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)

Formation of thick, stable foams and scums on activated sludge wastewater treatment plants is a worldwide problem, and to better understand what causes this foam and to cure it, there is a need to identify and quantify the bacteria present there. Fluorescence in situ hybridisation (FISH) overcomes the difficulties experienced with microscopic methods of identification for the mycolic-acid-containing actinomycetes (the mycolata), which are present in foams, where many share the morphotype of right-angled branching filaments. However, the presence of hydrophobic mycolic acids in their cell wall makes this group of bacteria particularly difficult to permeabilise, which greatly reduces the usefulness of FISH. While several permeabilisation treatments have been described, none appear to adequately permeabilise all genera of the mycolata. In this study several protocols for permeabilisation were assessed with both pure cultures of selected genera of the mycolata and foam samples. Combining mild acid hydrolysis with enzyme treatments (either mutanolysin/lysozyme or lipase/proteinase K) was found to be the most effective method, although other evidence presented here suggests that negative FISH results can not always be explained in terms of cell permeability to the probes.
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PMID:Improved permeabilization protocols for fluorescence in situ hybridization (FISH) of mycolic-acid-containing bacteria found in foams. 1567 95

The cells of Streptomyces sp. YB-1 adsorbed 4-6 mg ytterbium (Yb) per g dry weight. The Yb contents of the cell wall fraction, cell-free extract, and cell membrane fraction were 11%, 2%, and 87%, respectively. The Yb content in the cell membrane fraction was 20-25 mg per g dry weight. The adsorbed Yb could be quantitatively desorbed by treating the cell membrane fraction with 1 mM EDTA and 1 M HCl at 37 degrees C for 4 h. Treatment with 1 M NaOH caused Yb desorption to some extent. Treatments with proteinase K, lysozyme, 0.5% Triton X-100, 0.4% sodium dodecyl sulfate, and 1 M NaCl did not cause Yb desorption. Elemental analysis of Yb-adsorbed materials after removal of proteins and then extraction of lipids from the membrane fraction revealed that the molar ratio of Yb and P in the materials was about 1:1. The cells and the membrane fraction could be used repeatedly as a bioadsorbent for Yb.
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PMID:Distribution of ytterbium (Yb) in cells of Streptomyces sp. YB-1 which can accumulate Yb, and reusability of cells and cell membrane as bioadsorbent for Yb. 1623 93

An efficient method for obtaining DNA from compost which contained high levels of organic matter was developed. The protocol consisted of washing with phosphate-EDTA before extraction, cell lysis with hot-SDS and enzymes (lysozyme, lywalzyme, proteinase K), removing humic acid and other inhibitors with PVPP and precipitation with PEG-8000. The compost total DNA was extracted from four different composts, the DNA yield was 63.54 +/- 12.08 to approximately 106.50 +/- 28.36 microg/g of dry compost. Molecular size of DNA obtained using this protocol was about 23kb and contained low protein and humic acid contamination with the A260/A280 ratios exceeding 1.6 and A260/A230 ratios reaching 1.8. Usually, additional purification steps such as agarose gel electrophoresis, gel permeation chromatography, or affinity chromatography were needed to get PCR-amplifiable DNA, but the DNA obtained using this protocol could directly be used to PCR-amplification and restriction enzyme digestion. Just like purity of DNA template, lower DNA yield also appears to introduce a bias towards lower community diversity. In this study compared the purified DNA the direct DNA reveals higher microbial community diversity assessed by denaturing gradient gel electrophoresis(DGGE) of amplified V3 region of 16S rDNA.
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PMID:[An efficient method for DNA extraction from compost]. 1657 88

A method based on 32P-labeling of DNA in short-term incubations was developed for estimating the growth rate of mixed rumen bacteria. A freeze/thaw procedure was optimized to quantitatively disrupt mixed rumen bacteria and extract bacterial DNA. The preliminary enzymatic lysis step, with lysozyme rather than proteinase K, sodium lauroyl sarcosine, and, to a lesser extent, sodium dodecyl sulfate (SDS) strongly improved cell disruption and DNA recovery rates. Sodium deoxycholate, CHAPS or Triton X-100 had no significant effect. Increasing the number of cycles or lowering the freezing temperature from -20 degrees C to -50 degrees C had no effect on DNA extraction efficiency while setting the thawing temperature at +60 degrees C rather than +37 degrees C slightly increased DNA yield but also increased its contamination with RNA. The method finally selected led to the lysis of at least 93% of cells and to the extraction of 85% of bacterial DNA. The kinetics of in vitro 32P incorporation into rumen bacteria DNA was then determined in batch incubations of strained rumen contents with no additional substrate. The curvilinear effects of the amount of 32P and the incubation time (5-15 min) on the DNA radioactivity were investigated by applying a Doehlert experimental design and fitting a second order polynomial model to data. The DNA radioactivity was linearly related to time (p<0.02) with other coefficients in the model being equal to zero (p>0.20). The incorporation of 32P into bacterial DNA was initiated approximately 70 s after the start of incubation. Taking into account the accuracy of scintillation counting, 10-15 min incubations, with 15 microCi 32P and 10 mL rumen contents per tube, appeared satisfactory for future studies.
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PMID:Estimation of the growth rate of mixed ruminal bacteria from short-term DNA radiolabeling. 1688 35

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.
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PMID:Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. 1689 88

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as alpha-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.
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PMID:Isolation and preliminary characterization of a bacteriocin produced by Lactobacillus plantarum N014 isolated from nham, a traditional Thai fermented pork. 1692 20

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.
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PMID:Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature. 1694 96

Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.
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PMID:[DNA extraction methods of compost for molecular ecology analysis]. 1711 21

There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.
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PMID:Development of a fast and reliable method for the assessment of microbial colonization and growth on textiles by DNA quantification. 1787 10

A series of experiments were performed in an attempt to extract genomic DNA from a small number of Eimerian oocysts. Sonication, ammonia, ethanol and lysozyme were all found to be unsuitable for the digestion of Eimeria oocysts. The chemicals and enzyme given were not capable of either disruption or digestion of oocysts for nucleic acid extraction. They had the capability of penetrating the oocyst wall but could not break-up the oocyst wall. It is impossible to obtain nucleic acid from Eimeria oocysts if the wall is not broken-up. In this study oocyst disruption was achieved using a simple but highly effective treatment regime involving sodium hypochlorite treatment, osmotic shock and proteinase K digestion. Following the disruption of the oocyst walls, a commercially available nucleic acid purification kit (Wizard DNA Purification Kit, Promega) can be used to prepare high quality nucleic acid.
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PMID:A novel procedure for total nucleic acid extraction from small numbers of Eimeria species oocysts. 1791 54


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