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)

We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.
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PMID:Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity. 169 33

1. Mucins were isolated from sputum from a patient with chronic bronchitis and subjected to two different preparation procedures. 2. In the first procedure, CsBr density-gradient centrifugation gave rise to two well-separated fractions. Mucins recovered in the high-density fraction still contained mucus proteinase inhibitor (MPI) and lysozyme (LSZ). 3. Mucins were purified after a second step of CsBr density-gradient centrifugation or after gel-filtration chromatography with a buffer of high ionic strength, containing 0.5 M NaCl. 4. In the second procedure, trichloroacetic acid treatment of whole sputum followed by cation-exchange chromatography allowed the obtention of a non-retained fraction composed of mucins. 5. Gel-filtration in buffer containing 0.5 M NaCl, allowed the release of MPI and LSZ from mucins, thus confirming that interactions still occurred between those components. 6. The chemical compositions of the mucins isolated by the two above procedures were quite similar. 7. These data support the hypothesis of the existence of ionic interactions between basic amino acid residues of MPI and LSZ and acid residues of the carbohydrate chains of mucins in the secretions of the large airways. 8. These interactions could play a role in the protection of mucins against proteolysis and consequently in the maintenance of rheological properties of the mucus gel in disease.
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PMID:Strong ionic interactions between mucins and two basic proteins, mucus proteinase inhibitor and lysozyme, in human bronchial secretions. 173 97

The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity. After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70%. The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems. SLPI proved to be a potent inhibitor of translation in vitro. When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75%. The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA-metabolizing enzymes, such as RNase. In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61. Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin. On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA. Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPI/DNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E. coli. These results indicate that, in general, high-level expression of basic recombinant proteins in E. coli may be problematic.
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PMID:Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. 246

Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.
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PMID:Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum. 277 94

Airway inflammation is often associated with the infiltration of activated neutrophils and subsequent protease release. Although aiding in the digestion and phagocytosis of foreign proteins and microorganisms, neutrophil proteases can indiscriminately damage healthy lung tissue. In the conducting airway, proteases, particularly neutrophil elastase, are counter-balanced by several antiproteases, including secretory leukocyte protease inhibitor (SLPI). SLPI can be produced locally by a number of cells including the airway epithelial cell. To examine the effects of neutrophil granule components on SLPI transcript levels, airway epithelial cells were treated (up to 96 h) with elastase, other proteases, or enzymes isolated from human sputum. We found that neutrophil elastase increased SLPI transcript levels in primary and transformed human airway epithelial cells in a time- and dose-dependent manner. Other neutrophil products, such as cathepsin G, myeloperoxidase, and lysozyme, had little or no effect on SLPI transcript levels. However, two nonneutrophil proteases, trypsin and pancreatic elastase, also increased SLPI transcript levels at higher doses than that required of neutrophil elastase. Two inflammatory cytokines, tumor necrosis factor-alpha and interleukin-8, produced little or no effect on SLPI transcript levels. This study demonstrates one way in which SLPI is regulated, via a protease that it inhibits, neutrophil elastase.
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PMID:Neutrophil elastase increases secretory leukocyte protease inhibitor transcript levels in airway epithelial cells. 810 97

The goal of our studies was to establish procedures for subculturing normal human tracheobronchial epithelial (NHTBE) cells without compromising their ability to differentiate into mucous and ciliated cells (i.e., differentiation competence) and to study the regulation of airway secretions by epidermal growth factor (EGF) and retinoic acid (RA). Primary NHTBE cells were obtained from a commercial source and subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for differentiation competence in air-liquid interface (ALI) cultures. The apical secretions of cultured NHTBE cells were characterized by immunoblotting, Western blotting, or enzyme-linked immunosorbent assay using a variety of antibodies. They contained mucin-like materials as well as lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI). We found that an EGF concentration of 25 ng/ml, which is commonly used in airway cell cultures, adversely affected growth, mucin production, and morphology of ALI cultures and that RA was essential for mucociliary differentiation. Without RA, the epithelium became squamous and mucin secretions decreased 300- to 900-fold. In contrast, secretion of lysozyme, lactoferrin, and SLPI was significantly increased in RA-depleted cultures. Cells of passage 2 (P-2) through P-4 remained competent to differentiate into mucous and ciliated cells when grown in ALI cultures. However, mucin secretion and ciliagenesis decreased in P-3 and P-4 cell cultures and P-3 but not P-4 cell cultures exhibited bioelectric properties characteristic of airway epithelium. We concluded that P-2 and P-3 NHTBE cell cultures retain many important features of normal airway epithelium. This enables one to conduct many studies of airway cell biology with a greatly expanded (6,000-fold) cell pool.
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PMID:Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. 853 81

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.
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PMID:Antibacterial activity of antileukoprotease. 889 Feb 1

The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells. Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth. We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions. Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures. Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased. Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated. LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected. Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products. MUC2 and MUC5AC steady state mRNA levels were not consistently affected. In contrast LZ and SLPI gene expression were reproducibly increased. Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.
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PMID:Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. 919 74

Secretory leukocyte protease inhibitor (SLPI) is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte-derived proteinases, has anti-HIV-1, antibacterial, and anti-fungal properties, and interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages. We now report that at both the mRNA and the protein level, SLPI shows inducible expression in a nonglandular epithelium. A weak expression of SLPI was found in the stratum granulosum of adult normal human epidermis; however, in lesional psoriatic epidermis and in migrating keratinocytes of healing wounds, a strong cytoplasmic staining was seen in the suprabasal keratinocytes. Remarkably, in the dermis adjacent to SLPI-expressing keratinocytes, SLPI was found extracellularly associated with elastin fibers, whereas the dermis in normal skin was negative. In cell culture, SLPI was hardly expressed in monolayers of proliferating keratinocytes. Differentiating cultures with a phenotype of normal skin expressed low levels of SLPI, whereas cultures with a regenerative/psoriatic phenotype expressed high levels. Functional studies with recombinant SLPI indicated that its antibacterial spectrum and potency are distinct from other anti-microbial peptides such as lysozyme and defensins. In view of the multiple functions of SLPI and the inducibility, we propose that it acts as an important first line defence mechanism in cutaneous injury.
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PMID:Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes. 985 7

Although an elevated sodium concentration in human milk is suggested to be an indicator of mastitis, it is unclear whether elevated sodium concentrations are associated with immunological and inflammatory mediators in human milk. We conducted a cross-sectional study to evaluate the relationships between elevated breast milk sodium concentrations and levels of lactoferrin, lysozyme, secretory leukocyte protease inhibitor (SLPI), interleukin-8 (IL-8), and RANTES (regulated on activation normal T cell expressed and secreted) in human milk at 6 weeks postpartum in 96 lactating women in Blantyre, Malawi. Mastitis, as indicated by an elevated breast milk sodium concentration, was present in 15.6% of the women. Women with and without mastitis had respective median levels of other factors as follows: lactoferrin, 1,230 versus 565 mg/liter (P < 0. 0007); lysozyme, 266 versus 274 mg/liter (P = 0.55); SLPI, 76 versus 15 microg/liter, (P < 0.0002); IL-8, 339 versus 25 ng/liter (P < 0. 0001); and RANTES, 82 versus 3 ng/liter (P < 0.0001). Elevated sodium concentrations in breast milk are associated with an increase in levels of some immunological and inflammatory factors in breast milk.
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PMID:Mastitis and immunological factors in breast milk of lactating women in Malawi. 1047 15


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