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)

It is shown that simultaneously to the unfolding of hen egg white lysozyme and horse heart cytochrome c the sequential conformational changes and molten globule states can be detected by the combination of proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This is demonstrated by the differences among the products and the time courses of native lysozyme as well as those unfolded in 1 and 3 M guanidine hydrochloride (GuHCl) when they were proteolyzed by proteinase K and analyzed by MALDI-MS. Due to the absence of disulfide bonds in the cytochrome c molecule, it is more sensitive to the disturbance of the denaturant. The partially unfolded state as detected at low concentrations of guanidine hydrochloride in our experiment resemble the molten globule state. One of the unique properties of the method described herein is to measure directly the peptide fragment liberated from proteolysis of the protein. It allows the identification of the sensitive sites susceptible to denaturation, which are subsequently cleaved by proteinase K proteolysis.
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PMID:Protein conformational changes determined by matrix-assisted laser desorption mass spectrometry. 952 57

We have extensively modified the published method for the lysis of gram-positive bacteria to isolate chromosomal DNA from only 1 ml of oral streptococcal overnight culture. Cells were incubated with lysozyme and R Nase A in the presence of polyethylene glycol. After centrifugation, cells were lysed with sodium dodecyl sulfate and proteinase K. Following ethanol precipitation, sodium dodecyl sulfate solution was added to the residue, and the pellet was completely dispersed by incubating at 65 degrees C. The chromosome was purified by extraction over phenol and chloroform. Two regions corresponding to the ribosomal RNA (rrn) operon and the glucosyltransferase gene were amplified using the chromosome from Streptococcus mutans and Streptococcus sobrinus by polymerase chain reaction (PCR). Genetic heterogeneity was assessed by restriction fragment-length polymorphism (PCR-RFLP). The PCR-RFLP analysis readily allowed us to subtype each strain, suggesting that the strategy presented here will provide a useful tool to verify epidemiological studies at the molecular level.
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PMID:Rapid isolation of chromosomal DNA from oral streptococci and polymerase chain reaction-oriented restriction fragment-length polymorphism analysis for genetic heterogeneity. 957 16

Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.
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PMID:Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins. 960 41

Antimutagenic and binding properties of 28 strains of Lactobacillus gasseri and 2 strains of Bifidobacterium longum on the mutagenicity of amino acid pyrolysates were investigated in vitro using a streptomycin-dependent (SD510) strain of Salmonella typhimurium TA 98. Four strains of L. acidophilus (SBT0274, SBT1703, SBT10239, and SBT10241) and 1 strain of B. longum (SBT 2928) exhibited the highest percentage of antimutagenicity and binding. These 5 strains were further optimized for other physical factors influencing the mechanism of binding, such as cell and mutagen concentration, pH, and incubation time. In all of the selected strains, 2 mg of cells bound with 88 to 95% of 0.2 mg of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole in 30 min at pH 7.0. Other amino acid pyrolysates, such as 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldi-pyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methyl-imidazo[4,5,f]quinoline, and 2-amino-3,4-dimethyl-imidazo[4,5,f]quinoline were also tested for the binding ability of these strains. We observed that the complexity of the mutagens greatly influenced the binding properties. The binding of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole to the purified cell walls was very high compared with that of the crude cell wall, peptidoglycan, or the cell extract. Binding was inhibited when the cell walls were subjected to treatment with metaperiodate or trichloroacetic acid but not when they were subjected to treatment with lysozyme, trypsin, or proteinase K, reflecting the role of the carbohydrate component as a binding site.
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PMID:Antimutagenicity and the influence of physical factors in binding Lactobacillus gasseri and Bifidobacterium longum cells to amino acid pyrolysates. 968 59

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

Soluble chitosan and poly-L-lysine are readily hydrolysed using lysozyme or chitosanase for chitosan, and trypsin, chymotrypsin or proteinase K for poly-L-lysine. For similar amounts of enzyme, chitosanase hydrolysed 57% of the chitosan, compared to 35% for lysozyme. In the case of poly-L-lysine, chymotrypsin and trypsin exhibited similar activities, hydrolysing approximately 41% of the polymer compared to proteinase K at only 16%. In contrast, chitosan and poly-L-lysine membranes, coating alginate beads, were almost totally inert to the respective hydrolytic enzymes. Less than 2% of the membrane weight was hydrolysed. It appears that either membrane material would be stable for in vivo application, and in particular in the protection of DNA during gastrointestinal transit. At chitosanase concentrations of 1.4 mg/ml and in the presence of sodium ions, 20% of the total double-stranded DNA was released from chitosan coated beads. An exchange of calcium for sodium within the bead liquefied the alginate core releasing DNA. The presence of calcium stabilized the alginate bead, retaining all the DNA. Highly pure DNA was recovered from beads through mechanical membrane disruption, core liquefaction in citrate and use of DNA spin-columns to separate DNA/alginate mixtures in a citrate buffer. DNA recovery efficiencies as high as 94% were achieved when the initial alginate/DNA weight ratio was 1000.
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PMID:Stability of chitosan and poly-L-lysine membranes coating DNA-alginate beads when exposed to hydrolytic enzymes. 997 4

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.
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PMID:Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species. 1039 13

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of beta-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP(Sc)) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.
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PMID:Upregulation of the genes encoding lysosomal hydrolases, a perforin-like protein, and peroxidases in the brains of mice affected with an experimental prion disease. 1059 Jan 30

A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-L-alanine amidase, the pneumococcal autolysin.
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PMID:Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae. 1061 14

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.
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PMID:Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. 1074 29


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