Induction Of Lysozyme Research Articles

Genotoxicity, oxidative stress and lysozyme induction in Clarias gariepinus chronically exposed to water-soluble fraction of burnt tire ash.

The safety of aquatic ecosystems has been compromised by numerous anthropogenic activities, especially leachates from non-point source toxicants, leaching into aquatic systems. This study evaluated the toxicity of the water-soluble fractions (WSFs) of burnt tire ash (BTA) on Clarias gariepinus via a battery of integrated biomarkers. Juvenile C. gariepinus were exposed to sublethal (0.56, 1.12, and 2.24 g/L) concentrations of BTA, derived from 11.2 g/L median lethal concentration (96 LC50), at duration intervals of 1, 14, and 28 days, followed by a recovery trial that lasted for 14 days. Serum biochemical parameters, antioxidant enzyme activities of the gill and liver, lysozymes activity and erythron profile were assessed. The findings of the present study revealed that BTA-WSF induced prominent alterations on biochemical parameters, lysozymes activity and antioxidant enzymes activities in the exposed fish. Furthermore, toxicant exposure promoted oxidative stress, cellular damage and genotoxicity (erythrocytic nuclear and cellular abnormalities) in the exposed fish. In general, a post-exposure trial showed partial recovery from the exposure effects of the toxicant, following the evident modifications of serum enzymes and erythron pathopathology in the experimental model. Biomonitoring of BTA, using sentinel aquatic species such as C. gariepinus, provides insights into the ecotoxicological potency of this toxicant.

The antimicrobial peptide lysozyme is induced after multiple trauma.

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction.

Haemozoin Induces Early Cytokine-Mediated Lysozyme Release from Human Monocytes through p38 MAPK- and NF-kappaB- Dependent Mechanisms

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1beta/MIP-1alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaBalpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1beta and MIP-1alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

Benzo[a]pyrene modulation of acute immunologic responses in red Sea bream pretreated with lipopolysaccharide

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM-hepc) gene and the activities of some immune-associated parameters in the lipopolysaccharide (LPS)-challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 μg L(-1) ) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM-hepc mRNA expression at 3, 6, and 12-h post-LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM-hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM-hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS-challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM-hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune-associated parameters.

The Bacillus subtilis Extracytoplasmic Function σ Factor σ V Is Induced by Lysozyme and Provides Resistance to Lysozyme

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that σ(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon σ(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.

Injury Is a Major Inducer of Epidermal Innate Immune Responses during Wound Healing

We examined the importance of injury for the epidermal innate immune response in human skin wounds. We found that injury, independent of infiltrating inflammatory cells, generated prominent chemotactic activity toward neutrophils in injured skin because of IL-8 production. Furthermore, injury was a major inducer of the expression of antimicrobial (poly)peptides (AMPs) in skin wounds. In human skin, these injury-induced innate immune responses were mediated by activation of the epidermal growth factor receptor (EGFR). Consequently, inhibition of the EGFR blocked both the chemotactic activity generated in injured skin and the expression of the majority of the AMPs. The importance of injury was confirmed in mouse experiments in vivo, in which injury independent of infection was a potent inducer of AMPs in skin wounds. To our knowledge, these data thereby provide a previously unreported molecular link between injury and neutrophil accumulation and identify the molecular background for the vast expression of IL-8 and AMPs in wounded epidermis. Conceptually, these data show that the growth factor response elicited by injury is important for the recruitment of neutrophils in skin wounds.

Innate immune defense of the sponge Suberites domuncula against gram-positive bacteria: induction of lysozyme and AdaPTin

Sponges are filter feeders that are exposed to large amounts of bacteria present in their surrounding aqueous milieu. The characteristic cell wall component of gram-positive bacteria, peptidoglycan (PPG), was used as a model molecule to study the responsiveness of cells from the marine demosponge Suberites domuncula toward gram-positive bacteria. The sponge lysozyme, which hydrolyzes PPG, was isolated from the living sponge; in addition its gene was cloned (SDLYS) and expressed in Escherichia coli. Antibodies were raised against the recombinant protein to demonstrate that in the Western blot both molecules give the same signal. In situ hybridization with SDLYS as a probe showed that cells in the mesohyl, the gray cells, strongly react with SDLYS. Subsequent immunofluorescence studies with antibodies raised against lysozyme revealed that only bacteria react with anti-lysozyme and only those that are scattered within the mesohyl of the tissue. An adaptor gene (AdaPTin-1) was isolated from the same sponge species that encodes a putative protein involved in endosome formation. Based on its differential expression we conclude that sponge cells react to PPG with a rapid activation of endocytosis, followed by the release of lysozyme.

Lung Mucin Production Is Stimulated by the Air Pollutant Residual Oil Fly Ash

Human and animal exposure to particulate air pollution is correlated with airway mucus hypersecretion and increased susceptibility to infection. Seeking clues to the mechanisms underlying this pathology, we examined the effect of the particulate air pollutant residual oil fly ash (ROFA) on production of the major component of mucus, mucin, and the major antibacterial protein of the respiratory tract, lysozyme. We found that following in vitro exposure to ROFA, epithelial cells showed an increase in mucin (MUC5AC) and lysozyme (LYS) steady state mRNA. This upregulation was controlled at least partly at the level of transcription as shown by reporter assays. Experiments testing the ability of the major components of ROFA to mimic these effects showed that vanadium, a metal making up 18.8% by weight, accounted for the bulk of the response. A screen of signaling inhibitors showed that MUC5AC and LYS induction by ROFA are mediated by dissimilar signaling pathways, both of which are, however, phosphotyrosine dependent. Recognizing that the ROFA constituent vanadium is a potent tyrosine phosphatase inhibitor and that mucin induction by pathogens is phophotyrosine dependent, we suggest that vanadium-containing air pollutants trigger disease-like conditions by unmasking phosphorylation-dependent pathogen resistance pathways.

Insect lysozymes.

Lysozymes, related to the chicken-type lysozymes in vertebrates, are ubiquitous components in the bacteriolytic armamentarium of insects. The enzyme is normally present in the blood, and together with other bactericidal factors lysozyme is often strongly induced when the insect is infected. This response is regulated by mechanisms that are related to those that activate inflammatory, acute-phase and immune responses in mammals, and the induction of lysozyme and other factors is now being investigated as a model for innate immune reactions in general. A special adaptation is seen in flies like Musca and Drosophila. These animals live on the microorganisms in decompositing matter, and they have developed a specialized set of lysozymes that are expressed in the alimentary tract. In Drosophila, at least seven different lysozyme genes are clustered in a small region on the third chromosome. The different genes are expressed in different parts of the digestive tract, and at different time points during development, and they are highly divergent in sequence. The major lysozymes in the fly gut have acidic isoelectric points and/or pH optima, and their evolution provides an interesting parallel to the ruminants.

Parallel induction of cecropin and lysozyme in larvae of the silkworm, Bombyx mori

Lysozyme activity in the hemolymph of Bombyx mori increased in parallel with cecropin activity after injection of the larvae with soluble peptidoglycan or UV-killed bacteria. The lysozyme and cecropin A genes were expressed in parallel in the fat body after injection of peptidoglycan as detected by northern blot hybridization. The elicitor specificity for lysozyme induction was identical to that for cecropin, suggesting a common mechanism for recognition of bacteria and following signal transduction introducing to the simultaneous synthesis of cecropin and lysozyme. Bacterial cells killed by UV-irradition were also effective as elicitor when added to the fat body culture, suggesting that phagocytosis of bacteria by hemocytes may not be an essential process for the induction of antibacterial protein synthesis in the silkworm.

Lysozyme activity in immunized and non-immunized hemolymph during the development of the silkworm, Bombyx mori

Lysozyme activity in normal hemolymph was determined during the larval-pupal development of the silkworm, Bombyx mori. The lysozyme level in young larvae was approx. 40 μg Bombyx lysozyme protein per ml of hemolymph, then increased to 100 μg/ml in mature larvae and decreased again to 60 μg/ml upon pupation. Immunization of larvae or pupae by the injection of soluble peptidoglycan (SPG) induced an increase in lysozyme activity within 24 hr to a level of approx. 440 μg/ml, which was almost constant independent of the developmental stages of the insect. Induction of cecropin by SPG was parallel to the induction of lysozyme.

Effects of environmental pollutants on immunological competency of the clam Mercenaria mercenaria: Impaired bacterial clearance

Subclinical doses of heavy metals and other environmental pollutants have been shown to be synergistic to infectious agents in mammals. The results of this study suggest that similar phenomena may be elicited in the bivalve mollusk Mercenaria mercenaria. Clams were exposed to benzo[ a]-pyrene, pentachlorophenol, or hexachlorobaizene for up to 18 wk at pollutant levels which produced no increased mortality or morbidity. The majority of the pollutant-exposed clams showed significantly impaired ability to clear a marine Flavobacterium sp. which had been injected into their hemolymph; some of these animais showed complete failure to clear. Untreated clams routinely cleared > 90% of the bacteria in the same period of time, none of these controls were completely unable to clear bacteria. These findings imply that resistance to bacterial infection in this common marine organism is decreased by exposure to xenobiotics. Intrahemocytic lysozyme was elevated in certain exposed clams. Lysozyme is considered to serve a protective antimicrobial function in invertebrates; generally, it is more active against gram-positive bacteria than against gram-negative forms like Flavobacterium. It appeared that hemocyte lysozyme induction was more pronounced at lower levels of pollutant exposure; however, the deficiency in bacterial clearance was directly proportional to tissue levels of the test pollutants.

Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3.

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by 1 alpha,25-dihydroxyvitamin D3, the active form of vitamin D3. The minimal concentration of 1 alpha,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM 1 alpha,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 microM dexamethasone, the most potent known stimulator. Among several markers of the differentiation by 1 alpha,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction of lysozyme and locomotive activities. Similar changes were also induced by 0.01-1 microM 1 alpha-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, 1 alpha, 25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.

Genetic dissociation of different cellular effects of interferon on myeloid leukemic cells

AbstractMutant clones of mouse myeloid leukemic cells have been used to study the effect of mouse interferon on the expression of different cellular genes. The cell mutants, MGI+D+, MGI+D− and MGI−D−, differed in their competence to be induced to undergo cell differentiation by the normal macrophage and granulocyte protein inducer MGI or the steroid hormone dexamethasone. The growth of all the clones tested was inhibited by interferon, but there was a 20‐fold difference in sensitivity to growth inhibition in different clones. This difference was not associated with the same difference in susceptibility to the cytotoxic effect of cytosine arabinoside and actinomycin D, or with cell competence for the induction of differentiation. The variation in cytotoxic effect of interferon on different malignant cell populations should, therefore, be taken into consideration in the possible clinical use of interferon as an anti‐tumor agent. Some MGI+D+ clones, but none of the MGI+D− or MGI−D− clones, showed, in addition to a high sensitivity to growth inhibition by interferon, a 14‐ and 2‐fold enhancement of the induction of lysozyme by dexamethasone and MGI, respectively, and a slight induction of Fc rosettes with dexamethasone. But interferon had no effect on induction of C3 rosettes, immune phagocytosis and differentiation to mature macrophages or granulocytes. The enhancing effect on lysozyme induction by interferon was also obtained with cordycepin, vinblastine, or 2‐deoxy‐D‐glucose, although neither interferon nor any of these other compounds alone induced any of the differentiation‐associated properties. A MGI+D− mutant, derived from an MGI+D+ clone with an enhancement of lysozyme induction by interferon, showed the same high sensitivity to growth inhibition by interferon as its parental MGI+D+ clone, but there was no enhancement of the induction of lysozyme by MGI or dexamethasone. The results indicate that interferon can exert a selective enhancing effect on the expression of some genes involved in differentiation in clones with the appropriate genotype and that, by using appropriate cell mutants, it is possible to genetically dissociate different cellular effects of interferon.

Control of lysozyme induction in the differentiation of myeloid leukemic cells

A system has been developed with clones of mouse myeloid leukemic cells in culture to study the induction of synthesis and secretion of lysozyme in relation to other steps in myeloid cell differentiation. Lysozyme was initially absent in all the clones studied. The different clones can be divided into three types according to their ability to be induced to undergo normal cell differentiation by the protein inducer MGI (macrophage and granulocyte inducer). One type of clone that can be induced by MGI to form Fc and C3 receptors and differentiate to mature macrophages and granulocytes (MGI +D +) was also induced by MGI to synthesize and secrete lysozyme. Lysozyme was induced after Fc and C3 receptors, and labeling with 35S-methionine has shown that the induced lysozyme was newly synthesized. MGI +D + clones were also induced to synthesize and secrete lysozyme by dexamethasone, prednisolone, cytosine arabinoside, or thymidine and in one of four clones by dimethylsulfoxide but not by sodium butyrate. Inhibition of cell multiplication by itself was not sufficient to induce lysozyme synthesis. A second type of clone which can be induced by MGI to form Fc and C3 receptors but not mature cells (MGI +D −) was more weakly inducible by MGI for lysozyme and was not inducible by any of the other compounds. A third type of clone (MGI −D −) is not inducible by MGI for receptors or mature cells. One of four MGI −D − clones was induced to synthesize but not secrete lysozyme by dexamethasone together with sodium butyrate, but there was no lysozyme induction by MGI or any of the other compounds separately. The different clones maintained their different properties for at least 6 months in culture. The results indicate that clones with different hereditary blocks in the ability to be induced to differentiate by specific compounds can be used to dissect the process of myeloid cell differentiation, that the sequence of differentiation is induction of Fc and C3 receptors→lysozyme→mature cells, and that there are separate controls for these developmental steps.

Progesterone induction of lysozyme and peptidase activities in the porcine uterus

When nonpregnant ovariectomized gilts were treated daily for 15 days with progesterone or progesterone plus estradiol, there was a tenfold increase in the amount of secreted protein that could be flushed from their uteri compared with control animals administered either no steroid or only estradiol. This increase was due to the appearance of a number of new proteins not present in the controls. In addition to a purple-colored protein with acid phosphatase activity, which has been described previously, there were large increases in lysozyme and leucine aminopeptidase activity. All three enzymes appeared to be induced by progesterone. By continuing the progesterone treatments for periods up to 60 days, it was possible to recover very high levels of each of these enzymes from the uterine flushings of the pigs. Cathepsin activities (B 1, D, and E) were also found to increase as progesterone treatment was prolonged. The levels of the peptidases does not seem to be coordinated since their activities change relative to each other when different animals are examined. Acid phosphatase (due entirely to the purple protein), lysozyme, and leucine aminopeptidase activities are also detectable in allantoic fluid after Day 30 of pregnancy and reach a maximum between Days 60 and 80. It is suggested that these enzymes may be maternal in origin.

Regulation of Protein Synthesis in Chick Oviduct: I. INDEPENDENT REGULATION OF OVALBUMIN, CONALBUMIN, OVOMUCOID, AND LYSOZYME INDUCTION

Egg white protein synthesis is induced and maintained in oviduct magnum of immature female chicks by administering gonadal steroid hormones. The relative rate of synthesis of four egg white proteins (ovalbumin, conalbumin, ovomucoid, and lysozyme) was measured by culturing magnum explants with radioactive amino acids followed by specific immunoprecipitation of each of the egg white proteins. Induction of these proteins was studied as a function of time, hormonal dose, and hormonal combination. A combination of estrogen, progesterone, and testosterone was found which would induce the relative rate of synthesis of each of the four egg white proteins to a level that closely approximates that observed in laying hens; this combination also promotes maximal magnum growth. The synthesis of these proteins is not strictly coordinated, since the rate of synthesis of one or more of these proteins can change relative to the others, although they are all synthesized in the same cell. Noncoordinate protein synthesis is exemplified by the changing ratio of conalbumin to ovalbumin synthesis during both primary and secondary stimulation with estrogen. The ratio of synthesis of these two proteins also changes as a function of the dosage of hormone administered. Furthermore, different combinations of hormones can produce noncoordinate synthesis; for example, the synthesis of ovomucoid and conalbumin is increased relative to ovalbumin when either progesterone or testosterone are administered along with estrogen. The most likely explanation for the change in the ratio of ovalbumin to conalbumin synthesis observed during hormonal stimulation is the 2-fold difference in the rates of degradation of their mRNAs. In contrast, changes in mRNA synthesis or activation best account for the preferential induction of conalbumin synthesis by low doses of estrogen, and the increased rates of conalbumin and ovomucoid synthesis which are produced by supplementing estrogen with either progesterone or testosterone. The results suggest that a single regulatory site for the control of egg white synthesis is unlikely since several of these proteins appear to be under independent control.