Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.
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PMID:Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy. 777 15

The proinflammatory cytokine and potent chemoattractant IL-8 is involved in regulation of infectious or inflammatory processes. Human vascular endothelial cells (EC) and smooth muscle cells (SMC) probably contribute to these responses by recognition and/or production of rIL-8. We demonstrate here in competitive binding studies with radiolabeled rIL-8 that EC and fibroblasts, but not SMC, specifically bind IL-8 with low affinity. The binding was not saturated by ligand concentrations up to 80 nM 125I-rIL-8. Unlabeled neutrophil-activating peptide-2 competed the binding of 125I-rIL-8, although less potently than unlabeled rIL-8, as reported previously for polymorphonuclear neutrophils. In contrast, connective tissue-activating peptide III, platelet factor 4, or lysozyme did not reduce binding of 125I-rIL-8 to EC or fibroblasts. In accordance with these binding studies, EC and fibroblasts, but not SMC, expressed human IL-8 receptor type I mRNA. Neither cell type expressed mRNA for IL-8 receptor type II. Stimulation with IL-1 alpha or LPS did not alter the results obtained in PCR or binding studies. Although SMC did not express specific binding sites for IL-8, Western blot experiments showed that IL-1 alpha-, TNF-, or LPS-stimulated SMC released two major immunoreactive isoforms of IL-8 in a time- and dose-dependent manner. The m.w. were similar to IL-8 isoforms released by EC or mononuclear cells. The differential capacity of EC and SMC to produce IL-8 and express IL-8 binding sites indicates that vascular cell-derived IL-8 may contribute to differential regulation of infectious and inflammatory responses in the vessel wall.
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PMID:IL-8 specifically binds to endothelial but not to smooth muscle cells. 786 4

Inflammatory reactions induce the production of reactive oxygen species (ROS): the reverse sequence of these events is also true. Moreover, many components of these reactions interact with a synergistic effect. In this short comprehensive review we analyze some of these interactions which may have pathological effects. Inflammatory reactions are triggered off by exogenous or endogenous aggressions and are characterized by cellular and vascular events. The activated leucocytes leave the circulating blood and reach the site of the aggression where they release a large amount of ROS as well as the content of their granules. The granular content is made in a large part by molecules with killing and degradative activities such as myeloperoxidase, defensins, elastase, collagenase, cathepsins and lysozyme. The inflammatory reaction is beneficial for humans when its effects are limited to the pathogens. The insufficiency of a component of the inflammatory reaction such as the production of ROS which is seen, for example in chronic granulomatous disease, leads to severe and recurrent bacterial infections. In other situations inflammatory reactions are deleterious because they are directed against normal tissues instead or in addition to pathogens. In some cases the behaviour of the phagocytes is modified because they have been primed by inflammatory molecules such tumor necrosis factor, LPS, interleukins or interferons. Priming often leads to a decreased speed of locomotion of the leucocytes with an increased susceptibility to their stimuli. The combination of these effects leads to a premature release by the phagocytes of their killing and degradative factors. Production of ROS such as that seen during irradiation, drug metabolism, or ischemia followed by reperfusion for example, induces inflammatory reactions with a secondary amplification of ROS production. Acute ROS production can also lead to thrombosis, whereas chronic ROS production can induce a chronic inflammatory reaction of the endothelium with atherosclerosis as a possible consequence. Some examples are also given to show that ROS might control positively or negatively the activity of inflammatory molecules. The multiplicity of the cross reactions between ROS and inflammation allows to suggest that drugs that disconnect these two events might be therapeutically used.
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PMID:[Reactive oxygen species and inflammation]. 801 8

Endotoxin (lipopolysaccharide [LPS]) released during gram-negative bacterial infection induces varieties of cytokines which directly and/or indirectly cause shock, disseminated intravascular coagulation, and death. We previously showed that lysozyme (LZM) was an LPS-binding protein and inhibited various immunomodulating activities of LPS. In this study, we examined the effect of LZM on the LPS-triggered septic shock model induced by carrageenan treatment and assessed by tumor necrosis factor production. The data presented in this report strongly suggest that LZM-LPS complex formation completely abrogates tumor necrosis factor production and the mortality caused by LPS and that LZM may be useful for the treatment of endotoxin shock.
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PMID:Binding of lysozyme to lipopolysaccharide suppresses tumor necrosis factor production in vivo. 813 23

It has been found previously that peritoneal exposure to hematoporphyrin derivative (HpD) photodynamic therapy (PDT) can induce systemic immunosuppression of contact hypersensitivity. We have now found that HpD-PDT also significantly prolongs survival of murine skin allografts. Normal A/J mice transplanted with BALB/c skin rejected the grafts within 10 +/- 0.9 days. Recipient mice treated 24 hr previously with HpD-PDT rejected skin allografts at 16 +/- 1.2 days. HpD alone or irradiation alone had no effect on skin graft survival, nor did HpD-PDT administered shortly after grafting. Flow cytometric analyses showed a nearly complete depletion of peritoneal lymphocytes 3 days after HpD-PDT. Lymphocyte levels were normal in the spleen, an organ not directly targeted by the PDT treatment, but the cells were totally unresponsive to Con A and LPS mitogens. Conversely, peritoneal HpD-PDT caused a striking enhancement in macrophage function as measured by phagocytosis of antibody-coated sheep erythrocytes. Humoral immunity to hen egg-white lysozyme was not significantly changed by HpD-PDT. These results demonstrate that HpD-PDT causes systemic immunosuppression of cellular immunity which, in turn, allows prolonged survival of allografts. Humoral immunity appears to remain largely unaffected by HpD-PDT and macrophages become activated, suggesting that this therapy might be more effective in specifically targeting T cell-mediated immunity than current immunosuppressive treatments.
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PMID:Enhanced skin allograft survival after photodynamic therapy. Association with lymphocyte inactivation and macrophage stimulation. 827 23

Using radioiodinated, photoactivable, reducible cross-linker conjugated bacterial endotoxic lipopolysaccharide (125I-ASD-LPS), we have demonstrated that LPS selectively binds to the S2 subunit of pertussis toxin (PT). Since LPS also interacts with the S2 subunit of the B-oligomer of the toxin, the binding of LPS to PT is not A-protomer (S1 subunit) dependent. The binding can be inhibited with native underivatized LPS and with purified lipid A, suggesting that the binding is mediated through the lipid A moiety of the LPS molecule. The binding of PT to LPS can be inhibited by bovine fetuin glycoprotein. Since PT has been demonstrated to interact specifically with N-linked oligosaccharide side chains of fetuin, the interaction of LPS with the S2 subunit of PT may involve carbohydrate-dependent interactions of the disaccharide backbone of lipid A with S2. Additional studies have documented that LPS binding to PT may be competitively inhibited by lysozyme but not by polymyxin B. Sequence analysis has allowed identification of a high degree of amino acid sequence similarity between the S2 subunit of PT and hen egg white lysozyme at the N-terminal 80-residue regions. Shared N-terminal sequence similarity between lysozyme, PT-S2, and a third LPS-binding protein alpha-lactalbumin allows tentative identification of a second family of LPS binding proteins.
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PMID:Lipopolysaccharide interaction with S2 subunit of pertussis toxin. 841 48

Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.
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PMID:Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. 842 97

A new procedure for quantifying the endotoxin-neutralizing capacity (ENC) of plasma or serum is described. Serially diluted samples were preincubated with endotoxin (lipopolysaccharide; LPS) and the sample dilution producing 50% inhibition of Tachypleus amebocyte lysate activation was measured by a Limulus peptide C enzyme-linked immunosorbent assay. The assay was not subject to interference from plasma or serum at a 500-fold dilution. The ENC of fresh sera from 120 healthy human donors, determined with Salmonella abortus LPS, had a median value of 7.7 kEU/ml (95% confidence limits 3-24 kEU/ml). Values for heparinized fresh plasma were close to those for the corresponding sera. Serum ENC varied greatly with different types of LPS. Neutralization of LPS by serum was rapid, heat-labile, and fully reversed by acidification. Addition of 2 mM EDTA to the serum diluent or pretreatment of LPS with 0.5% deoxycholate enhanced the ENC of serum about 25-fold or 10-fold respectively. The neutralization of LPS by polymyxin B or lysozyme could be demonstrated by the ENC assay, while that by human serum albumin, fibronectin or anti-LPS immunoglobulins was only detected in the presence of 2 mM EDTA. The kinetic changes of LPS and ENC during rabbit endotoxemia were also determined. The ENC assay may be used to study the significance of plasma ENC in Gram-negative infections and to identify the components contributing to plasma ENC.
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PMID:Quantification of the endotoxin-neutralizing capacity of serum and plasma. 853 31

The effect of her egg-white lysozyme (HEWL) on immune response was evaluated by measuring antibody-producing cells and circulating antibodies in mice inoculated with the test antigen (SRBC or BSA) and HEWL at the same time but in a separate body area. HEWL caused a premature decline in SRBC-specific plaque forming cells (PFC) and a reduction in the total amount of these cells. HEWL inhibited antibody production against BSA in the primary response, but was devoid of any effect on the secondary response elicited in the same mice by a second inoculation of the test antigen. The inhibitory effect of HEWL was dose-dependent, being maximal with 300 micrograms, required an enzymatically active protein and was not shown by other basic proteins. HEWL also abolished the enhancing effect of LPS and CFA on anti-BSA antibody production. The inhibitory activity of HEWL was further increased by hydrolyzed peptidoglycan. These results suggest that HEWL modulates the immune response in mice and performs this function through activation of non-specific suppression mechanisms.
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PMID:Modulatory effects of hen egg-white lysozyme on immune response in mice. 867 48

Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis. Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells. Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested. The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level. Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h. Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation. Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG). Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens. These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.
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PMID:Macrophage function in deer. 867 37


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