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)

Oridonin (Ori) is an active principle isolated from Rabdosia rubescens. The cytotoxic effect of the combination of Ori and cisplatin was tested by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay. IC50 of cisplatin to cultured S180 cells in 24 h was 9.38 micrograms.ml-1. When the cells were treated with cisplatin plus Ori 0.5 and 1 microgram.ml-1, the IC50 were 1/3.4 and 1/6.7, respectively, of that with cisplatin alone. Modified alkaline elution was used to detect the DNA interstrand cross-link and DNA-protein cross-link in S180 cells induced by the 2 drugs. A greater amount of DNA cross-link was detected when the cells were treated with cisplatin plus Ori than with cisplatin alone (P < 0.05). After lysis by proteinase K, a reduction in DNA cross-link was seen, which suggested that the drugs could produce both kinds of DNA cross-link.
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PMID:Synergistic effect of oridonin and cisplatin on cytotoxicity and DNA cross-link against mouse sarcoma S180 cells in culture. 801 60

A primer-directed DNA amplification polymerase chain reaction assay for detection of seed contaminated with highly virulent Leptosphaeria maculans was developed. The primers were derived from a 5,238-bp repetitive sequence present only in the highly virulent isolates of the fungus. A procedure for isolating DNA from organisms infesting germinating seed was also developed. Seeds were added to liquid fungal minimal medium, and the culture was incubated for 3 days at room temperature with shaking. The organisms were collected from the cultures by centrifugation and lysed with a combination of sodium dodecyl sulfate and proteinase K. The DNA was extracted with organic solvents and with a high-salt-cetyltrimethylammonium bromide solution. It was also precipitated with a low-salt-cetyltrimethylammonium bromide solution. The extensive treatments used for minimizing polysaccharide contamination greatly improved the reliability of the assay. The minimum contamination level (2 of 1,000 seeds) that was tested was successfully detected with this DNA isolation procedure. The reliability of the assay was 96% at the 1 to 2% level of seed contamination. The described method is less laborious and requires only 4 to 5 days for completion in comparison to the 11 to 22 days required for the currently employed methods. In addition, large sample sizes can be easily handled, thus reducing the probability of contaminated seed escaping detection.
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PMID:A simple, sensitive, and rapid method for detecting seed contaminated with highly virulent Leptosphaeria maculans. 828 76

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.
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PMID:DNA recovery from soils of diverse composition. 859 35

Domain I of ovoinhibitor was isolated by subjecting the protein to specific chemical cleavage by cyanogen bromide followed by repeated gel filtration. The first domain of ovoinhibitor was found to be homogeneous by the criteria of gel chromatography, SDS-PAGE and PAGE. Mr values by gel filtration (10900) and SDS-PAGE (8300) were slightly higher than that computed from amino-acid sequence. This discrepancy has been attributed to the glycoprotein nature of domain I as it was found to contain 10% neutral carbohydrate and 2% sialic acid. Fluorescence spectral properties showed the presence of tryptophan in domain I. The amino-acid composition of domain I isolated in this study was in very good agreement with that computed from amino-acid sequence. Gel filtration behaviour of the first domain was consistent with a Stokes radius of 1.6 nm and a frictional ratio of 1.2 suggesting asymmetry and/or excessive hydration. Domain I was found to be a potent inhibitor of bovine trypsin but was virtually devoid of activity against chymotrypsin, elastase and proteinase K. The equilibrium association constant for domain I-trypsin complex was computed to be 6.6x10(8)M-1.
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PMID:Isolation and characterization of domain I of ovoinhibitor. 865 16

DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.
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PMID:Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. 886 37

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.
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PMID:A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen. 900 95

With the objective of monitoring xenobiotic degrading bacteria in soil, a method for rapid extraction of DNA from soil, amenable to amplification by PCR, was developed. The method was based on lysis by freeze-thawing and subsequent addition of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide and proteinase K. The extraction method required 2 h and was tested on six different soils differing in organic content, water holding capacity and pH, including ones from which DNA extraction is difficult. DNA yields from the soils ranged from 6.1 to 54.0 micrograms of DNA per g soil. The efficiency and reproducibility of the DNA extraction method were evaluated by competitive PCR. The organic content in the soils was a major factor affecting the amount of obtained DNA amenable for amplification by PCR. A PCR primer-pair was designed on the basis of the known nucleotide sequences of several catechol 2,3-dioxygenase genes. The specificity of the primer-pair was demonstrated on different sequenced catechol 2,3-dioxygenase genes and on site-specific bacterial isolates from polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The concentration of catechol 2,3-dioxygenase DNA in PAH-contaminated sediment undergoing an ex situ compost process was quantified by competitive PCR over a period of 16 weeks. The concentration of PAHs and catechol 2,3-dioxygenase DNA in the soil samples, was found to correlate.
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PMID:DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types. 908 10

The major outer membrane protein of Campylobacter jejuni (MOMP, 43 kDa), supposed to be one of the structures responsible for adhesion to INT 407 cells, was isolated from the crude outer membrane preparation by treatment with n-octyl-beta-D-glucopyranoside followed by preparative SDS-polyacrylamide gel electrophoresis. By cleavage of the isolated protein with cyanogen bromide and proteolytic enzymes, peptides were generated, separated by reverse phase high pressure liquid chromatography, and sequenced by automatic Edman degradation. The protein was aligned by identification of overlapping peptides. Treatment of bacteria with proteinase K prior to preparation of the outer membrane yielded a truncated MOMP with an apparent molecular mass of 25 kDa consisting of the C-terminal part of the protein. The isolated MOMP was functionally characterized by significant binding activity towards INT 407 cell membranes when isolated by preparative native gel electrophoresis, however, no binding activity was detected when the protein was isolated in the presence of SDS.
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PMID:Primary structure analysis and adhesion studies on the major outer membrane protein of Campylobacter jejuni. 916 18

Our laboratory described a ca. 34-kDa protein of the HeLa S cell surface that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl-6-noneamide (capsaicin) also blocked these same enzymatic activities. Using capsaicin inhibition as the criterion, the drug-responsive oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer. Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.
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PMID:A drug-responsive and protease-resistant peripheral NADH oxidase complex from the surface of HeLa S cells. 975 Jan 73

The aims of the study were to compare polymerase chain reaction PCR with nucleic acid hybridisation HC in the routine diagnosis of HPV infections. Smears collected for PCR were digested for 24 hours using proteinase K. After DNA extraction 174 samples were tested by PCR with human bglobin primers PG04-GH20. The PCR products were separated in 2% agarose gel electrophoresis stained with ethidium bromide. In 80.6% of the samples 256 base pair DNA fragments were observed in the gel in UV light. These samples were tested by PCR with HPV primers MY09-MY11. In 40% of the samples the presence of HPV DNA was confirmed. Next we carried out PCR using a mixture of two pairs of primers bglobin PG04-GH20 and HPV MY09-MY11. DNA for this study was extracted from 24 samples in which the presence of human DNA was not confirmed in the first PCR test and from 7 untested samples. In 21 cases HPV DNA was found to be present in gel electrophoresis. The presence of HPV DNA was confirmed in 44.75% of the samples.
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PMID:[Papillomaviruses and herpesviruses as selected risk factors in the etiopathogenesis of cervix neck cancer. II. Use of PCR for isolating DNA of human papillomavirus and Herpes simplex]. 1080 69


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