Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.
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PMID:Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99. 877 61

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H.pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not alpha- or beta-galactosidase, heparitinase,lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 microg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H.pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble 125I-labeled heparin and other sulfated carbohydrate compounds.
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PMID:Sulfatides inhibit binding of Helicobacter pylori to the gastric cancer Kato III cell line. 909 25

Reverse genetics was used to modify the influenza virus genome by inserting the p46-63 sequence of hen egg lysozyme (HEL) into the neuraminidase stalk of the virus. The resulting virus, HEL-Flu, contained the epitopes recognized by CD4+ T cells from 3A9-TCR transgenic mice (C3HTg). Here, we show that HEL-Flu was infectious in the respiratory tract of both C3H and C3HTg mice, the latter animals showing an early, transient morbidity. Splenic dendritic cells and certain cloned populations of splenic macrophages and brain microglia constitutively presented infectious and inactivated HEL-Flu to the T cells in an Ag-specific and MHC class II-restricted manner. These results demonstrate the utility of HEL-Flu in assessing the APC activity for naive T cells; they also extend the previous studies showing that discrete populations of macrophages and microglia constitutively process and present Ag to naive T cells.
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PMID:HEL-Flu: an influenza virus containing the hen egg lysozyme epitope recognized by CD4+ T cells from mice transgenic for an alphabeta TCR. 930 Jun 73

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.
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PMID:Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water. 1160 32

During its normal life cycle, Diplostomum spathaceum cercariae attach to and invade fish intermediate hosts. They are also known to attach to various other aquatic animals in response to water currents, touch and carbon dioxide. The purpose of this study was to identify the specific stimuli used by D. spathaceum cercariae to recognise the appropriate fish host. We characterised the host cues which stimulate them to remain on the host (enduring contact) and to penetrate the skin. Cercariae were exposed to animal skin tissues and fish skin surface mucus, their extracts and chemical modifications integrated into agar or offered via membrane filters. Enduring contact was stimulated by hydrophilic extracts Mr<3kDa, which were sensitive to oxidation of carbohydrates. The stimulating cues are probably small molecular carbohydrates, as monosaccharides stimulated enduring contacts, but amino acids, urea, electrolytes and peptides did not. Penetration was stimulated by hydrophilic macromolecules, Mr>30kDa, and by lipids. The hydrophilic stimuli were protease resistant and precipitable with Alcian blue and they were sensitive to alkaline cleavage, to digestion with lysozyme and neuraminidase as well as to oxidation of sialic acids. They were considered to be glycoproteins with O-glycosidically linked carbohydrate chains and bound sialic acids as signal structures. The lipophilic penetration stimuli were contained exclusively in the fatty acid fractions, and the stimulating characteristics of these fatty acids resembled the stimulating penetrations in other cercarial species. Diplostomum spathaceum cercariae respond to a unique profile of cues in their sequence of host-recognition phases. These cues differ from those used in other fish parasites studied to date and underline the diversity of fish recognition strategies.
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PMID:Diplostomum spathaceum cercariae respond to a unique profile of cues during recognition of their fish host. 1211 97

From saline extracts of Phytolacca esculenta (shoriku) roots, two phytomitogenes were isolated by salting out with (NH4),SO4 and chromatography on DEAE-cellulose and Sephadex G-100 columns. Both fractions were homogeneous on disc electrophoresis and on immunoelectrophoresis. One of these (Fraction E-2) was shown to be similar to pokeweed mitogen in respect to mol. wt (32,000) and amino acid composition. The other (Fraction E-3) was a protein of 18,000 mol. wt. Both fractions had similar biological activities to pokeweed mitogen in their ability to stimulate pig blood lymphocytes in vitro to incorporate tritiated thymidine, and to induce blastoid transformation. Both fractions contained an unusually large amount of cystine, i.e., 18 half-cystine residues % for Fraction E-2 and 22 residues % for Fraction E-3. Although these mitogens were resistant to deproteinizing procedures such as perchloric acid treatment and Sevag's procedure, the DNA synthesis-stimulating activity was inactivated by digestion with Pronase E and Nagarse, but resistant to trypsin, chymotrypsin, deoxyribonuclease, ribonuclease, lysozyme and neuraminidase. The activity was stable at acidic and neutral pH (4-7) but unstable at alkaline pH. The activity at pH 7.3 was stabilized by the addition of Ca2+ or Mg2+. On the addition of more than 2 mM of Ca2+, precipitation of mitogen occurred. From the above results the molecular basis of the mitogenic activity of shoriku mitogen is discussed.
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PMID:Isolation and characterization of pokeweed mitogen-like phytomitogens from Shoriku, Phytolacca esculenta. 1999 19

Numerous studies have addressed the nature of antibody-antigen interaction, but only recently have three-dimensional structures of complexes of antibodies with protein antigens been reported, one with lysozyme and one with the influenza virus antigen neuraminidase. Both structures show that there are epitopes involving about 16 amino acids on surface loops of the antigen. In the lysozyme complex the interaction of the components is rigid, but there is a degree of structural flexibility in the formation of the complex with neuraminidase. In this article, Peter Colman, Graeme Laver and their colleagues discuss some speculative implications of the results currently available.
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PMID:How antibodies recognize virus proteins. 2529 Oct 54


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