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Active Efflux of Ciprofloxacin from J774 Macrophages through an MRP-Like Transporter. Jean-Michel Michot, 2004.The accumulation and efflux kinetics of ciprofloxacin have been examined by using murine J774 macrophages . Accumulation (at equilibrium) was increased (three- to fourfold) (i) when cells were incubated with high extracellular drug concentrations (typically 200 mg/liter) as opposed to clinically meaningful concentrations (10 mg/liter or lower), (ii) during ATP- depletion and at acid pH, and (iii) during coincubation with probenecid, gemfibrozil and the preferential multidrug resistance-related protein (MRP) inhibitor MK571 . All these conditions were also associated with a marked decrease in ciprofloxacin efflux (half-lives increased from <2 min in controls to up to 10 min) . Monensin (a proton ionophore), verapamil, and the preferential P-glycoprotein (P-gp) inhibitor GF120918 had no or only minimal effect, while cyclosporin A, which is not specific for P-gp but also acts on MRP, had an intermediate effect . Short-term uptake studies showed that the influence of the modulators on the apparent drug influx was almost immediate (delay of  1 min) . Cells made resistant to probenecid and showing a marked overexpression of MRP1 (by Western blot analysis and confocal microscopy) accumulated ciprofloxacin to almost the same extent as did control cells, but efflux was inhibited less by probenecid, gemfibrozil, and MK571 . We conclude that ciprofloxacin is subject to constitutive efflux in J774 macrophages through the activity of an MRP-related transporter which is probably distinct from MRP1 . We also suggest that the cellular accumulation of ciprofloxacin in wild-type cells is constitutively impaired at therapeutically meaningful concentrations .
Detection and Quantification of Airborne Conidia of Fusarium circinatum, the Causal Agent of Pine Pitch Canker, from Two California Sites by Using a Real-Time PCR Approach Combined with a Simple Spore Trapping Method. Wolfgang Schweigkofler, 2004.Pinus radiata (Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production . A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by Fusarium circinatum (Hypocreales) . We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air . The assay is also useful for diagnosing the presence of the pathogen on plants . The test is based on the F . circinatum specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon . Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F . circinatum spores . The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium . A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002 . Spore counts were in the range of ca . 1 x 103 to ca . 7 x 105/m2, with the highest spore counts in the fall, suggesting a seasonal fluctuation . Pseudoknot-Dependent Translational Coupling in repBA Genes of the IncB Plasmid pMU720 Involves Reinitiation. J. Praszkier, 2002.Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB . The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence . A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB . The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence . Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB . The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed . Interfering with Different Steps of Protein Synthesis Explored by Transcriptional Profiling of Escherichia coli K-12. Jeffrey Sabina, 2003.Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level . A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents . Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response . Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response . Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons . Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents . Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts . However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain . Structures of the Mating-Type Loci of Cordyceps takaomontana. Eiji Yokoyama, 2003.Nucleotide sequences of the mating-type loci MAT1-1 and MAT1-2 of Cordyceps takaomontana were determined, which is the first such report for the clavicipitaceous fungi . MAT1-1 contains two mating-type genes, MAT1-1-1 and MAT1-1-2, but MAT1-1-3 could not be found . On the other hand, MAT1-2 has MAT1-2-1. A pseudogene of MAT1-1-1 is located next to MAT1-2 .
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