Comparative in vitro activities of linezolid, quinupristin-dalfopristin, moxifloxacin, and trovafloxacin against erythromycin-susceptible and -resistant streptococci.
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The in vitro activities of the new agents linezolid, quinupristin-dalfopristin, moxifloxacin, and trovafloxacin were determined and compared with those of penicillin, clindamycin, and four macrolides against 53 erythromycin-resistant Streptococcus pneumoniae, 117 S. pyogenes (64 erythromycin-susceptible and 53 -resistant), and 101 S. agalactiae (53 erythromycin-susceptible and 48 -resistant) isolates. Differentiation of macrolide resistance phenotypes was performed by the double-disk method. The genetic basis for macrolide resistance in 52 strains was also determined. The M phenotype was found in 84.9, 6.3, and 1.9% of S. pyogenes, S. agalactiae, and S. pneumoniae isolates, respectively. These strains were susceptible to miocamycin and clindamycin. Strains with the inducible phenotype accounted for 27.1% of S. agalactiae isolates and 9.4% each of S. pyogenes and S. pneumoniae isolates. All erythromycin-resistant isolates were also resistant to the 14- and 15-membered macrolides tested. Strains with all three phenotypes were susceptible to +info)
In vitro activities of linezolid alone and in combination with amoxicillin, clarithromycin, and metronidazole against Helicobacter pylori.
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Linezolid was tested against 70 strains of Helicobacter pylori by the agar dilution method. The MIC range and MICs at which 50 and 90% of strains were inhibited were 8 to 64, 16, and 32 microgram/ml, respectively. With minimum and maximum fractional inhibitory concentration summation values of 0.31 and 2.50, respectively, the combination of linezolid with amoxicillin, clarithromycin, or metronidazole showed either partial synergy or indifference for the majority of strains. (+info)
Metabolism of the influenza neuraminidase inhibitor prodrug oseltamivir in the rat.
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The metabolism of [2-acetyl-(14)C]oseltamivir (GS4104, Ro 64-0796), the prodrug of the novel influenza neuraminidase inhibitor GS4071 (Ro 64-0802), was examined in rats after oral dosing. Intact oseltamivir was observed only in lung and urine, accounting for 37 and 15% of the total radioactivity in these samples, respectively. GS4071 was the major metabolite in plasma, tissues, and urine, and accounted for 32 to 56% of the radioactivity present in these samples. The second most abundant peak in these samples (13-24% of radioactivity) was a novel metabolite (M3). This metabolite was purified from urine of rats dosed orally with oseltamivir and was identified by liquid chromatography-mass spectrometry and NMR as the (R)-omega-carboxylic acid metabolite of oseltamivir. The omega-carboxylic acid metabolite of oseltamivir could not be produced in vitro. However, omega-hydroxylated products of oseltamivir were produced by rat liver microsomes. Both the (R)- and (S)-omega-hydroxylated products were observed, but formation of the (R)-isomer predominated. These data indicated that in the rat, oseltamivir was primarily metabolized to the active influenza neuraminidase inhibitor GS4071 and, to a lesser extent, to an (R)-omega-carboxylic acid metabolite. (+info)
Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes.
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Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH(2) and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales. (+info)
Steric hindrance regulation of the Pseudomonas aeruginosa amidase operon.
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Expression of the amidase operon of Pseudomonas aeruginosa is controlled by AmiC, the ligand sensor and negative regulator, and AmiR the transcription antitermination factor activator. We have titrated out AmiC repression activity in vivo by increased AmiR production in trans and shown AmiC regulation of the antitermination activity of AmiR by a steric hindrance mechanism. In the presence of the co-repressor butyramide we have isolated a stable AmiC.AmiR complex. Addition of the inducing ligand acetamide to the complex trips the molecular switch, causing complex dissociation and release of AmiR. The AmiC.AmiR butyramide complex exhibits acetamide-dependent, sequence-specific RNA binding activity and a K(d) of 1.0 nm has been calculated for the AmiR.RNA interaction. The results show that amidase operon expression is controlled by a novel type of signal transduction system in which activity of a site-specific RNA binding activator is regulated via a sequestration mechanism. (+info)
Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes.
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To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions. (+info)
kappa -opioid receptor agonists modulate visceral nociception at a novel, peripheral site of action.
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kappa-opioid receptor agonists (kappa-ORAs) have been shown to modulate visceral nociception through an interaction with a peripheral, possibly novel, kappa-opioid-like receptor. We used in the present experiments an antisense strategy to further explore the hypothesis that kappa-ORA effects in the colon are produced at a site different from the cloned kappa-opioid receptor (KOR). An antisense oligodeoxynucleotide (ODN) to the cloned rat KOR was administered intrathecally (12.5 microg, twice daily for 4 d) to specifically knock-down the cloned KOR. Efficacy of the KOR antisense ODN treatment was behaviorally evaluated by assessing the antinociceptive effects of peripherally administered kappa- (EMD 61, 753 and U 69,593), mu- (DAMGO) and delta- (deltorphin) ORAs in the formalin test. Intrathecal antisense, but not mismatch ODN blocked the actions of EMD 61,753 and U 69,593 without affecting the actions of DAMGO or deltorphin; a complete recovery of antinociceptive actions of the kappa-ORA EMD 61,753 was observed 10 d after the termination of antisense ODN treatment. In contrast, the ability of EMD 61,753 to dose-dependently attenuate responses of pelvic nerve afferent fibers to noxious colonic distension was unaffected in the same rats in which the antisense ODN effectively knocked-down the KOR as assessed in the formalin test. Additionally, Western blot analysis demonstrated a significant downregulation of KOR protein in the L4-S1 dorsal root ganglia of antisense, but not mismatch ODN-treated rats. The present results support the existence of a non-kappa-opioid receptor site of action localized in the colon. (+info)
Actions of the anticonvulsant remacemide metabolite AR-R12495AA on afferent-evoked spinal synaptic transmission in vitro and on models of acute and chronic inflammation in the rat.
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The effects of the anticonvulsant remacemide [(+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)-acetamide hydrochloride] and its metabolite AR-R12495AA [(+/-)-1-methyl-1, 2-diphenylethylamine-monohydrochloride] on primary afferent synaptic transmission were assessed in the young rat spinal cord in vitro. Stimulation of dorsal roots at A- and C-afferent intensity elicited a dorsal root-evoked ventral root potential (DR-VRP) with a slowly decaying phase. Repetitive stimuli (2 Hz) produced summation of slow potentials and a cumulative ventral root depolarization (CVRD), a form of wind-up. Remacemide and AR-R12495AA antagonized the DR-VRP slow peak t(1/2) decay and slow phase total duration at drug concentration of > or =25 microM. AR-R12495AA was approximately 2-fold more potent than remacemide. The most potent action was against the slow phase duration with IC(50) values of 157 and 60 microM for remacemide and AR-R12495AA, respectively. Both drugs at concentrations of > or =100 microM attenuated the DR-VRP fast peak amplitude (IC(50) = 253 and 142 microM, respectively). The amplitude of CVRD was reduced by remacemide and AR-R12495AA (IC(50) = 195 and 111 microM, respectively). MK-801 reduced DR-VRP fast peak amplitude (IC(50) = 58 microM), slow peak t(1/2) decay (IC(50) = 60 microM), slow phase duration (IC(50) = 50 microM), and CVRD amplitude (IC(50) = 91 microM). In behavioral studies, AR-R12495AA (i.p.) reduced the mechanical hyperalgesia and paw swelling that followed hind paw injection of carrageenan or Freund's complete adjuvant. These electrophysiological and behavioral data indicate further studies should be conducted on the efficacy of remacemide and AR-R12495AA as putative analgesics. (+info)