Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recombinant plasmid pRI203 carries a Yersinia pseudotuberculosis chromosomal gene that makes E. coli K-12 HB101 strain able to synthetize an outer membrane protein, invasin, which interacts with integrin receptors of eukaryotic cells, enabling this microorganism to penetrate human cultured animal cells. In this study we evaluated the involvement of HeLa cell membrane structural components in the early phases of the invasive pathway of E. coli HB101 (pRI203). When HeLa cell monolayers were treated with several enzymes we showed that trypsin-, proteinase K- and neuraminidase-sensitive components are required for bacterial invasion. Comparison of the ability of simple and complex carbohydrates to inhibit bacterial invasion indicated that N-acetyl neuraminic acid, N-acetyl glucosamine and mucin were the most effective competitive inhibitors. Among glycolipids, gangliosides enhanced bacterial entry in HeLa cells. The results obtained suggest that N-acetyl neuraminic acid and N-acetyl glucosamine-containing glycoproteins and/or glycolipids participate as putative HeLa cell binding sites for the penetration process of E. coli HB101 (pRI203).
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PMID:Involvement of membrane carbohydrates of HeLa cells in the E. coli HB101 (pRI203) invasive pathway. 160 81

A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.
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PMID:Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. 197 20

A member of the high molecular weight glycoproteins of human milk and breast cancer was isolated from the sera, ascites and breast carcinoma tissue of patients with breast cancer using monoclonal antibody 3E1.2. The 3E1.2 defined antigen, termed mammary serum antigen (MSA) was obtained by immunoaffinity chromatography and a solid phase immuno-precipitation technique (SPIT) from serum of patients with metastatic breast cancer. MSA was found to be a high molecular weight glycoprotein with a Mr greater than 300,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a native Mr approximately 1 x 10(6) by gel filtration chromatography; in accord with the published Mr of other high molecular weight glycoproteins obtained from human milk and breast cancer. A high degree of glycosylation of MSA molecule was shown by its poor staining with Coomassie blue but good staining in a PAS-silver stain. In addition, MSA contained N-acetyl neuraminic acid and N-acetyl glucosamine as indicated by its binding to wheat-germ agglutinin. The epitope defined by antibody 3E1.2 is sensitive to treatment by sodium periodate and neuraminidase, implying that both carbohydrate and sialic acid are required for binding of antibody 3E1.2. Sandwich immunoassays demonstrated that MSA+ molecules are likely to express repeated 3E1.2 defined epitopes. Furthermore, MSA was susceptible to degradation by pronase, subtilisin and proteinase K and gave a different peptide profile from that of the PAS-O glycoprotein of human milk. MSA+ molecules were found to carry epitopes for a number of other monoclonal antibodies which were reactive with the PAS-O glycoprotein. It is suggested that MSA has the same core protein as is recognised by antibody DF3 which has been used to clone the same cDNA as was cloned with antibodies HMFG-1, HMFG-2 and SM-3. However, the epitope detected by the 3E1.2 antibody is either absent or weakly expressed on human milk, human milk-fat globule membrane (HMFGM) or deglycosylated HMFGM--all of which react strongly with various anti-HMFG antibodies. The antibody 3E1.2 thus recognises a unique epitope of the high molecular weight glycoproteins of human milk and breast cancer, being found in cancer tissue, serum and ascitic fluid of patients with breast cancer but weakly expressed or absent in human milk.
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PMID:Purification and biochemical characterisation of a novel breast carcinoma associated mucin-like glycoprotein defined by antibody 3E1.2. 246 54

Analyses of chemical composition in whole cells of Rickettsia tsutsugamushi were performed and compared with those of the other rickettsiae and gram-negative bacteria. The results indicated that R. tsutsugamushi does not contain detectable amounts of 3-deoxy-D-mannooctulosonic acid, heptose, muramic acid, or glucosamine (less than 2, less than 2, less than 3, and less than 3 nmol/mg, respectively). The microorganism was found to contain four kinds of fatty acids (16:0, 18:0, 18:1, and 18:2), but not hydroxy fatty acids. Furthermore, in analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver or Coomassie blue staining, lipopolysaccharide bands were not detected in preparations treated with proteinase K. It is concluded that R. tsutsugamushi has little or no peptidoglycan or lipopolysaccharide.
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PMID:Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi. 311 50

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.
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PMID:Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro. 368 Feb 8

Leptospiral lipopolysaccharides (LPS) are the main antigens responsible for immunity in leptospirosis. In this investigation we studied the nature of the antigenic determinants of LPS extracted from Leptospira interrogans serovar hardjo (reference strain Hardjoprajitno). The reactions of anti-LPS monoclonal antibodies (mAbs) MUM/F1-4/hardjo (IgM) and MUM/F1-6/hardjo (IgG) with whole cell lysates in Western immunoblotting analysis were unaffected by proteinase K treatment. Periodate treatment of the LPS destroyed the binding of MUM/F1-6/hardjo but preserved that of MUM/F1-4/hardjo. Alkaline phosphatase decreased significantly the binding of MUM/F1-4/hardjo to the LPS but only slightly that of MUM/F1-6/hardjo. On the other hand, phosphodiesterase totally destroyed the binding capacity of both monoclonal antibodies in enzyme immunoassays (EIA). A number of mono- and oligosaccharides was used in EIA inhibition studies. Mannose-6-phosphate and galactose-6-phosphate inhibited the binding of MUM/F1-4/hardjo (50% inhibition at a concentration of 5 mM) to the antigen, but glucose-6-phosphate did not. Galactosamine and mannosamine inhibited the binding of MUM/F1-6/hardjo (50% inhibition at a concentration of 3-4 mM), whereas only a weak inhibition was observed with glucosamine. In contrast, N-acetylated amino sugars did not show any inhibition. An O-acetyl group also appears to be involved in the antigen-antibody binding process.
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PMID:Immunochemical studies of opsonic epitopes of the lipopolysaccharide of Leptospira interrogans serovar hardjo. 751 91

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
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PMID:Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin. 787 96

Recent advances in melanogenesis have focused on the role of dihydroxyindole-2-carboxylic acid[(HO)2IndCOOH]. For example, it has been shown that formation of (HO)2IndCOOH from dopachrome is catalyzed by dopachrome tautomerase, that the melanogenic protein tyrosinase-related protein (TRP)-1 can oxidize (HO)2IndCOOH to its indole quinone, that (HO)2IndCOOH-melanins can be synthesized chemically, that mammalian melanins are naturally rich in (HO)2IndCOOH subunits, and that (HO)2IndCOOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)2IndCOOH and other precursors become incorporated into melanins in vivo. Accordingly, an activity was partially purified that catalyzed melanin formation with (HO)2IndCOOH as a substrate. Analyses of the (HO)2IndCOOH polymerization factor from Cloudman melanoma cells revealed the following: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N-acetyl glucosamine; it was located predominantly in the melanosomal fraction of cell homogenates; the activity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)2IndCOOH polymerization reaction was inhibited by superoxide dismutase. In addition, the activity was found with the mouse pmel 17/silver locus protein immunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice compared to extracts from Si/Si melanocytes. In summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide-dependent polymerization of (HO)2IndCOOH to melanin in vitro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus.
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PMID:Polymerization of 5,6-dihydroxyindole-2-carboxylic acid to melanin by the pmel 17/silver locus protein. 861 63

A macromolecule with a molecular weight of 90-100 kDa was purified from normal human pregnancy urine. The molecule was proved to be the Tamm-Horsfall glycoprotein (THG) by Western blot analysis. The macromolecule contains carbohydrate as detected by an enzyme immunoassay. Functionally, the glycoprotein can adhere to and stimulate the thymidine incorporation of human mononuclear cells (MNC) in modest degree via its membranotropic property. In addition to MNC, the protein can also bind to the surface of human polymorphonuclear neutrophils (PMN), red blood cells (RBC) and rat glomerular mesangial cells (RMC). Western blot analysis of various cell lysates with/without proteinase K pretreatment before cell lysis revealed that a 60 kDa and a molecule larger that 94 kDa on the surface of PMN, a 60 kDa protein on MNC and a 32 kDa protein on RBC are the binding molecules for THG. In contrast, many proteins on the surface of RMC could be bound by THG. Immunoprecipitation of membranous iodinated MNC lysates also confirmed that the 60 kDa molecule on MNC is the binding protein for THG. A number of monosaccharide including N-acetylneuraminic acid, N-acetyl-galactosamine, N-acetyl-glucosamine and alpha-methyl-D-mannoside could not inhibit the mitogenic effect of THG on human mononuclear cells. These results suggest that THG is capable of reacting with surface membrane proteins on different cells, but not through the specific carbohydrate-containing lectin-like receptors on the cell surface.
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PMID:Tamm-Horsfall glycoprotein (THG) is a binder for surface membrane proteins on blood cells and glomerular mesangial cells. 904 37

Nine strains of oral Fusobacterium were examined for their ability to coaggregate in vitro with four strains of the oral yeast. Candida albicans. All of the Fusobacterium nucleatum strains and Fusobacterium periodontium and Fusobacterium sulci coaggregated to various degrees with all of the Candida strains. Fusobacterium alocis, Fusobacterium mortiferum and Fusobactrium simiae strains did not coaggregate with any of the Candida strains. Exposure of the coaggregating Fusobacterium strains but not the Candida strains to heat, trypsin, and proteinase K eliminated coaggregation. Amphotericin B or trichodermin treatment of the yeast species had no effect. The reactions were inhibited by addition of 0.1 M mannose, glucosamine and alpha-methyl mannoside. All coaggregating pairs were disaggregated by the addition of sodium dodecyl sulfate, but nonionic detergents had no effect. The addition of 2.0 M urea completely reversed coaggregation. Candida strains were sensitive to periodate oxidation, whereas the Fusobacterium strains were stable to this treatment. All coaggregations occurred in the presence of saliva and appeared stronger than in buffer. These data suggest that the coaggregations involve either a protein or glycoprotein on the Fusobacterium surface, which may interact with carbohydrates or carbohydrate-containing molecules on the surface of the Candida. These observations expand the known range of intergeneric coaggregations occurring between human oral microbes and indicate that coaggregation of C. albicans and Fusobacterium species may be an important factor in oral colonization by this yeast. The authors believe this to be the first description of coaggregation concerning a carbohydrate component on the yeast cell and a protein component on the oral bacterial cell.
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PMID:Coaggregation of Candida albicans with oral Fusobacterium species. 946 3


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