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)

The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 were metabolically labeled with [3H]-cis-9-octadecenoic acid and extracted with the non-ionic detergent Triton X-114. The extract was phase separated, precipitated with acetone and delipidated to remove non-covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]-cis-9-octadecenoic acid incorporation in bands with apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last band resolved into a 53 kDa [3H]-cis-9-octadecenoic acid band upon heating for 10 min, at 100 degrees C. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa protein. Proteinase K degraded the [3H]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]-fatty acid labeled compound found in the water phase following phenol-water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [3H]-cis-9-octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8-8 micrograms/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1 microM) resulted in a synergistic activation of LDCL. At 21 micrograms/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotactic peptide at 1 microM. In addition, dLPP (21 micrograms/ml) increased additively the release of lysozyme caused by 1 microM FMLP. The release of beta-glucuronidase was not affected. The modulation of neutrophil activity was abolished by preincubation of dLPP with proteinase K. The purified 14 kDa had no effect on either LDCL or exocytosis of lysosomal enzymes of PMN. These data strongly suggest that T. denticola possesses several lipoproteins including outer sheath major oligomeric polypeptides (113-234 kDa) and a lipooligosaccharide of molecular mass of 14 kDa. In addition, an enriched lipoprotein fraction from this oral spirochaete modulates oxygen dependent and independent mechanisms for controlling microorganisms by human PMN.
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PMID:Lipoproteins of Treponema denticola: their effect on human polymorphonuclear neutrophils. 926 97

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

A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced.
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PMID:A method for DNA extraction from the desert cyanobacterium chroococcidiopsis and its application to identification of ftsZ 975 40

Lacrimal fluid plays a very significant role in maintaining proper functions of conjuctivas, cornea and eyelids. The fluid is secreted by the main lacrimal gland and additional glands. It produces the so called preocular lacrimal film. A number of clinical tests, such as Chirmer's tests I and II, break-up-time (BUT), lysozyme, and flow tests are used in quantitative and qualitative analyses, as well as in the determination of the lacrimal film stability. The aim of work was to utilize these in assessing the lacrimal secretion and the lacrimal film stability in workers chronically exposed to petroleum derivatives. Fifty three workers from departments of acetobenzene, benzene and butadiene, phenol and acetone, sewage waters, asphalt oxidas, polyethylene and polypropylene, were eligible for the study (group I). During previous examinations, acquired disorders in colour perception were diagnosed in all the subjects by means of the Mansuella-Fansworth 100-Hue test. The age range was 25 to 56 years, with a mediane of 44.1 years +/- 6.5. Mean duration of employment was 22 years (SD +/- 8.25). The control group (group II) was composed of 28 men aged between 24 and 60 years with a median of 42.7 years +/- 6.3, never employed under conditions of exposure to toxic chemicals. On the right eye of each subject Schirmer's test was performed after instilling into the conjunctival sac 1-2 drops of Alcain solution according to Whitcher. Five min following anesthesia of the conjunctival sac, a standardised belt of blotting-paper with colour dampness markers Vidisic (Dr Mann Pharma GMBH, Germany) was placed in the vicinity of the external angle of the eye. After 5 min the degree of the belt dampness was measured in millimetres. After 30 min the break-up-time test was performed on the left eye. Fluorescein was released to conjunctival sac from a sterile belt of blotting-paper (Haag-Strait Co.). A slit lamp with cobalt filter was used to calculate time (in sec) that elapsed between opening of the lid slit and the first symptom of breaking-up the lacrimal film. The results obtained were presented in the form of arithmetic means and standard deviation values +/- SD. Schirmer's test was 13.40 +/- 7.43 mm in group 1, and 22.54 +/- 8.25 mm in the control group, mean values differed significantly, p < 0.01. Lacrimal film break-up-time was 16.30 +/- 6.19 sec in group 1, and 31.48 +/- 7.96 sec in the control group, mean values differed significantly, p < 0.01. In persons chronically exposed to petroleum derivatives, statistically significant decrease in lacrimal secretion, as well as shortening of lacrimal film break-up-time were found when compared with the control group.
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PMID:[Lacrimation disorders in workers chronically exposed to petroleum derivatives]. 1039 14

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.
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PMID:Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. 1054 76

Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 microM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 microM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion (K(d)) from monensin-selected cells was 16-fold greater than "unadapted" wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a K(d) of 100 nM, and these inocula could not grow in batch culture when the monensin concentration was greater than 300 nM. Continuous cultures treated with monensin nearly washed out, but the surviving cells had a K(d) of 1,300 nM. When wild-type cells were transferred in batch culture with 10 microM monensin, the K(d) did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings). K(d) declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and K(d) indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type cultures that were washed in Tris buffer (pH 8.0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type P. bryantii cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.
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PMID:Selection of a highly monensin-resistant Prevotella bryantii subpopulation with altered outer membrane characteristics. 1054 82

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.
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PMID:Comparison of different methods for the isolation and purification of total community DNA from soil. 1057 2

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.
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PMID:An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria. 1101 74

Mergenhagen, Stephan E. (National Institute of Dental Research, Bethesda, Md.), and George R. Martin. Properties of a lysozyme-dissociated endotoxic fraction from Escherichia coli. J. Bacteriol. 88:1169-1174. 1964.-Treatment of a phenol-water preparation of C(14)-labeled Escherichia coli O91-H21 endotoxin of low solubility with lysozyme at pH 5.0 or 8.0 effected a dissociation of the preparation. Such products of dissociation were equally distributed in the chloroform and water phases after extraction. beta-Glucosidase, but not beta-galactosidase, significantly dissociated this endotoxin also. Concomitant with dissociation, recoverable endotoxin after lysozyme treatment had a reduced content of bound lipid, and dissolved easily in aqueous media to yield a clear solution. Examination of lysozyme-treated endotoxin in an analytical ultracentrifuge revealed that it sedimented as a single major boundary with a sedimentation coefficient of 13.3. Lysozyme-treated endotoxin was more potent than was the conventional endotoxin as evidenced by lethal activity in rabbits and pertussis-sensitized mice. Agar-gel diffusion analysis indicated that the higher molecular weight component associated with conventional endotoxin was dissociated by lysozyme treatment. In immunoelectrophoresis, lysozyme-treated endotoxin was observed as a single sharp band of precipitation which migrated toward the cathode.
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PMID:PROPERTIES OF A LYSOZYME-DISSOCIATED ENDOTOXIC FRACTION FROM ESCHERICHIA COLI. 1421 34


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