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 efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp.
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PMID:Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. 891 55

Seven chemicals, three buffers, and a salt solution known to affect bacterial attachment were tested to quantify their abilities to enhance the penetration of Alcaligenes paradoxus in porous media. Chemical treatments included Tween 20 (a nonionic surfactant that affects hydrophobic interactions), sodium dodecyl sulfate (an anionic surfactant), EDTA (a cell membrane permeabilizer that removes outer membrane lipopolysaccharides), sodium PPi (a surface charge modifier), sodium periodate (an oxidizer that cleaves surface polysaccharides), lysozyme (an enzyme that cleaves cell wall components), and proteinase K (a nonspecific protease that cleaves peptide bonds). Buffers included MOPS [3-(N-morpholino)propanesulfonic acid], Tris, phosphate, and an unbuffered solution containing only NaCl. Transport characteristics in the porous media were compared by using a sticking coefficient, alpha, defined as the rate at which particles stick to a grain of medium divided by the rate at which they strike the grain. Tween 20 reduced alpha by 2.5 orders of magnitude, to alpha = 0.0016, and was the most effective chemical treatment for decreasing bacterial attachment to glass beads in buffered solutions. Similar reductions in alpha were achieved in unbuffered solutions by reducing the solution ionic strength to 0.01 mM. EDTA, protease, and other treatments designed to alter cell structures did not reduce alpha by more than an order of magnitude. The number of bacteria retained by the porous media was decreased by treatments that made A. paradoxus more hydrophobic and less electrostatically charged, although alpha was poorly correlated with electrophoretic mobility and hydrophobicity index measurements at lower alpha values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media. 764 12

Transformation of cercariae of Schistosoma mansoni into schistosomula is accompanied by release of a soluble 28-kDa serine protease (s28) from the acetabular glands. The postulated activities of s28 include cleavage of skin connective tissue proteins (elastin, etc.), release of the cercarial glycocalyx, and cleavage of complement proteins. Our previous results demonstrated the presence of an antigenically cross-reactive protein on the surface of mechanically transformed schistosomula. As shown here, schistosomula express on their surface a 28-kDa serine protease (m28) which can be immunoprecipitated with anti-s28 antibodies. m28 eluted from the schistosomular tegumental membrane with NP-40 was purified to homogeneity in one step by adsorption on a chymotrypsin inhibitor column: 6-aminocaproyl-D-tryptophan methyl ester-Sepharose. Proteolytic activity of m28 was completely inhibited by the chymotrypsin inhibitor N-succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone. Efficient removal of m28 from schistosomula was achieved with NP-40, deoxycholate, cholate, Tween 20, and phospholipases A2 and C, but not with papain, trypsin, pronase, or proteinase K. Furthermore, treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) followed by hydroxylamine also released m28. Anti-cross-reactive determinant antibodies which recognize a neo epitope exposed in glycosyl phosphatidyl inositol-containing molecules cleaved by PI-PLC bind to purified m28. The latter results suggest that m28 is anchored to the tegumental membrane of schistosomula by a lipid anchor and that perhaps some of the m28 molecules are bound via glycosylphosphatidyl inositol. Based on inhibitor sensitivity and antigenic cross-reactivity, it is conceivable that s28 and m28 are related, if not identical, proteins. Finally, m28 was detected antigenically also on lung-stage and adult worms of S. mansoni.
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PMID:Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula. 865 54

Recent reports suggest an association between Chlamydia pneumoniae and Helicobacter pylori bacteria and atherosclerosis. We studied 51 patients (mean age, 68.3 years) who underwent abdominal aortic aneurysm surgery. For each patient we performed a microimmunofluorescence test for immunoglobulin G (IgG), IgA, and IgM antibodies to C. pneumoniae specific antigen (TW-183). Anti-H. pylori antibodies were determined by means of an EIA-G test. Each aortic aneurysm surgical specimen was sampled into multiple sections of 0.3 cm2 each and frozen at -20 degrees C. Two samples of each aneurysm were used for a nested PCR with two sets of C. pneumoniae and two sets of H. pylori specific primers. Specimens were treated with a solution containing 20 mM Tris-HCl, Tween 20-Nonidet P-40 (0.5% [vol/vol] each), and 100 micrograms of proteinase K per ml and incubated at 60 degrees C for 1 h and at 98 degrees C for 10 min. DNA was extracted twice with phenol-chloroform-isoamylic alcohol and precipitated with sodium acetate-ethanol by standard methods. Forty-one patients were seropositive for C. pneumoniae with past-infection patterns in 32 patients (16 < or = IgG < 512; 32 < or = IgA < 256) and high antibody titers in 9 patients (IgG > or = 512). In 26 of 51 patients, C. pneumoniae DNA was detected in aortic aneurysm plaque specimens. Of these patients, 23 had a serologic past-infection pattern, 2 had an acute reinfection pattern, and 1 was seronegative. Forty-seven of 51 patients were seropositive for H. pylori. In all cases PCR showed no evidence of H. pylori presence in plaque specimens. This study provides data on a possible C. pneumoniae involvement in the pathogenesis of aortic aneurysm and additional evidence for an association between this agent and atherosclerosis. Conversely, notwithstanding a high H. pylori seroprevalence observed, our results tend to rule out the possibility of a direct involvement of H. pylori in atherosclerosis.
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PMID:Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. 889 80

A newly modified non-RI, PCR-SSCP method is presented and applied to K-ras analysis of colorectal tumors. It comprises five steps: (1) sampling of tiny pieces of fresh solid tissue by scraping with disposable bamboo combs (thin bars made of bamboo, 3 x 3 x 120 mm in size); (2) one-step DNA extraction with lysis buffer containing proteinase K, Nonidet P-40 and Tween 20; (3) PCR with 108 bp, c-ki-ras 2 gene primers; (4) SSCP analysis with 10% formamide-added polyacrylamide gels; and (5) detection with silver staining. In comparison with conventional RI- or non-RI-PCR-SSCP, It can give reliable and clear results in a much shorter time (within a total of 6 h). This novel approach should allow more frequent use of molecular diagnosis in biopsies and surgical pathology.
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PMID:Simplified rapid non-radioactive PCR-SSCP method applied to K-ras mutation analysis. 891 53

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa-stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze-thaw method, boiling in 10% Chelex-100 resin solution, proteinase K/Tween 20/NP-40 method coupled with simplified phenol/ chloroform/isoamyl alcohol protocol or salting-out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen. Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa-stained bone marrow slides.
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PMID:DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods. 960 34

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.
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PMID:Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples. 1095 71

A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products. Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin. DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C. The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B. cepacia. The B. cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene. No DNA amplification was observed in samples that were not spiked with B. cepacia. However, all contaminated samples showed the specific 209-bp fragment for B. cepacia. There was a 100% correlation between standard methods and the PCR assay. Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h. PCR detection of B. cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.
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PMID:Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products. 1099 22

Molecular methodologies such as adenosine triphosphate (ATP) bioluminescence and polymerase chain reaction (PCR)-based assays provide rapid quality control analysis of cosmetic and pharmaceutical finished products and raw materials. Using a single enrichment broth for bacteria, yeast, and mold, ATP bioluminescence detected microbial contamination within 27 h. Samples were automatically lysed to release microbial ATP and light production was quantitated using the Celsis Optocomp. However, to maintain the detection time to within 27 h, different enrichment broths were required for neutralization of antimicrobial ingredients in finished products and to provide specific nutrients for growth optimization. To perform the PCR reaction, bacterial DNA was extracted using a Tris-EDTA-Tween 20-proteinase K buffer at 35 degrees C while yeast and mold DNA were extracted using a Tris-EDTA-SDS buffer at 95 degrees C. Extracted microbial DNA was added to Ready-To-Go PCR beads and specific DNA primers. The primers were targeted to amplify specific regions within Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Burkholderia cepacia, Candida albicans, and Aspergillus niger. Furthermore, conserved bacterial ribosomal DNA sequences have also been used for sterility testing of samples. The results from these studies indicate that both ATP bioluminescence and PCR assays provide rapid, reliable, and cost effective methods for quality evaluation. This will ultimately result in faster product release and production optimization.
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PMID:Molecular diagnosis of microbial contamination in cosmetic and pharmaceutical products: a review. 1141 29

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.
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PMID:Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR. 1168 10


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