Subject(s)
Rectovaginal Fistula , Surgical Flaps , Female , Humans , Rectovaginal Fistula/etiology , Rectovaginal Fistula/surgerySubject(s)
Anal Canal/surgery , Anus, Imperforate/surgery , Fecal Incontinence/surgery , Plastic Surgery Procedures/methods , Postoperative Complications/surgery , Anus, Imperforate/complications , Fecal Incontinence/congenital , Female , Humans , Middle Aged , Postoperative Complications/congenitalSubject(s)
Formaldehyde/administration & dosage , Gastrointestinal Hemorrhage/drug therapy , Nerve Block/adverse effects , Proctitis/drug therapy , Radiation Injuries/drug therapy , Aged , Gastrointestinal Hemorrhage/etiology , Humans , Proctitis/etiology , Pudendal Nerve , Radiation Injuries/etiologyABSTRACT
BACKGROUND: We have identified a novel class 1 integron (1503 bp), named In671 in a clinical Pseudomonas aeruginosa isolate. Integron sequence analysis revealed two gene cassettes, one coding for a new OXA-type ß-lactamase designated as OXA-205 and the other coding for the aadB gene that is responsible for aminoglycoside resistance. The 266 amino acid sequence of OXA-205 revealed that this ß-lactamase belongs to the Ambler class D showing highest sequence homology to the OXA-2 sub-lineage. Our objective was to purify and characterize ß-lactamase OXA-205. METHODS: Escherichia coli cells were transformed with a plasmid containing cloned bla OXA-205 gene from P. aeruginosa. Purification of overproduced OXA-205 consisted of a single ion-exchange chromatography step. SDS-PAGE and isoelectric focusing were performed to determine the molecular mass and pI, respectively. Size-exclusion chromatography was undertaken to determine the OXA-205 oligomerization state. Substrate hydrolysis reactions were employed to assess enzyme kinetic parameters. RESULTS: Purification of OXA-205 yielded the enzyme with >95 % purity (as verified by SDS-PAGE). Approximate yield of the protein was estimated to be 20 mg per liter of culture. OXA-205 had a pI at 8.1, molecular mass of 26 kDa and a monomeric native structure. Kinetic analysis revealed that OXA-205 hydrolyzed narrow spectrum substrates, including ampicillin, carbenicillin, oxacillin, penicillin G, cefazolin and cefuroxime. Additionally, we observed a substrate inhibition profile towards carbenicillin and oxacillin, but not with ampicillin or penicillin G. Our results also show that OXA-205 conferred unusually high (among class D ß-lactamases) resistance towards inhibition by NaCl. CONCLUSIONS: OXA-205 can be considered a narrow spectrum monomeric ß-lactamase that demonstrates unusually high resistance profile towards inhibition by NaCl.
Subject(s)
Pseudomonas aeruginosa/enzymology , beta-Lactamases/isolation & purification , beta-Lactamases/metabolism , Amino Acid Sequence , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Hydrolysis , Integrons , Isoelectric Focusing , Isoelectric Point , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Protein Conformation , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Sequence Alignment , Substrate Specificity , beta-Lactamases/chemistry , beta-Lactams/metabolismABSTRACT
The authors analyze the results of studies on the effect produced by the immunization of calves with paratyphoid vaccine on the production of agglutinins, changes in the blood serum immunoglobulin levels, and the specificity of IgM to Salmonella dublin and Salmonella typhimurium antigens. The highest level of agglutinins to paratyphoid antigens in the blood sera of the immunized calves was registered on days 14-21 after immunization, changes in the levels of different immunoglobulin classes being insignificant. The agglutination test and the enzyme immunoassay have revealed that antibodies to S. dublin anb S. typhimurium antigens belong to IgM.
