RESUMO
Recombinant methioninase (rMETase) is an enzyme that has antitumor activity. In this work, METase gene from Pseudomonas putida ATTCC 8209 was cloned to pT7-7 plasmid (yielded, PT7-METase-R7 clone) and expressed in E. coli strain BL21 (DE3). A protein band with a molecular massof 42 kDa was visualized by SDS-PAGE. The applied protocol yielded a total protein of 3.13 g with a recovery of 66.89% and a specific activity of 18.59 U mg-1 which considered as a low yield. However, when the METase gene was cloned to the vector (pTrc99A, clone: pTrc99A-MET-3) cells of E. coli JM109 yielded a total protein of 32.63 g with a recovery of 41.62% and a specific activity of 54.86 U mg-1 which revealed that the enhancement of METase gene expression by trc promoter was more than the T7 RNA polymerase promoter. The t1/2 of the rMETase was 2 h asanalyzed in mice by IV injection. Antitumor efficacy of rMETase was studied in five human cancer cell lines. At 1 U mL-1 the growth rate of treated colon cancer cell lines, Colo205 and SW620, with rMETase was 46 and 32% relative to control, respectively. With the ovarian cancer cell line (A2780) rMETase produced an inhibition effect of 54% at 1.5 U mL-1. In addition, the growth rate was reduced to 45 and 53% with the skin cancer cell line (A375) and the breast cancer cell line (MCF-7), respectively. These results indicate the feasibility of rMETase for use as a potent antitumor agent.
RESUMO
PURPOSE: Vancomycin-resistant enterococci (VRE) pose an emerging problem in hospitals worldwide. The present study was undertaken to determine the occurrence, species prevalence, antibacterial resistance, and phenotypic and genetic characteristics of VRE isolated in Riyadh hospitals, KSA. MATERIALS AND METHODS: Two hundred and six isolates of enterococcal species were obtained from clinical samples. The antibiotic susceptibility of isolates and minimum inhibitory concentration (MIC) tests for vancomycin and teicoplanin were determined. Molecular typing of VRE isolates was carried out by using pulsed field gel electrophoresis (PFGE) and the resistance genotype was determined by polymerase chain reaction (PCR). RESULTS: VRE accounted for 3.9% of the isolates and were detected mostly in urine, wound and blood specimens isolated from ICU, internal medicine and surgical wards. All strains were identified to species level and were found to consist of E. faecalis (69.2%), E. faecium (11.3%), E. avium (2.1%), E. hirae (0.8%), E. casseliflavus (1.3%) and E. gallinarum (1.3%) species. According to the susceptibility data obtained, 8 (3.9%) out of 206 isolates were found to be VRE (MICs > 32 µg/ml). The vanA, vanB and vanC gene fragments of E. faecalis, E. faecium and E. gallinarum were amplified from isolates and were detected. PFGE patterns of the VRE isolates revealed homogenous patterns with dominant clone suggesting that the strains intrinsic resistance is independent. CONCLUSIONS: This study shows an emergence of VRE along with increased rate of multidrug-resistant enterococci in the area of the study. Regular surveillance of antimicrobial susceptibilities should be done regularly and the risk factors should be determined.