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)

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.
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PMID:Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers. 238 Mar 80

Crude ribosomes were isolated from Listeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice against Listeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment, but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction II, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part non-specific, because both preparations also protected mice against L. monocytogenes serotype 3, Streptococcus pneumoniae and Pseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations from P. aeruginosa.
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PMID:RNase-sensitive and RNase-insensitive protective components isolated from Listeria monocytogenes. 241 92

In this study we investigated the relation between enhanced resistance and delayed hypersensitivity (DH) induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyldioctadecylammonium bromide (DDA). Ribosomal RNA as well as cell envelope fragments (fraction I) protected mice against lethal Listeria infection. However, only fraction I induced DH against killed Listeria. For the induction of protection with fraction I or RNA as well as for the induction of DH with fraction I, preparations had to be administered in combination with DDA. Fraction I elicited a DH response in mice immunized with viable Listeria, but RNA did not. These observations pointed to a dissociation between DH and enhanced resistance induced with RNA, and to a dissociation between fraction I and RNA with respect to their ability to induce or elicit DH. Also DH and enhanced resistance induced with fraction I could be dissociated. Intracutaneous administration of fraction I induced high levels of DH without concomitant induction of protection against lethal challenge with Listeria. On the other hand, intraperitoneal administration of fraction I fully protected mice against lethal infection, but only induced a moderate DH response. DH induced with fraction I was largely specific, whereas enhance resistance induced with this preparation was nonspecific. Finally, proteinase K-sensitive proteins were found to be essential for the induction of DH but not for the induction of protection with fraction I.
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PMID:Dissociation between enhanced resistance and delayed hypersensitivity induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyl-dioctadecyl-ammonium bromide. 242 34

A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.
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PMID:Rapid method for the purification of DNA from subgingival microorganisms. 262 68

Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples.
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PMID:Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms. 263 97

A novel replicating agent (IFDO) was isolated from ileal fluid. Growth occurred in vitro under aerobic and anaerobic conditions, and was faster at 37 degrees C than at room temperature. The doubling time was 15.8 min. Colonies were dark brown in colour and occurred beneath the surface of agar after conventional surface inoculation. Provisional data indicate that the agent may be a normal intestinal commensal. The agent was remarkably resistant to inactivation by steam at 134 degrees C, formaldehyde and glutaraldehyde; it was relatively resistant to ionising radiation, and it was filterable through membranes with a nominal pore diameter of 10 nm. Such properties, with the exception of growth in cell-free medium, are shared by "unconventional agents" such as those of Creutzfeldt-Jakob disease and scrapie. Further comparison of the properties of the intestinal agent and of slow viruses revealed additional shared characteristics, including resistance to proteinase K and trypsin, and inactivation by guanidine thiocyanate, diethyl pyrocarbonate, phenol and sodium hydroxide. The agent differs from that of scrapie in being inactivated by ethidium bromide, zinc nitrate, EDTA, hydroxylamine in the presence Sarkosyl, and, under certain circumstances, by ribonuclease. Broth cultures of the agent contained particles possessing considerable size heterogeneity. The smaller filterable particles were generally more susceptible to inactivation, did not survive autoclaving, and were inactivated by papaya protease and lipase. It is possible that the replicating agent may be formed by crystallisation from constituents of the medium, and not by a biological process. This does not exclude the postulated relationship to slow viruses.
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PMID:A novel replicating agent isolated from the human intestinal tract having characteristics shared with Creutzfeldt-Jakob and related agents. 265 97

Hen oviduct N alpha-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified N alpha-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. N alpha-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureus nuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2 digest of the nucleic acid was composed of four 3'-ribomononucleotides and completely separated from 3'- and 5'-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in N alpha-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that N alpha-acetyltransferase consisted of one molecule of 7 S RNA (Mr 83,000) and two identical Mr 79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of N alpha-acetyltransferase estimated by Sepharose 6B gel filtration.
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PMID:Hen oviduct N alpha-acetyltransferase is a ribonucleoprotein having 7 S RNA. 275 10

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis.
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PMID:A new and fast method for preparing high quality lambda DNA suitable for sequencing. 296 28

A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure E. coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-Cl chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 micrograms for pBR-322 DNA, and 250-500 micrograms for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation does not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days.
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PMID:A rapid and inexpensive method for preparing E. coli plasmid-DNA. 301 51

The sedimentation of nucleoids from thymic and splenic cells of rats was tested following total-body X-irradiation (TBI) with doses ranging from 24 to 1520 cGy. The principal results may be summarized as follows: 1) The nucleoid sedimentation of the cells was reduced immediately after TBI with doses of greater than or equal to 760 cGy. In the following postirradiation period, an enhancement of sedimentation rate has been observed which could be neutralized by addition of proteinase K to the nucleoid preparation. 2) When nucleoids were prepared 6 h after TBI with doses greater than or equal to 190 cGy, beside the main nucleoid band a smaller nucleoid fraction appeared in the ethidium bromide containing saccharose gradient. This fraction was of less sedimentability than the main nucleoid peak and could not be distinguished from pure, high molecular DNA. From the present results it is suggested that the reduction of the nucleoid sedimentation immediately following high doses of TBI is the result of primary (non-repaired) DNA lesions whereas the changes detectable some hours later are due to the secondary enzymatic changes connected with the interphase death of the cells. With respect to the detection of in vivo effects of X-irradiation, the nucleoid sedimentation has to be regarded much less sensitive than some biochemical and/or cytomorphological methods.
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PMID:[Sedimentation of nucleoids from rat thymus and spleen cells following whole-body irradiation]. 328 84


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