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In Vitro Susceptibilities of Madurella mycetomatis to Itraconazole and Amphotericin B Assessed by a Modified NCCLS Method and a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5- Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H- Tetrazolium Hydroxide (XTT) Assay. Abdalla O. A. Ahmed, 2004.Susceptibilities of Madurella mycetomatis against amphotericin B and itraconazole in vitro were determined by protocols based on NCCLS guidelines (visual reading) and a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay for fungal viability . The XTT assay was reproducible and sensitive for both antifungals . Itraconazole (MIC at which 50% of the isolates tested are inhibited [MIC50]) of 0.06 to 0.13 mg/liter) was superior to amphotericin B (MIC50 of 0.5 to 1.0 mg/liter) . Comparison of Total Culturable Virus Assay and Multiplex Integrated Cell Culture-PCR for Reliability of Waterborne Virus Detection. Hwa Kyung Lee, 2004.The total culturable virus assay (TCVA) and an integrated cell culture-PCR (ICC-PCR) were compared in parallel to evaluate their detection reliability . Source, finished, and tap water samples from three drinking water treatment plant systems were analyzed by TCVA, and every cell culture dish was subsequently examined by reverse transcription (RT) multiplex PCR using enterovirus- and adenovirus-specific primers . Twenty-seven of 180 (15%) inoculated dishes exhibited cytopathic effects (CPE) . Virus concentrations for source water ranged from 3.3 to 21.0 most probable numbers of infectious units (MPN) per 100 liters . No finished or tap water samples were positive . On the other hand, 38 (21%) of the dishes were positive in multiplex ICC-PCR . Virus concentrations ranged from 4.5 to 10.2 MPN/100 liters for source water and 0 to 0.9 MPN/100 liters for finished and tap water . In spite of its superior sensitivity, the ICC-PCR assay resulted in lower virus concentration values than the TCVA for two of the source water sites . Retest of the CPE-positive dishes using reovirus-specific RT-PCR revealed that 24 of the 27 (89%) dishes were also positive for reoviruses . These observations suggested that the detection reliability of ICC-PCR is restricted by the primer sets that are integrated in the reaction mixture . The observation of an uneven distribution of PCR-positive culture dishes in a given sample raises an additional caution that simple extrapolation of the ICC-PCR result from the analysis of a limited fraction of collected samples should be avoided to minimize possible over- and underestimation of the amount of virus . Identification of fur and fldA Homologs and a Pasteurella multocida tbpA Homolog in Histophilus ovis and Effects of Iron Availability on Their Transcription. Andrew Ekins, 2002.tbpA, fur, and fldA homologs from two strains (9L and 3384Y) of the sheep pathogen Histophilus ovis were sequenced . The predicted TbpA proteins of these strains are homologs of the Pasteurella multocida TbpA protein and collectively represent the second example of a new subfamily of TonB-dependent receptors . tbpA transcripts were readily detected by reverse transcription (RT)-PCR with RNA isolated from strain 9L grown under iron-restricted conditions in the presence or absence of bovine transferrin (Tf) . However, with strain 3384Y and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was from cells grown under iron-restricted conditions in the presence of bovine Tf . In both strains, the fldA homolog was found to be immediately upstream of fur and, based on RT-PCR, these genes are transcribed as a single unit; the availability of iron and the presence or absence of bovine Tf in the growth medium had no apparent effect on the relative amounts of the fldA-fur transcripts . Substrate Specificity and Expression of Three 2,3-Dihydroxybiphenyl 1,2-Dioxygenases from Rhodococcus globerulus Strain P6. David B. McKay, 2003.Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases . In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R . globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined . Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl . More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1 . In R . globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions . Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R . globerulus P6 .
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