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)

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.
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PMID:Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria. 946 57

Although rare, detection of Mycobacterium bovis infection of captive or free-ranging elk (Cervus elaphus) elicits serious concern due to regulatory and zoonotic implications. Few studies, however, have evaluated the immune response of elk to M. bovis or other pathogens. To model natural infection, elk were vaccinated with live M. bovis bacillus Calmette Guerin (BCG, Pasteur strain) for evaluation of immune responsiveness to this attenuated live vaccine. Peripheral blood mononuclear cells (PBMC) of vaccinated elk proliferated in response to stimulation with a soluble mycobacterial antigen preparation (i.e. M. bovis purified protein derivative, PPDb). Greater numbers of sIgM(+) cells (i.e. B cells) proliferated in this response than did either CD4(+), gammadeltaTCR(+) or CD8(+) cells. The in vivo response (i.e. delayed type hypersensitivity, DTH) to PPDb by vaccinated elk exceeded both the response by non-vaccinated elk and BCG-vaccinated cattle at 24, 48, and 72h post-administration of PPD. In vivo responses to PPDb by vaccinated elk diminished after 72h as compared to responses at 24 and 48h. Serum was also collected periodically and evaluated by ELISA for immunoglobulin (i.e. IgG heavy and light chains) reactivity to crude mycobacterial antigens. Two weeks post-vaccination and throughout the duration of the study, serum immunoglobulin reactivity to PPDb and to a proteinase K-digested whole cell sonicate of BCG exceeded that of serum from non-vaccinated elk. Intradermal administration of PPD for measurement of hypersensitive responses boosted the serum antibody response. These findings demonstrate that BCG vaccination of elk induces a serum antibody response to crude M. bovis antigens, a B cell in vitro proliferative response, and in vivo trafficking of mononuclear cells to sites of mycobacterial antigen administration (i.e. delayed type hypersensitivity). A predominant B cell in vitro proliferative response by elk PBMC to crude mycobacterial test antigens will likely impact the development of improved diagnostic tests of tuberculosis infection for this species.
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PMID:Immune responses of elk to Mycobacterium bovis bacillus Calmette Guerin vaccination. 1261 49

The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis.
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PMID:Identification of outer membrane proteins of Mycobacterium tuberculosis. 1843 72