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 present study was designed to obtain further information regarding the molecular nature of the corneal receptor(s) facilitating Pseudomonas aeruginosa adhesion to cornea. Scarified adult mouse corneas in organ culture were treated for 10 or 60 min with a panel of lipase-free proteases [each at 20 micrograms ml-1 or 0.22 Units (U) ml-1, activity] including trypsin, chymotrypsin, V8 protease, elastase, subtilisin A, pronase protease and proteinase K. All of these, except proteinase K treatment (20 micrograms ml-1 for 60 min), either significantly elevated or had no effect (proteinase K 20 micrograms ml-1 for 10 min) on subsequent bacterial adhesion at 60 min following topical application of the inoculum to the scarified corneal surface. Enzyme treatment times of 10, 30 or 60 min at a higher concentration (50 micrograms ml-1) of proteinase K, significantly decreased binding at 60 min after bacterial application for each enzyme treatment time. The combined effects of proteases and lipase on bacterial binding also was examined. Eyes treated with proteinase K (20 micrograms ml-1 for 1 hr) or protease-free lipase (50,000 U ml-1 for 1 hr) alone or in combination, all reduced bacterial binding, but the effect was not additive. Trypsin or lipase alone significantly enhanced or decreased binding, respectively. In contrast, trypsin (20 micrograms ml-1 for 1 hr) followed by lipase treatment (50,000 U ml-1 for 1 hr) resulted in binding which was not significantly different than phosphate-buffered saline (PBS) control binding, indicating that the trypsin exposed receptor was lipase sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteinase K decreases Pseudomonas aeruginosa adhesion to wounded cornea. 148 4

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.
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PMID:Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors. 244 12

A novel replicating agent (IFDO) was isolated from ileal fluid. Growth occurred in vitro under aerobic and anaerobic conditions, and was faster at 37 degrees C than at room temperature. The doubling time was 15.8 min. Colonies were dark brown in colour and occurred beneath the surface of agar after conventional surface inoculation. Provisional data indicate that the agent may be a normal intestinal commensal. The agent was remarkably resistant to inactivation by steam at 134 degrees C, formaldehyde and glutaraldehyde; it was relatively resistant to ionising radiation, and it was filterable through membranes with a nominal pore diameter of 10 nm. Such properties, with the exception of growth in cell-free medium, are shared by "unconventional agents" such as those of Creutzfeldt-Jakob disease and scrapie. Further comparison of the properties of the intestinal agent and of slow viruses revealed additional shared characteristics, including resistance to proteinase K and trypsin, and inactivation by guanidine thiocyanate, diethyl pyrocarbonate, phenol and sodium hydroxide. The agent differs from that of scrapie in being inactivated by ethidium bromide, zinc nitrate, EDTA, hydroxylamine in the presence Sarkosyl, and, under certain circumstances, by ribonuclease. Broth cultures of the agent contained particles possessing considerable size heterogeneity. The smaller filterable particles were generally more susceptible to inactivation, did not survive autoclaving, and were inactivated by papaya protease and lipase. It is possible that the replicating agent may be formed by crystallisation from constituents of the medium, and not by a biological process. This does not exclude the postulated relationship to slow viruses.
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PMID:A novel replicating agent isolated from the human intestinal tract having characteristics shared with Creutzfeldt-Jakob and related agents. 265 97

Using sucrose density gradient centrifugation in a vertical rotor, we have separated three major binding components contained in hepatic cytosols from C57BL/6 mice and Sprague-Dawley rats. Using this preparative method we have obtained, after a 3-h run of 2.4 ml of crude cytosol from 1,4-bis[2-(3,5-dichlorodipyridyloxy)]benzene-treated C57BL/6 mice (approximately 50 mg of protein: 10,000 fmol of Ah receptor) 50 and 75% yields of isolated Ah receptor and carcinogen-binding protein (4 S binding protein), respectively. Both binding components may be kept at -70 degrees C for several months without loss of activity. A third binding component, which did not sediment in a sucrose density gradient (5-20%), even after a 4-h run at 63,000 rpm, was recovered from the top fractions of gradients. When applied to Sephacryl S-300 columns this component was eluted in the void fraction. Resistant to the direct degradative action of nucleases and proteases, this large complex was sequentially converted to its subcomponents by lipoprotein-lipase, proteinase K, and phospholipases. Only the phospholipases are able to abolish the binding capacity of this light density component (LDC) for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin: hence, we conclude that phospholipids are the true binders of this radioligand. In vitro, this lipoprotein irreversibly binds many hydrophobic radioligands (2,3,7,8-tetrachlorodibenzo-p-dioxin,3-methylcholanthrene, benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and dexamethasone). Using single vertical spin density gradient ultracentrifugation, the major part (80%) of LDC was characterized as a very low-density lipoprotein, and a minor part (20%) as a low-density lipoprotein. This conclusion was supported by the size of LDC particles (about 25-75 nm) observed in electron microscopy.
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PMID:The binding components for 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. Separation from the rat and mouse hepatic cytosol and characterization of a light density component. 355 72

This study determined the presence of two hemolytic activities in the oral treponeme, Treponema denticola, strains ATCC 35404 (TD-4), ATCC 33520, GM-1, and MS25. These activities, referred to as hemolytic and hemoxidative (HeA, HeO, respectively), were found to be both secreted into the extracellular environment, and cell associated. The extracellular activity was associated with small molecules with relative molecular weights of < 1000 Da, and its activity was cysteine independent; the cell-associated HeA and HeO activities were associated with a molecular weight fraction > 10 kDa, and were cysteine dependent. The HeO activity of the fractionated material observed was due to the oxidation of hemoglobin to methemoglobin, and preceded the HeA lysis of the RBCs by approximately 2 h. Heating at 80 degrees C and treatment with proteinase K resulted in the complete destruction of these activities in the fraction > 10 kDa, while lipase at high concentration (800 micrograms/ml) reduced the HeA and HeO activities in the extracellular fraction by approximately 50%. Proteinase inhibitors had a variable effect on HeA and HeO activities in both extracellular and cell-associated fractions. Scanning and transmission electron microscopy revealed a progressive destruction of the RBC membrane, with membrane protrusions formed early in the interaction, which progressed to irregular holes in the membrane, and the complete loss of membrane integrity.
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PMID:Characterization of hemolysis and hemoxidation activities by Treponema denticola. 809 77

For the first time reactivation of cell extract of three strains of Propionibacterium shermanii in UV inactivated not filament-forming strain Escherichia colli AB 1157 is shown. Reactivation was demonstrated in preincubated and postincubated test-culture and increased as survival of E. coli decreased in a range 1.8-0.006%. The factor (factors) of defense is dialysable, thermolabile and is present as in a fraction of nucleoproteins and nucleic acids so in a fraction of soluble proteins. The extracts were inactivated by incubation with proteinase K and trypsin, partly decreased activity by incubation with alpha-amylase and selected nuclease but not with lipase. Polypeptide nature of reactivating factor is supposed.
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PMID:[Reactivation of Escherichia coli inactivated by ultraviolet light by cell extracts of propionic acid bacteria]. 811 46

Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121 degrees C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and alpha-chymotrypsin but insensitive to alpha-amylase, lysozyme, catalase, and lipase. The 4.5-kDa bacteriocin was purified and characterized and designated lactobin A. Lactobin A was isolated as a floating pellicle from culture supernatant brought to 35% saturation with ammonium sulfate. Upon this ammonium sulfate treatment, crude lactobin A was incorporated, together with Tween 80 as a major contaminant, in high-molecular-mass complexes sized at approximately 670 kDa by gel filtration chromatography. Contaminating fatty acids were removed from these micelles by a simple one-step methanol-chloroform extraction without loss of activity. Both inhibitory peptides were separated in an isocratic isopropanol gradient on a PepRPC 5/5 reversed-phase column, and both peptides retained activity towards Lactobacillus helveticus ATCC 15009 upon separation. Lactobin A has a molecular mass determined by electrospray mass spectrometry of 4,879 +/- 0.69 Da. Its peptide chain contains 50 unmodified amino acids, of which 26% are glycine residues and 40% are hydrophobic residues (A, V, L, I, and P). It displays the highest structural homology (42% identity and 28% similarity) with the lafX gene product, encoded by the second open reading frame of the lactacin F operon. These data strongly indicate that lactobin A belongs to the class IIb bacteriocins according to the classification of Klaenhammer.
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PMID:Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139. 897 34

Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE. The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes. These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (<5.0). They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme. These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin. Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin. The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is discussed.
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PMID:Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables. 926 41

An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYT-EXP1 (pT1) expression vector downstream of the temperature-inducible lambda promoter PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000-9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660,000 soluble lipase U/g cells was produced, whereas, with E. coli DH5 alpha and BL321, production levels of 30,000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage and ultrafiltration allowed the isolation of 1.15 x 10(6) units of 90% pure mature lipase/wet cells.
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PMID:High-level expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli. 961 82

Five stains of Bifidobacterium bifidum (ATCC 11863 and 29591, and NCFB 1453, 1454, 1455) were examined for production of bacteriocins in MRS broth with 0.05% cysteine. Only strain NCFB 1454 excreted a bacteriocin into the broth: it was designated bifidocin B. Bifidocin B was sensitive to several proteolytic enzymes (protease IV, pronase E, protease XVII, proteinase K, trypsin, alpha-chymotrypsin, papain, and pepsin), but was resistant to catalase, peroxidase, lipase, lysozyme, cellulase, ribonuclease A, and amylases. It was also resistant to organic solvents such as ethyl alcohol, acetone, hexane, chloroform, methanol, and ether, and to heating at 90 degrees C for 15, 30, and 60 min or at 121 degrees C for 15 min. Bifidocin B remained active after storage at -20 or -7 degrees C for 3 months and retained biological activity after exposure to pH values of 2 to 10. Bifidocin B was active against some food-borne pathogens and food spoilage bacteria such as Listeria, Enterococcus, Bacillus, Lactobacillus, Leuconostoc, and Pediococcus species but was not active against the other gram-positive and gram-negative bacteria tested. Bifidocin B was produced during exponential phase, reaching a maximum activity of 3,200 AU/ml at early stationary phase. Bifidocin B had a molecular mass of about 3.3 kDa as analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:Characterization and antimicrobial spectrum of bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454. 970 52


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