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)

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.
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PMID:Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide. 216 86

Modification of the alkaline lysis at elevated temperature technique is proposed isolation of plasmid DNA from lactobacilli. Modification consists of colorimetric control of culture phase during the biomass growth, pH control at the probes treatment with lysozyme and alkaline solution of natrium dodecylsulfate by adding the indicator bromcrezolpurple into the medium for biomass growth. The high concentration of lysozyme is used (10 mkg.ml-1). Lactobacilli are lysed at 2 min incubations of the probes with the lytic solution in the boiling water bath. The treatment of the probes by proteinase K, by the mixture of chloroform:phenol:isoamyl spirit (25:24:1 vol/vol/vol) and by diethylpirocarbonate increased considerably the quality of the obtained DNA preparations. The modified technique is suitable for isolation of the plasmid DNA from lactobacilli of different species, enterococci, streptococci and other lactic bacteria. The connection of antibiotic resistance marker and the plasmid profile of lactobacilli under different conditions with the presence of the plasmid DNA- protein complex is discussed.
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PMID:[Optimization of the method of isolation of microamounts of plasmid DNA from lactobacilli]. 236

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations.
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PMID:Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose. 283 4

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.
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PMID:Rapid extraction of plasmids from Clostridium perfringens. 287 Jun 80

We demonstrated by spectral analysis a method to enzymatically cleave the chromophore precursor from nutritionally deficient streptococcal cell walls by treatment with lysozyme. The peak absorbances without and with lysozyme pretreatment were 511.4 +/- 2.02 nm and 513.1 +/- 0.69 nm, respectively. Extraction yields varied among strains and were found to be growth phase dependent. A secondary peak of absorbance (mean of 477 nm) was found in only five of eight strains. The chromophore at a neutral pH undergoes a reaction with phenol consistent with that of a furan, indicating its carbohydrate composition.
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PMID:Enzymatic extraction and spectral analysis of the chromophore from cell walls of nutritionally deficient streptococci. 291 29

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.
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PMID:Isolation and restriction endonuclease analysis of mycobacterial DNA. 301 65

Phenol has been added to the Coomassie Brilliant Blue G-250 dye reagent used in the standard Bradford protein assay and its effect upon the reagent blank and assay response of fourteen proteins investigated. Phenol can enhance or impair colour yield depending upon its concentration and the amount and type of protein assayed. Four characteristic protein responses to increasing assay concentrations of phenol have been observed. These indicate a complex influence of phenol upon the protein assay. Dye reagent containing 0.5% phenol gave optimal colour yield with most of the proteins investigated and an improved assay response of ovalbumin, ribonuclease, lysozyme, insulin, pepsin and chymotrypsinogen-A relative to bovine albumin.
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PMID:Phenol addition to the Bradford dye binding assay improves sensitivity and gives a characteristic response with different proteins. 378 18

Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60 micrograms) from the ear isolate also inhibited bactericidal activity in the respective immune serum. LPSs exhibited minimal inhibition (greater than 110 micrograms). Three human sera (two normal, one immune) were selectively depleted of 80% of antibody activity against OMPs (measured by enzyme-linked immunosorbent assay) by affinity chromatography using OMPs from the pulmonary isolate coupled to a solid phase. These OMP antibody-depleted sera also showed an 88% reduction of bactericidal activity against this strain. Immunopurified antibody against OMPs eluted from the solid phase was bactericidal.
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PMID:Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins. 387 75

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.
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PMID:Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins. 406 99

Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of Bacillus cereus T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C(15) and C(17) and of normal C(16) acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl(2). The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl(2) concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl(2) concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments.
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PMID:Chemical composition and ultrastructure of native and reaggregated membranes from protoplasts of Bacillus cereus. 420 99


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