Endogenous Lysozyme Research Articles

Enhancing the decomposition and volatile fatty acids production of excess sludge: Synergistic pretreatment by endogenous lysozyme and rhamnolipid

This study investigated a green and efficient pretreatment strategy for enhancing excess sludge (ES) decomposition using combined pretreatment with endogenous lysozyme (EL) and biosurfactant rhamnolipid (RL). The employed lysozyme was produced by the strains isolated from the ES. The ES hydrolysis performance was strengthened in EL + RL system as the RL dosage increased from 0 to 0.5 g/gSS, and the optimized synergistic effect was achieved at 0.3 g/gSS RL dosage. The additional released SCOD, protein and polysaccharide in EL + RL pretreatment system over those of sole RL pretreatment system reached around 2796.1, 1174.4 and 277.7 mg/L at 8 h, respectively. Moreover, the bacteria and archaea decomposition degree reached around 80.3% and 93.4%. Both sole EL pretreatment and EL + RL pretreatment were conducive to the production of volatile fatty acids (VFAs), with the highest VFAs concentrations reaching 2.5 and 3.0 fold of control, respectively. Although sole EL pretreatment was more appropriate for the survival of volatile fatty acids (VFAs) producing bacteria, the EL + RL pretreatment gained more VFAs generation. Tepidiphilus with a relative abundance of 59.8%-76.7%, replaced Dechloromonas and became the predominant bacteria.

Bacterial killing is enhanced by exogenous administration of lysozyme in the lungs

ObjectiveLysozyme, a 14-kDa protein, is one of the most abundant antimicrobials in the lungs. Its concentration in airway surface sufficient to kill several bacterial pathogens in vitro. The purpose of this study was to determine if administration of exogenous lysozyme would further enhance bacterial killing in vivo. MethodsTo assess the effect of acute lung infection on endogenous lysozyme protein levels, mice were infected by intratracheal instillation of Pseudomonas aeruginosa and bronchoalveolar (BAL) fluid assessed for lysozyme concentration and for muramidase activity. In order to inform in vivo testing, species-specific bacterial killing efficacy was determined by incubating mucoid P. aeruginosa with 2×105 units of chicken lysozyme, human lysozyme or with vehicle at 37°C for 2hours. Subsequently, mice challenged with intratracheally-administered mucoid P. aeruginosa, were reintubated and injected with 2×105 Units of native human lysozyme, recombinant human lysozyme or with vehicle. Lung bacterial burden was enumerated subsequently. ResultsThe concentration of lysozyme protein in BAL fluid from mice challenged with mucoid clinical isolate of P. aeruginosa was increased 4-fold at 6hours post-infection. Quantitative culture showed that the number of recoverable bacteria was significantly decreased by both chicken and human lysozyme compared to vehicle but human lysozyme was significantly more effective than chicken egg lysozyme. In vivo, 24hours post-infection quantitative culture of lung homogenates showed that the number of viable bacteria recovered from mice treated with either native or recombinant lysozyme was decreased with 0.76±0.25×104 and 0.84±0.16×104, respectively, vs. 7.0±2.52×104 CFU/g protein in mice treated with HBSS, both P<0.05. ConclusionsThese results indicate that endogenous lysozyme is increased during acute lung infection and that early administration of exogenous lysozyme further enhances bacterial killing in vivo.

Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy.

To visualize intravital immune cell dynamics in the subconjunctival tissue during the wound-healing process using multiphoton microscopy. Gene-targeted mice expressing enhanced green fluorescent protein under the control of the endogenous lysozyme M promoter (LysM-eGFP mice) were anesthetized with isoflurane, and injured by a 10-0 nylon conjunctival suture. Vessels were visualized by intravenous injection of 70 kDa rhodamine-conjugated dextran. Using a multiphoton microscope, the three-dimensional images of the subconjunctival tissue were acquired every minute for 20 minutes before and 0.5, 3, 6, and 72 hours after injury. Raw imaging data were processed for four-dimensional images and analyzed for the number and the velocity of the LysM-eGFP-positive cells using Imaris software. The intravital LysM-eGFP-positive cells and the red-labeled vessels were successfully visualized using a multiphoton microscope. The conjunctival and scleral collagen fibers were detected as secondary harmonic generation signals, which were colored blue. Compared with mice without injury, the number of LysM-eGFP-positive cells in the subconjunctival tissue after conjunctival surgery increased in a time-dependent manner. The cell velocities significantly increased until 3 hours after surgery (5.9 ± 3.2 μm/min; P < 0.0001) and the elevated level was sustained until 72 hours after injury (5.9 ± 3.3 μm/min). This is the first report to visualize and evaluate intravital cellular dynamics during inflammation in the subconjunctival tissue using multiphoton microscopy. This technique may be a useful tool to characterize the molecular mechanisms of the wound-healing process after various ocular injuries, such as glaucoma surgery.

Deficiency of IκB Kinase β in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Intravital imaging of the cellular dynamics of LysM-positive cells in a murine corneal suture model

BackgroundCorneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent...

Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.

Enhanced Antimicrobial Activity of Engineered Human Lysozyme

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.

Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes

The -2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the -2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a lambda bacteriophage vector (lambdaDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blastodermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.

Myeloid Elf-1-like Factor, an ETS Transcription Factor, Up-regulates Lysozyme Transcription in Epithelial Cells through Interaction with Promyelocytic Leukemia Protein

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

Presentation on class II MHC molecules of endogenous lysozyme targeted to the endocytic pathway.

We compared the processing and presentation of the model Ag, hen-egg white lysozyme (HEL) expressed in C3.F6 APC as a fusion protein to three different acid hydrolases: cathepsin D, to an unglycosylated form of cathepsin D, and to pepsinogen. As expected from the biology of mannose 6-phosphate (Man-6-P)-containing enzyme, cathepsin D-HEL was delivered to the endosomal/lysosomal system. In contrast, the unglycosylated cathepsin D-HEL was retained in ER/Golgi and some was found in lysosomes. Most of pepsinogen-HEL was rapidly secreted from the APC. All transfectants presented HEL epitopes to T cell hybridomas. Regardless of the main route of traffic of the proteins, the strong I-Ak binding epitope HEL 48-62 was well presented by all. The biochemical forms of this epitope were identical for all. Three other epitopes of HEL that bind I-Ak with less affinity were processed equally well by unglycosylated cathepsin D-HEL and HEL-Ld. The glycosylated cathepsin D-HEL was less efficient in generating the 114-129 epitope. Pepsinogen-HEL was the less efficient of all transfectants in presenting these subdominant epitopes. Soluble cathepsin D-HEL recovered from culture supernatant was strongly immunogenic when added to C3.F6. The uptake was inhibited by free Man-6-P, indicating that the surface Man-6-P receptor can effectively deliver proteins to the class II MHC system.

Protein absorption by tubular mesonephric and metanephric structures in the human embryo. An immunohistochemical study.

The presence of different serum proteins in the cells of the proximal tubule of both meso- and metanephric nephrons in human embryos (7th-12th week of intrauterine life) was investigated using immunohistochemistry. Endogenous lysozyme, alpha-1-antitrypsin and ferritin were detected in mesonephric proximal tubules and, starting from the 8th week, also in metanephric proximal tubules. Our observations provide information concerning the appearance and distribution of tubular protein reabsorption during the early stages of development.

Rubus hispidus callus cultured in vitro and endogenous lysozyme: Growth processes

Relationships between growth and endogenous lysozyme content were studied in tissues of Rubus hispidus cultured in vitro (solid medium). The enzyme level was found to be closely related to the growth rate of culture. A growth regulator (abscisic acid) which reduced cell growth also decreased the endogenous lysozyme level. In the first few days of culture, bacteriolytic activity was strongly correlated to the age of the callus from which the culture was obtained.

Nephrotoxic effects of aminoglycoside treatment on renal protein reabsorption and accumulation.

To quantify the effects of gentamicin, kanamycin and netilmicin on renal protein reabsorption and accumulation, these drugs were administered to rats intraperitoneally (30 mg/kg/day) for 7, 14 or 21 days. Scanning electron microscopy of the glomerular endothelia, urinary measurements of sodium, potassium, endogenous lysozyme, N-acetyl-beta-D-glucosaminidase (NAG) as well as clearance and accumulation experiments after i.v. administration of egg-white lysozyme and measurements of inulin clearance (GFR) were done in each treatment group. Gentamicin administration decreased diameter, density and shape of endothelial fenestrae. Kanamycin and netilmicin appeared to have no effect at the dose used. All three aminoglycosides decreased GFR and increased urinary excretion of sodium and potassium. While gentamicin and kanamycin decreased the percentage reabsorption and accumulation of lysozyme after i.v. administration of egg-white lysozyme netilmicin had no effect. Daily excretion of total protein, endogenous lysozyme and NAG increased only after treatment with kanamycin and gentamicin. Thus, aminoglycosides may act as nephrotoxicants at glomerular and/or tubular level inducing impairment of renal reabsorption and accumulation of proteins.

Renal handling of endogenous lysozyme in the rat.

Quantitative studies of endogenous lysozyme (low molecular weight protein) were performed in rats. Urine and plasma concentrations of lysozyme and inulin were measured spectrophotometrically. An improved lysozyme assay (standard curve established by using egg white-lysozyme) enabled us to determine the mean plasma concentration of endogenous lysozyme (4.4 micrograms X ml-1) and the urinary concentrations of endogenous lysozyme (between 0.1 and 3.8 micrograms X ml-1. The urinary concentrations of endogenous lysozyme were found to be dependent on urinary flow rate. High urinary concentrations (ULy) were found at low urinary flow rates (V). The excreted amount of endogenous lysozyme (ULy X V) was independent of urinary flow rate and yielded a constant value of 0.02 micrograms X min-1. Mean glomerular filtration rate (GFR) was 1.2 ml X min-1 while clearance of endogenous lysozyme averaged 0.0039 ml X min-1. Inhibition of endogenous lysozyme reabsorption by cytochrome c was used to estimate the glomerular sieving coefficient of endogenous lysozyme in clearance experiments. CLy/GFR increased from a mean value of 0.0053 in control rats to 0.8 at maximal inhibition of tubular reabsorption of endogenous lysozyme by cytochrome c. Knowing the glomerular sieving coefficient, GFR and the lysozyme concentrations in plasma and urine samples, the filtered, excreted and reabsorbed lysozyme amounts could be calculated: 0.5% excreted and 99.5% reabsorbed. Reabsorbed endogenous lysozyme is stored in the kidney in high amounts (1,983 micrograms X g-1 kidney).

Endogenous lysozyme inactivation in EMIT-dau assays.

Though extraction is probably the best method to remove interfering endogenous lysozyme activity, other methods have been suggested. Micropore filtering, 65 degrees C heat treatment and methanol precipitation, or a combination of these methods are ineffective. Heating urine samples to 85 degrees C for 30 minutes removed endogenous lysozyme activity, but unpredictably alters the responsiveness of the various drugs of abuse urine assays. It cannot be recommended for removal of endogenous lysozyme activity for all EMIT immunoassays in urine.

A Comparison of Enzyme Immunoassay and Gas Chromatography/Mass Spectrometry in Forensic Toxicology

A series of 137 urine samples were analyzed for drugs of abuse by enzyme immunoassay (EMIT) and by gas chromatography/mass spectrometry (GC/MS). Agreement between these methods was excellent and ranged from 93.4% for benzodiazepines to 98.5% for propoxyphene. EMIT false negative were traced to the presence of elevated endogenous lysozyme or other interfering materials. In the case of moderate amounts of lysozyme the use of a blank would lead to correct results. Disagreement in the identification of nine benzodiazepine samples was found to be due to a low recovery of benzodiazepine metabolites from urine. Recovery could be improved by incubation of the urine sample with the enzyme beta-glucuronidase.

Drug screening by enzyme immunoassay with the American Monitor KDA.

The methods for barbiturate, opiate, methadone, and amphetamine have been modified for use with the American Monitor KDA. The modification, which incorporates automatic correction for endogenous lysozyme activity, was evaluated by comparing results obtained with the KDA for human urine samples containing known amounts of drug(s) with results obtained with the procedure recommended by Syva for the Gilford 3500. There was 98% agreement bewteen the two systems. Six calibrators and 40 samples can be assayed for all four drugs in about 2.5 h. The procedure has proven to be reliable for screening urine samples obtained from clients at a methadone treatment center.

Studies on biotransformation of lysozyme III. Comparative studies on biotransformation of exogenous and endogenous lysozyme in rats

Exogenous hen lysozyme or endogenous rat lysozyme labeled with 131I was intravenously injected to rats with the same dosage, respectively, and the uptake and degradation of injected 131I-labeled rat lysozyme in liver and kidney were studied in comparison with those of 131I-labeled hen lysozyme. 1. 1.|Although the serum levels of both enzymes injected were almost identical during the first 6 h, the liver uptake of 131I-labeled hen lysozyme was 2.2-fold more than that of 131I-labeled rat lysozyme at the peak time of 5 min after injection. The uptake and clearance of 131I-labeled rat lysozyme in the kidney were exclusively slow as compared with those of 131I-labeled hen lysozyme. 2. 2.|The intracellular distribution in the liver and kidney were examined by the differential centrifugation after injection of each lysozyme. The protein-bound radioactivity of each subcellular fraction was found to be the highest in the 12 000 × g (10 min) fraction in the liver and the 19 600 × g (20 min) fraction in the kidney. The relative specific activity of 12 000 × g fraction of the liver after injection increased with the time lapse. On the other hand, the relative specific activity of 105 000 × g (1 h) fraction of the liver attained a maximum within 5 min after injection and thereafter decreased. It was assumed that the mechanism of the uptake of injected 131I-labeled rat lysozyme in the liver and kidney was similar to that of 131I-labeled hen lysozyme. 3. 3.|The degradation of exogenous or endogenous lysozyme in subcellular particles was examined. From the effect of pH, activator and inhibitor on the degradation, the proteolytic enzyme to degrade the injected 131I-labeled hen lysozyme was indicated to be mainly cathepsin B1, with the optimal pH of about 5.0, and the injected 131I-labeled rat lysozyme was mainly degraded by cathepsin D, with the optimal pH of about 3.5. The in vitro degradation of exogenous and endogenous lysozymes showed a tendency similar to the in vivo clearance from the liver and kidney.

The urinary excretion of lysozyme in dogs.

1. Twelve dogs under light anaesthesia were infused with creatinine, lysozyme and other proteins, and the femoral arterial and urinary concentrations of the infused substances were measured. In some experiments renal venous blood was withdrawn through a catheter inserted by laparotomy. 2. The glomerular permeability to lysozyme was estimated to be 38% of the permeability to creatinine, but there was wide variation and the standard deviation was 11%. 3. The maximum tubular reabsorptive capacity for lysozyme was estimated to be 1·0 mg/100 ml glomerular filtrate, SD 0·44 mg/100 ml. 4. The renal threshold for lysozyme was about 1·0 mg/100 ml. 5. There was evidence supporting the hypothesis that reabsorbed lysozyme is catabolized and no evidence of reabsorption of intact lysozyme into the blood stream. 6. Infusion of myglobin, Bence Jones protein and ovalbumin had no effect on the excretion of endogenous or exogenous (infused) lysozyme. 7. Lysozyme infusions had no conclusive effect on the excretion of (infused) Bence Jones protein or ovalbumin. 8. Corrections, based on the recovery in vitro of lysozyme activity from dog serum and urine, do not significantly alter the results.

RELATIONSHIP OF ENDOGENOUS PYROGEN TO LYSOZYME.

SummaryEndogenous pyrogen (EP) and lysozyme concentrations in serum paralleled one another when dogs were challenged with varying doses of endotoxin, were made tolerant to the action of the toxin, and were given endotoxin along with aminopyrine, adrenal steroids, and hypothermia. Despite these biologic parallels, there is no evidence that endogenous pyrogen and lysozyme are the same biochemical substance.