragonal lysozyme, our observation on the dependence of lysozyme growth kinetics on step sources and impurities has been summarized in a manuscript which was accepted for publication in the Journal of Crystal Growth; (3) Numerical modelling of the interaction between bulk transport and interface kinetics, for a detailed summary of this work see the manuscript which was accepted for publication in the Journal of Crystal Growth; and (4) Light scattering studies, this work has been summarized in a manuscript that has been submitted for publication to the Journal of Chemical Physics.

Crystallization of lysozyme with ( R)-, ( S)- and ( RS)-2-methyl-2,4-pentanediol

DOE PAGES

Stauber, Mark; Jakoncic, Jean; Berger, Jacob; ...

2015-03-01

Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with ( R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with ( R)-MPD and ( RS)-MPD the crystal contacts are made by ( R)-MPD, demonstrating that there ismore » preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.« less

Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions

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

Babcock, Abigail S.; Anderson, Amy L.; Graduate Program in Environmental Toxicology, Clemson University, Clemson, SC 29634

2013-01-01

Indirubin is a deep-red bis-indole isomer of indigo blue, both of which are biologically active ingredients in Danggui Longhui Wan, an ancient Chinese herbal tea mixture used to treat neoplasia and chronic inflammation and to enhance detoxification of xenobiotics. Multiple indirubin derivatives have been synthesized and shown to inhibit cyclin-dependent kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of potency. Several indirubins are also aryl hydrocarbon receptor (AhR) agonists, with AhR-associated activities covering a wide range of potencies, depending on molecular structure. This study examined the effects of indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin with potent STAT3 inhibitory properties,more » on basal and LPS-inducible activities in murine RAW264.7 macrophages. Using a focused commercial qRT-PCR array platform (SuperArray®), the effects of E804 on expression of a suite of genes associated with stress and toxicity were determined. Most genes up-regulated by LPS treatment were suppressed by E804; including LPS-induced expression of pro-inflammatory cytokines and receptors, apoptosis control genes, and oxidative stress response genes. Using qRT-PCR as a follow up to the commercial arrays, E804 treatment suppressed LPS-induced COX-2, iNOS, IL-6 and IL-10 gene expression, though the effects on iNOS and COX-2 protein expression were less dramatic. E804 also inhibited LPS-induced secretion of IL-6 and IL-10. Functional endpoints, including iNOS and lysozyme enzymatic activity, phagocytosis of fluorescent latex beads, and intracellular killing of bacteria, were also examined, and in each experimental condition E804 suppressed activities. Collectively, these results indicate that E804 is a potent modulator of pro-inflammatory profiles in LPS-treated macrophages. -- Highlights: ► RAW 264.7 macrophages were treated with 1 μM Indirubin E804, 1 μg/ml LPS, or both. ► E804 suppresses LPS-induced expression of iNOS, IL-6, COX-2, and IL-10. ► E804 suppresses LPS-induced iNOS and lysozyme activity, and IL-6 and IL-10 secretion. ► E804 suppressed phagocytosis and intracellular killing by macrophages.« less

Regulation of dietary glutamine on the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus japonicus (Selenka).

PubMed

Yu, Haibo; Gao, Qinfeng; Dong, Shuanglin; Lan, Ying; Ye, Zhi; Wen, Bin

2016-03-01

The present study examined the effects of dietary glutamine (Gln) on the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus japonicus (Selenka). The specific growth rate, intestinal morphology, activity of digestive enzymes, activity and gene expression of lysozyme and antioxidative enzymes of the sea cucumbers were determined after feeding 5 experimental diets with additions of increasing levels of Gln (at 0%, 0.4%, 0.8%,1.2% and 1.6%, respectively) for 60 days. We discovered that the specific growth rate of the sea cucumbers in 0.4%, 0.8% and 1.2% groups increased 35.3%, 27.3% and 24.1%, respectively, compared to the control (0%) group with significant differences. Dietary Gln can improve the intestinal function of the sea cucumbers by increasing the activities of trypsin and lipase in the intestine and the villus height and villus density of the intestine, eventhough significant differences were not observed in some groups. 0.4%-0.8% of dietary Gln can significantly increase the activity of lysozyme (LSZ) in the coelomic fluid of the sea cucumbers. Significant improvements were observed on the SOD activity in coelomic fluid of the sea cucumbers fed diets supplemented with 0.4%-1.6% of Gln compared to the control group. Similarly, the CAT activity in coelomic fluid of the sea cucumbers significantly increased in 0.8%, 1.2% and 1.6% groups compared to the control and 0.4% groups. Change pattern of the activity of CAT was consistent with the change pattern of the expression of CAT gene, indicating the dietary Gln can up-regulate the expression of CAT gene and consequently promote the secretion of CAT. However, the down-regulation of the expression of SOD gene by dietary Gln were observed in almost all of the treatment groups, which is in contrast with the change pattern of the activity of SOD, indicating the negative feedback regulation of the secretion of SOD on the expression of SOD gene. In summary, the suitable supplementation levels of Gln in diets of sea cucumber A. japonicus are 0.4%-0.8%, based on the effectiveness of dietary Gln on the growth, intestinal function, immunity and antioxidant capacity of the sea cucumbers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Newly identified invertebrate-type lysozyme (Splys-i) in mud crab (Scylla paramamosain) exhibiting muramidase-deficient antimicrobial activity.

PubMed

Zhou, Jian; Zhao, Shu; Fang, Wen-Hong; Zhou, Jun-Fang; Zhang, Jing-Xiao; Ma, Hongyu; Lan, Jiang-Feng; Li, Xin-Cang

2017-09-01

Lysozymes are widely distributed immune effectors exerting muramidase activity against the peptidoglycan of the bacterial cell wall to trigger cell lysis. However, some invertebrate-type (i-type) lysozymes deficient of muramidase activity still exhibit antimicrobial activity. To date, the mechanism underlying the antimicrobial effect of muramidase-deficient i-type lysozymes remains unclear. Accordingly, this study characterized a novel i-type lysozyme, Splys-i, in the mud crab Scylla paramamosain. Splys-i shared the highest identity with the Litopenaeus vannamei i-type lysozyme (Lvlys-i2, 54% identity) at the amino acid level. Alignment analysis and 3D structure comparison show that Splys-i may be a muramidase-deficient i-type lysozyme because it lacks the two conserved catalytic residues (Glu and Asp) that are necessary for muramidase activity. Splys-i is mainly distributed in the intestine, stomach, gills, hepatopancreas, and hemocytes, and it is upregulated by Vibrio harveyi or Staphylococcus aureus challenge. Recombinant Splys-i protein (rSplys-i) can inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio alginolyticus, Vibrio parahemolyticus, and Escherichia coli), Gram-positive bacteria (S. aureus, Bacillus subtilis, and Bacillus megaterium), and the fungus Candida albicans to varying degrees. In this study, two binding assays and a bacterial agglutination assay were conducted to elucidate the potential antimicrobial mechanisms of Splys-i. Results demonstrated that rSplys-i could bind to all nine aforementioned microorganisms. It also exhibited a strong binding activity to lipopolysaccharide from E. coli and lipoteichoic acid and peptidoglycan (PGN) from S. aureus but a weak binding activity to PGN from B. subtilis and β-glucan from fungi. Moreover, rSplys-i could agglutinate these nine types of microorganisms in the presence of Ca 2+ at different protein concentrations. These results suggest that the binding activity and its triggered agglutinating activity might be two major mechanisms of action to realize the muramidase-deficient antibacterial activity. In addition, rSplys-i can hydrolyze the peptidoglycan of some Gram-positive bacteria because it exhibits weak isopeptidase activities in salt and protein concentration-dependent manner. This result indicates that such an isopeptidase activity may contribute to the muramidase-deficient antimicrobial activity to a certain degree. In conclusion, Splys-i is upregulated by pathogenic bacteria, and it inhibits bacterial growth by binding and agglutination activities as well as isopeptidase activity, suggesting that Splys-i is involved in immune defense against bacteria through several different mechanisms of action. Copyright © 2017 Elsevier Ltd. All rights reserved.

PPAR-γ contributes to immunity by cancer vaccines that secrete GM-CSF.

PubMed

Goyal, Girija; Wong, Karrie; Nirschl, Christopher J; Souders, Nicholas; Neuberg, Donna; Anandasabapathy, Niroshana; Dranoff, Glenn

2018-04-18

Peroxisome proliferator activated receptor-γ (PPARγ) is a lipid-activated nuclear receptor that promotes immune tolerance through effects on macrophages, dendritic cells (DCs), and regulatory T cells (Tregs). Granulocyte-macrophage colony stimulating factor (GM-CSF) induces PPARγ expression in multiple myeloid cell types. GM-CSF contributes to both immune tolerance and protection, but the role of PPARγ in these pathways is poorly understood. Here we reveal an unexpected stimulatory role for PPARγ in the generation of antitumor immunity with irradiated, GM-CSF-secreting tumor-cell vaccines (GVAX). Mice harboring a deletion of PPARγ in lysozyme M (LysM)-expressing myeloid cells showed a decreased ratio of CD8+ T effectors to Tregs and impaired tumor rejection with GVAX. Diminished tumor protection was associated with altered dendritic cell responses and increased production of the Treg attracting chemokines CCL17 and CLL22. Correspondingly, the systemic administration of PPARγ agonists to vaccinated mice elevated the CD8+ T effector to Treg ratio through effects on myeloid cells and intensified the antitumor activity of GVAX combined with cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antibody blockade. PPARγ agonists similarly attenuated Treg induction and decreased CCL17 and CCL22 levels in cultures of human peripheral blood mononuclear cells (PBMCs) with GM-CSF-secreting tumor cells. Together, these results highlight a key role for myeloid cell PPARγ in GM-CSF stimulated antitumor immunity and suggest that PPARγ agonists might be useful in cancer immunotherapy. Copyright ©2018, American Association for Cancer Research.

Identification of 10 Candidate Biomarkers Distinguishing Tuberculous and Malignant Pleural Fluid by Proteomic Methods.

PubMed

Lee, Chang Youl; Hong, Ji Young; Lee, Myung Goo; Suh, In Bum

2017-11-01

Pleural effusion, an accumulation of fluid in the pleural space, usually occurs in patients when the rate of fluid formation exceeds the rate of fluid removal. The differential diagnosis of tuberculous pleurisy and malignant pleural effusion is a difficult task in high tuberculous prevalence areas. The aim of the present study was to identify novel biomarkers for the diagnosis of pleural fluid using proteomics technology. We used samples from five patients with transudative pleural effusions for internal standard, five patients with tuberculous pleurisy, and the same numbers of patients having malignant effusions were enrolled in the study. We analyzed the proteins in pleural fluid from patients using a technique that combined two-dimensional liquid-phase electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. We identified a total of 10 proteins with statistical significance. Among 10 proteins, trasthyretin, haptoglobin, metastasis-associated protein 1, t-complex protein 1, and fibroblast growth factor-binding protein 1 were related with malignant pleural effusions and human ceruloplasmin, lysozyme precursor, gelsolin, clusterin C complement lysis inhibitor, and peroxirexdoxin 3 were expressed several times or more in tuberculous pleural effusions. Highly expressed proteins in malignant pleural effusion were associated with carcinogenesis and cell growth, and proteins associated with tuberculous pleural effusion played a role in the response to inflammation and fibrosis. These findings will aid in the development of novel diagnostic tools for tuberculous pleurisy and malignant pleural effusion of lung cancer. © Copyright: Yonsei University College of Medicine 2017

Tetragonal Lysozyme Nucleation and Crystal Growth: The Role of the Solution Phase

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Sumida, John; Maxwell, Daniel; Gorti, Sridhar; Curreri, Peter A. (Technical Monitor)

2002-01-01

Experimental evidence indicates a dominant role of solution phase interactions in nucleating and growing tetragonal lysozyme crystals. These interactions are extensive, even at saturation, and may be a primary cause of misoriented regions in crystals grown on Earth. Microgravity, by limiting interfacial concentrations to diffusion-controlled levels, may benefit crystal quality by also reducing the extent of associated species present at the interface.

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.

Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

PubMed Central

Gill, Christina; Blow, Frances; Darby, Alistair C.

2016-01-01

Background Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. Results After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. Conclusions An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies. However, we recommend that the same method is used on all samples within a particular study. PMID:27643503

Protein Crystal Growth Dynamics and Impurity Incorporation

NASA Technical Reports Server (NTRS)

Chernov, Alex A.; Thomas, Bill

2000-01-01

The general concepts and theories of crystal growth are proven to work for biomolecular crystallization. This allowed us to extract basic parameters controlling growth kinetics - free surface energy, alpha, and kinetic coefficient, beta, for steps. Surface energy per molecular site in thermal units, alpha(omega)(sup 2/3)/kT approx. = 1, is close to the one for inorganic crystals in solution (omega is the specific molecular volume, T is the temperature). Entropic restrictions on incorporation of biomolecules into the lattice reduce the incorporation rate, beta, by a factor of 10(exp 2) - 10(exp 3) relative to inorganic crystals. A dehydration barrier of approx. 18kcal/mol may explain approx. 10(exp -6) times difference between frequencies of adding a molecule to the lattice and Brownian attempts to do so. The latter was obtained from AFM measurements of step and kink growth rates on orthorhombic lysozyme. Protein and many inorganic crystals typically do not belong to the Kossel type, thus requiring a theory to account for inequivalent molecular positions within its unit cell. Orthorhombic lysozyme will serve as an example of how to develop such a theory. Factors deteriorating crystal quality - stress and strain, mosaicity, molecular disorder - will be reviewed with emphasis on impurities. Dimers in ferritin and lysozyme and acetylated lysozyme, are microheterogeneous i.e. nearly isomorphic impurities that are shown to be preferentially trapped by tetragonal lysozyme and ferritin crystals, respectively. The distribution coefficient, K defined as a ratio of the (impurity/protein) ratios in crystal and in solution is a measure of trapping. For acetylated lysoyzme, K = 2.15 or, 3.42 for differently acetylated forms, is independent of both the impurity and the crystallizing protein concentration. The reason is that impurity flux to the surface is constant while the growth rate rises with supersaturation. About 3 times lower dimer concentration in space grown ferritin and lysozyme crystals might be examples explaining higher quality of the space grown protein crystal. Depletion of solution with respect to isomorphic impurities around a growing crystal may be K times deeper than with respect to the crystallizing protein.

The immune responses and expression of metallothionein (MT) gene and heat shock protein 70 (HSP 70) in juvenile rockfish, Sebastes schlegelii, exposed to waterborne arsenic (As3+).

PubMed

Kim, Jun-Hwan; Kang, Ju-Chan

2016-10-01

Juvenile rockfish, Sebastes schlegelii (mean length 16.4±1.9cm, and mean weight 71.6±6.4g) were exposed for 20days with the different levels of waterborne arsenic concentration (0, 50, 100, 200 and 400μg/L). The plasma cortisol of S. schlegelii was significantly increased by the waterborne arsenit exposure. In the immune responses, the immunoglobulin M (Ig M) and lysozyme activity of S. schlegelii were significantly increased by the waterborne arsenic exposure. The acetylcholinesterase (AChE) activity of S. schlegelii was inhibited by the waterborne arsenic exposure. The substantial increases in the gene expression such as metallothionein (MT) and heat shock protein 70 (HSP 70) were observed by the waterborne arsenic exposure. The results demonstrated that waterborne arsenic exposure can induce the significant alterations in the immune responses and specific gene expression of S. schlegelii. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of random PEGylation reactions by means of high throughput screening.

PubMed

Maiser, Benjamin; Dismer, Florian; Hubbuch, Jürgen

2014-01-01

Since the first FDA approval of a PEGylated product in 1990, so called random PEGylation reactions are still used to increase the efficacy of biopharmaceuticals and represent the major technology of all approved PEG-modified drugs. However, the great influence of process parameters on PEGylation degree and the PEG-binding site results in a lack of reaction specificity which can have severe impact on the product profile. Consequently, reproducible and well characterized processes are essential to meet increasing regulative requirements resulting from the quality-by-design (QbD) initiative, especially for this kind of modification type. In this study we present a general approach which combines the simple chemistry of random PEGylation reactions with high throughput experimentation (HTE) to achieve a well-defined process. Robotic based batch experiments have been established in a 96-well plate format and were analyzed to investigate the influence of different PEGylation conditions for lysozyme as model protein. With common SEC analytics highly reproducible reaction kinetics were measured and a significant influence of PEG-excess, buffer pH, and reaction time could be investigated. Additional mono-PEG-lysozyme analytics showed the impact of varying buffer pH on the isoform distribution, which allowed us to identify optimal process parameters to get a maximum concentration of each isoform. Employing Micrococcus lysodeikticus based activity assays, PEG-lysozyme33 was identified to be the isoform with the highest residual activity, followed by PEG-lysozyme1 . Based on these results, a control space for a PEGylation reaction was defined with respect to an optimal overall volumetric activity of mono-PEG-lysozyme isoform mixtures. © 2013 Wiley Periodicals, Inc.

Influence of protein deposition on bacterial adhesion to contact lenses.

PubMed

Subbaraman, Lakshman N; Borazjani, Roya; Zhu, Hua; Zhao, Zhenjun; Jones, Lyndon; Willcox, Mark D P

2011-08-01

The aim of the study is to determine the adhesion of Gram positive and Gram negative bacteria onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials with and without lysozyme, lactoferrin, and albumin coating. Four lens types (three SH-balafilcon A, lotrafilcon B, and senofilcon A; one CH-etafilcon A) were coated with lysozyme, lactoferrin, or albumin (uncoated lenses acted as controls) and then incubated in Staphylococcus aureus (Saur 31) or either of two strains of Pseudomonas aeruginosa (Paer 6294 and 6206) for 24 h at 37 °C. The total counts of the adhered bacteria were determined using the H-thymidine method and viable counts by counting the number of colony-forming units on agar media. All three strains adhered significantly lower to uncoated etafilcon A lenses compared with uncoated SH lenses (p < 0.05). Lysozyme coating on all four lens types increased binding (total and viable counts) of Saur 31 (p < 0.05). However, lysozyme coating did not influence P. aeruginosa adhesion (p > 0.05). Lactoferrin coating on lenses increased binding (total and viable counts) of Saur 31 (p < 0.05). Lactoferrin-coated lenses showed significantly higher total counts (p < 0.05) but significantly lower viable counts (p < 0.05) of adhered P. aeruginosa strains. There was a significant difference between the total and viable counts (p < 0.05) that were bound to lactoferrin-coated lenses. Albumin coating of lenses increased binding (total and viable counts) of all three strains (p < 0.05). Lysozyme deposited on contact lenses does not possess antibacterial activity against certain bacterial strains, whereas lactoferrin possess an antibacterial effect against strains of P. aeruginosa.

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants.

PubMed

Cabrefiga, Jordi; Montesinos, Emilio

2017-02-17

Fire blight is an important disease affecting rosaceous plants. The causal agent is the bacteria Erwinia amylovora which is poorly controlled with the use of conventional bactericides and biopesticides. Antimicrobial peptides (AMPs) have been proposed as a new compounds suitable for plant disease control. BP100, a synthetic linear undecapeptide (KKLFKKILKYL-NH 2 ), has been reported to be effective against E. amylovora infections. Moreover, BP100 showed bacteriolytic activity, moderate susceptibility to protease degradation and low toxicity. However, the peptide concentration required for an effective control of infections in planta is too high due to some inactivation by tissue components. This is a limitation beause of the high cost of synthesis of this compound. We expected that the combination of BP100 with lysozyme may produce a synergistic effect, enhancing its activity and reducing the effective concentration needed for fire blight control. The combination of a synhetic multifunctional undecapeptide (BP100) with lysozyme produces a synergistic effect. We showed a significant increase of the antimicrobial activity against E. amylovora that was associated to the increase of cell membrane damage and to the reduction of cell metabolism. Combination of BP100 with lysozyme reduced the time required to achieve cell death and the minimal inhibitory concentration (MIC), and increased the activity of BP100 in the presence of leaf extracts even when the peptide was applied at low doses. The results obtained in vitro were confirmed in leaf infection bioassays. The combination of BP100 with lysozyme showed synergism on the bactericidal activity against E. amylovora and provide the basis for developing better formulations of antibacterial peptides for plant protection.

Dehydration decreases saliva antimicrobial proteins important for mucosal immunity.

PubMed

Fortes, Matthew B; Diment, Bethany C; Di Felice, Umberto; Walsh, Neil P

2012-10-01

The aim of the study was to investigate the effect of exercise-induced dehydration and subsequent overnight fluid restriction on saliva antimicrobial proteins important for host defence (secretory IgA (SIgA), α-amylase, and lysozyme). On two randomized occasions, 13 participants exercised in the heat, either without fluid intake to evoke progressive body mass losses (BML) of 1%, 2%, and 3% with subsequent overnight fluid restriction until 0800 h in the following morning (DEH) or with fluids to offset losses (CON). Participants in the DEH trial rehydrated from 0800 h until 1100 h on day 2. BML, plasma osmolality (Posm), and urine specific gravity (USG) were assessed as hydration indices. Unstimulated saliva samples were assessed for flow rate (SFR), SIgA, α-amylase, and lysozyme concentrations. Posm and USG increased during dehydration and remained elevated after overnight fluid restriction (BML = 3.5% ± 0.3%, Posm = 297 ± 6 mosmol·kg⁻¹, and USG = 1.026 ± 0.002; P < 0.001). Dehydration decreased SFR (67% at 3% BML, 70% at 0800 h; P < 0.01) and increased SIgA concentration, with no effect on SIgA secretion rate. SFR and SIgA responses remained unchanged in the CON trial. Dehydration did not affect α-amylase or lysozyme concentration but decreased secretion rates of α-amylase (44% at 3% BML, 78% at 0800 h; P < 0.01) and lysozyme (46% at 3% BML, 61% at 0800 h; P < 0.01), which were lower than in CON at these time points (P < 0.05). Rehydration returned all saliva variables to baseline. In conclusion, modest dehydration (~3% BML) decreased SFR, α-amylase, and lysozyme secretion rates. Whether the observed magnitude of decrease in saliva AMPs during dehydration compromises host defence remains to be shown.

In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography.

PubMed

Großhans, Steffen; Rüdt, Matthias; Sanden, Adrian; Brestrich, Nina; Morgenstern, Josefine; Heissler, Stefan; Hubbuch, Jürgen

2018-04-27

Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q 2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q 2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Impact of UV-B radiation on the digestive enzymes and immune system of larvae of Indian major carp Catla catla.

PubMed

Sharma, Jaigopal; Rao, Y Vasudeva; Kumar, S; Chakrabarti, Rina

2010-03-01

Ultraviolet radiation is a potent threat to the aquatic animals. Exposure to such stressor affects metabolic and immunological processes. The present investigation aims to study the effect of UV-B radiation on digestive enzymes and immunity of larvae of Catla catla. Larvae were exposed to ultraviolet-B (UV-B, 280-320 nm) radiation (145 microW/cm(2)) for three different exposure times of 5, 10 and 15 min on every other day. After 55 days, important digestive enzymes were assayed. For immunological study, lysozyme, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels were measured. Then the fish were kept for one month without radiation and lysozyme level was measured. Protein concentration varied directly with the duration of exposure and was highest among fish that had received the 15 min UV-B irradiation. Significantly higher amylase, protease, trypsin and chymotrypsin activities were found in 5 min exposed fish compared to others. Lysozyme level was significantly higher in control group compared to the UV-B treated fish. The lysozyme level decreased with the increasing duration of UV-B radiation. When fish were kept without UV-B radiation for one month, lysozyme level was brought to the normal level in all treatments, except 15 min exposed fish. The GOT and GPT levels were significantly higher in the 15 min exposed group than others. The effects of UV-B radiation on the digestive physiology and immune system of catla have been clearly observed in the present study. The decreased enzyme activities in UV-B radiated fish results into improper digestion and poor growth.

[Microbiological and biochemical characteristics of inflammatory tissues in the periodontium].

PubMed

Surna, Algimantas; Sakalauskiene, Jurgina; Vitkauskiene, Astra; Saferis, Viktoras

2008-01-01

To investigate bacterial populations in subgingival and supragingival plaque samples of patients with inflammatory periodontal diseases and activities of the lysosomal enzymes--lysozyme, alkaline phosphatase, and beta-glucuronidase--in peripheral venous blood, in gingival crevicular fluid, and mixed nonstimulated saliva. The study included 60 patients with inflammatory periodontal diseases without any internal pathology and 24 periodontally healthy subjects. Molecular genetic assay (Micro-IDent plus, Germany) for complex identification of additional six periodontopathic bacteria was applied. The activity of lysozyme was determined turbidimetrically, the activity of alkaline phosphatase--spectrophotometrically with a "Monarch" biochemical analyzer, the activity beta-glucuronidase--according to the method described by Mead et al. and modified by Strachunskii. A statistically significant association between clinical and bacteriological data was found in the following cases: gingival bleeding in the presence of Eubacterium nodatum, Eikenella corrodens, Capnocytophaga spp. (P<0.01); pathological periodontal pockets in the presence of Peptostreptococcus micros (alpha< or =0.05 and beta< or =0.2), Fusobacterium nucleatum (alpha< or =0.05 and beta< or =0.2), Campylobacter rectus (alpha< or =0.05 and beta< or =0.2), and Capnocytophaga spp. (P<0.05); and satisfactory oral hygiene in the presence of all microorganisms investigated (P<0.05). The activity of lysozyme in gingival crevicular fluid and mixed nonstimulated saliva indicates the severity of periodontal inflammation. Based on clinical data, in assessing the amount of lysozyme in mixed nonstimulated saliva, sensitivity and specificity of 100% was found. Increased activities of lysozyme, alkaline phosphatase, and beta-glucuronidase were found in peripheral venous blood of patients with inflammatory periodontal disease as compared to control group. The main principles of the treatment of periodontal inflammatory diseases should be based on microorganism elimination, creation of individual treatment means affecting microflora in the mouth and immune system of macroorganisms.

Microscopic mechanism of protein cryopreservation in an aqueous solution with trehalose

PubMed Central

Corradini, Dario; Strekalova, Elena G.; Stanley, H. Eugene; Gallo, Paola

2013-01-01

In order to investigate the cryoprotective mechanism of trehalose on proteins, we use molecular dynamics computer simulations to study the microscopic dynamics of water upon cooling in an aqueous solution of lysozyme and trehalose. We find that the presence of trehalose causes global retardation of the dynamics of water. Comparing aqueous solutions of lysozyme with/without trehalose, we observe that the dynamics of water in the hydration layers close to the protein is dramatically slower when trehalose is present in the system. We also analyze the structure of water and trehalose around the lysozyme and find that the trehalose molecules form a cage surrounding the protein that contains very slow water molecules. We conclude that the transient cage of trehalose molecules that entraps and slows the water molecules prevents the crystallisation of protein hydration water upon cooling. PMID:23390573

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

PubMed

Bradshaw, J G; Peeler, J T; Twedt, R M

1977-09-01

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

PubMed Central

Bradshaw, J G; Peeler, J T; Twedt, R M

1977-01-01

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators. PMID:199113

Energy Minimization of Molecular Features Observed on the (110) Face of Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Perozzo, Mary A.; Konnert, John H.; Li, Huayu; Nadarajah, Arunan; Pusey, Marc

1999-01-01

Molecular dynamics and energy minimization have been carried out using the program XPLOR to check the plausibility of a model lysozyme crystal surface. The molecular features of the (110) face of lysozyme were observed using atomic force microscopy (AFM). A model of the crystal surface was constructed using the PDB file 193L, and was used to simulate an AFM image. Molecule translations, van der Waals radii, and assumed AFM tip shape were adjusted to maximize the correlation coefficient between the experimental and simulated images. The highest degree of 0 correlation (0.92) was obtained with the molecules displaced over 6 A from their positions within the bulk of the crystal. The quality of this starting model, the extent of energy minimization, and the correlation coefficient between the final model and the experimental data will be discussed.

Microscopic mechanism of protein cryopreservation in an aqueous solution with trehalose.

PubMed

Corradini, Dario; Strekalova, Elena G; Stanley, H Eugene; Gallo, Paola

2013-01-01

In order to investigate the cryoprotective mechanism of trehalose on proteins, we use molecular dynamics computer simulations to study the microscopic dynamics of water upon cooling in an aqueous solution of lysozyme and trehalose. We find that the presence of trehalose causes global retardation of the dynamics of water. Comparing aqueous solutions of lysozyme with/without trehalose, we observe that the dynamics of water in the hydration layers close to the protein is dramatically slower when trehalose is present in the system. We also analyze the structure of water and trehalose around the lysozyme and find that the trehalose molecules form a cage surrounding the protein that contains very slow water molecules. We conclude that the transient cage of trehalose molecules that entraps and slows the water molecules prevents the crystallisation of protein hydration water upon cooling.

Study of lysozyme mobility and binding free energy during adsorption on a graphene surface

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

Nakano, C. Masato; Ma, Heng; Wei, Tao, E-mail: twei@lamar.edu

Understanding protein adsorption is a key to the development of biosensors and anti-biofouling materials. Hydration essentially controls the adsorption process on hydrophobic surfaces, but its effect is complicated by various factors. Here, we present an ideal model system to isolate hydration effects—lysozyme adsorption on a flat hydrophobic graphene surface. Our all-atom molecular dynamics and molecular-mechanics/Poisson-Boltzmann surface area computation study reveal that lysozyme on graphene displays much larger diffusivity than in bulk water. Protein's hydration free energy within the first hydration shell is dominated by the protein-water electrostatic interactions and acts as an energy barrier for protein adsorption. On the othermore » hand, the surface tension, especially that from the hydrophobic graphene, can effectively weaken the barrier to promote adsorption.« less

An improved 96-well turbidity assay for T4 lysozyme activity

DTIC Science & Technology

2015-05-13

enzyme present in the reaction, resulting in a measure of activity in Unitsmg1. 10. To improve the reliability of the activity values, perform the...quantification of lysozyme activity with significantly lower enzyme concentrations, and the signal intensity can be enhanced by using greater amounts of... enzyme at the expense of a shorter linear reaction time. Several parameters of the assay are critical for obtaining reproducible activity