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Liquid Chromatography Assay for Routine Monitoring of Cellular Ribavirin Levels in Blood. Yoichi Inoue, 2004.Ribavirin-induced hemolytic anemia is one cause for cessation of combination therapy with alpha interferon 2b and ribavirin for hepatitis C infection . Determining cellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, because the metabolites accumulate in erythrocytes . We simplified an assay method developed previously to make it suitable for routine monitoring of cellular ribavirin . Whole blood diluted with a sixfold volume of ice-cold distilled water was subjected to acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin . The resulting mixture, spiked with an internal standard, was treated by phenyl boronic acid column extraction, followed by reverse-phase high-performance liquid chromatography analysis . The calibration curve for ribavirin levels in whole blood was linear at concentrations of 5.3 to 1,024 µM (r2 = 0.9999) . Validation coefficients of variation for intra- and interday assays were 2.9 to 5.8% and 4.3 to 8.3%, respectively . We tested this method by monitoring blood ribavirin concentrations in two hepatitis C patients receiving alpha interferon 2b-plus-ribavirin combination therapy . Role of ß-Lactamases and Porins in Resistance to Ertapenem and Other ß-Lactams in Klebsiella pneumoniae. George A. Jacoby, 2004.High-level resistance to ertapenem was produced by ß-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36 . From a wild-type strain producing ACT-1 ß-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 µg/ml and expression of outer membrane proteins was diminished could be selected . Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S . Environmental Protection Agency Method 1623. Donna S. Francy, 2004.U.S . Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts . Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency . A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample . Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts . Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter . Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters . Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters . Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts . The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different . Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures . Structural Analysis and Evidence for Dynamic Emergence of Bacillus anthracis S-Layer Networks. Evelyne Couture-Tosi, 2002.Surface layers (S-layers), which form the outermost layers of many Bacteria and Archaea, consist of protein molecules arranged in two-dimensional crystalline arrays . Bacillus anthracis, a gram-positive, spore-forming bacterium, responsible for anthrax, synthesizes two abundant surface proteins: Sap and EA1 . Regulatory studies showed that EA1 and Sap appear sequentially at the surface of the parental strain . Sap and EA1 can form arrays . The structural parameters of S-layers from mutant strains (EA1- and Sap-) were determined by computer image processing of electron micrographs of negatively stained regular S-layer fragments or deflated whole bacteria . Sap and EA1 projection maps were calculated on a p1 symmetry basis . The unit cell parameters of EA1 were a = 69 Å, b = 83 Å, and  = 106°, while those of Sap were a = 184 Å, b = 81 Å, and
= 84° . Freeze-etching experiments and the analysis of the peripheral regions of the cell suggested that the two S-layers have different settings . We characterized the settings of each network at different growth phases . Our data indicated that the scattered emergence of EA1 destabilizes the Sap S-layer .
Quinolone Resistance Due to Reduced Target Enzyme Expression. Dilek Ince, 2003.We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus . A single-step mutant of wild-type S . aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type . Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G  A) 13 bp upstream of the start codon of parE . Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance . Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed . The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18 . The steady-state levels of topA transcripts were similar in the various strains . Growth competition experiments performed at 30, 37, and 41°C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41°C . However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts . Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype .
Molecular Detection and Quantitation of the Red Tide Dinoflagellate Karenia brevis in the Marine Environment. M. Gray, 2003.A real-time reverse transcription-PCR method targeting the rbcL gene was developed for the detection and quantitation of the Florida red tide organism, Karenia brevis. The assay was sensitive to less than 1 cell per reaction, did not detect rbcL from 38 nontarget taxa, and accurately quantitated K. brevis organisms in red tide samples from around Florida . These studies have resulted in a sensitive and specific method for K. brevis detection in the marine environment .
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