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In Vitro Interactions of Approved and Novel Drugs against Paecilomyces spp.. Montserrat Ortoneda, 2004.We have evaluated the in vitro activity of 15 combinations of antifungal drugs (amphotericin B, itraconazole, voriconazole, albaconazole, ravuconazole, terbinafine, and micafungin) against four isolates of Paecilomyces variotii and three of P . lilacinus . The interaction of terbinafine with the four azoles was synergistic for 53% of the combinations, while the interactions of both amphotericin B and micafungin with the rest of antifungal agents were mainly indifferent . Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters. M. H. Josefsen, 2004.As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens . Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000 . The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples . In the RotorGene, a positive PCR response was detected in 40 samples of the 66 . This was in complete agreement with the enriched ISO culture . The ABI-PRISM 7700 missed one culture-positive sample . Positive samples contained 102 to 107 CFU/ml after enrichment in Bolton broth . In the enriched samples a detection probability of 95% was obtained at levels of 1 x 103 and 2 x 103 CFU/ml in the RotorGene and ABI-PRISM, respectively . The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 101 to 1 x 107 (R2 = 1.00) for the RotorGene and 103 to 107 (R2 = 0.99) for the ABI-PRISM . In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively . The results showed a correlation (R2) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment . Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1) . The method will be taken further and validated in an international collaborative trial with regard to standardization . Energetics of Helicobacter pylori and Its Implications for the Mechanism of Urease-Dependent Acid Tolerance at pH 1. Kerstin Stingl, 2002.In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis . To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H . pylori were determined at various pH values . In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5 . In the presence of urea, H . pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2 . At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside . The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions . Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization . Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm . The urease activity of intact cells increased nearly exponentially with decreasing external pH . This activation was not due to enhanced gene expression at low external pH values . In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer . Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values . The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H . pylori is discussed . Newly Identified Cytochrome c Oxidase Operon in the Nitrogen-Fixing Cyanobacterium Anabaena sp . Strain PCC 7120 Specifically Induced in Heterocysts. Kathryn M. Jones, 2002.Two operons have been cloned from Anabaena sp . strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases . The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit . One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation . Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts . The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells . Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen . It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp . strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts .
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