Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.
(1/96)
We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics. (+info)
Effect of polymorphisms on ligand binding by mouse major urinary proteins.
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Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx-binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed. (+info)
The micronucleus assay in human exfoliated urothelial cells: application in a genotoxicity study of workers exposed to a mineral jelly containing sodium nitrite and N-phenyl-1-naphthylamine.
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Exposure to certain chemical agents in occupational settings has been identified as carcinogenic to the human bladder. Micronucleus (MN) analysis in exfoliated urothelial cells is an interesting method for biomonitoring genetic damage in human populations. However, few studies have been performed in an occupational context. The aim of this study was to examine whether the occupational use of a mineral jelly induced a genotoxic risk for workers employed at a single factory producing bearings using the MN test on exfoliated urothelial cells. The prevalence of micronucleated exfoliated urothelial cells (MNC) was determined in 35 female workers with dermal exposure to the jelly and 41 female controls. The mean percentage of MNC (expressed as percent cells with MN per 1000 cells scored) observed in the exposed worker group was 0.46 +/- 0.11% (range 0-2.8) and in the control group 0.14 +/- 0.03% (range 0-0.8). There is a significant job effect (P = 0.0018, MANCOVA) on the prevalence of MNC, whereas age and smoking habit had no significant effect (P = 0.90 and 0.91, respectively). There is no interaction between job and smoking habit (P = 0.4421). Exposure to the mineral jelly appeared to be the main factor inducing the increased prevalence of MNC. This may be due to the presence of mutagens/carcinogens in the jelly: an aromatic amine, N-phenyl-1-naphthylamine (CAS no. 90-30-2), which is carcinogenic in mice, or sodium nitrite (CAS no. 7632-00-0), which is genotoxic in human cell systems. In conclusion, these results suggest that use of the mineral jelly could present a genotoxic risk for workers. We think that the MN assay on exfoliated cells could be valuable for biological monitoring purposes in occupational contexts as a marker of significant exposure to bladder mutagenic/carcinogenic agents. (+info)
Specific interactions of the antimicrobial peptide cyclic beta-sheet tachyplesin I with lipopolysaccharides.
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The cyclic beta-sheet antimicrobial peptide tachyplesin I (T-SS) was found to show 280-fold higher affinity for lipopolysaccharides (LPS) compared with acidic phospholipids, whereas the linear alpha-helical peptide F5W-magainin 2 (MG2) could not discriminate between LPS and acidic phospholipids. The recognition site was the lipid A moiety and the cyclic structure was crucial to this specific binding. The cyclic structure also endowed the peptide with very rapid outer membrane (OM) permeabilization. (+info)
Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa.
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The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid. (+info)
Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release.
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The effect of EDTA on Salmonella enterica serovar Typhimurium was studied in different growth phases with cells grown with or without Ca(2+) and Mg(2+) supplementation. EDTA affected the outer membrane much more strongly in the early exponential phase than in the mid- or late exponential phase, as indicated by uptake of 1-N-phenylnaphthylamine (a nonpolar hydrophobic probe, M(r) 219), and detergent (SDS) susceptibility. This effect was, however, not paralleled by LPS release (determined by measuring LPS-specific fatty acids or 14C-labelled LPS in cell-free supernatants, per a standardized cell density), which remained unchanged as a function of the growth curve. The conclusion from these results is that in the early exponential phase the effect of EDTA in S. enterica involves a component that is independent of LPS release. (+info)
Neuroendocrine response to clonidine and 8-OH-DPAT in rats following chronic administration of desipramine or sertraline.
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1. Rats were administered either desipramine (DMI) or sertraline daily at doses 7.5 mg kg-1 or 10 mg kg-1, i.p., respectively and the effects on the functional state of hypothalamic neuroendocrine control mechanisms assessed by measurements of plasma hormones following acute drug challenge. The effects of treatment on gross behaviour and brain adrenoceptor density were also determined. 2. Both DMI and sertraline caused significant reduction in activity measured as ambulation and rearing at 14 days of treatment. 3. All animals were chronically cannulated after 14 days of treatment and tested for neuroendocrine response to acute i.v. clonidine (50 micrograms kg-1) or 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 250 micrograms kg-1) after 21 or more days of treatment. 4. Rats treated with DMI but not sertraline showed a virtually complete suppression of the growth hormone (GH) secretion elicited by clonidine in controls, while the secretion of corticosterone was augmented. 5. Treatment with DMI but not sertraline led to a significantly greater 8-OH-DPAT-induced secretion of prolactin than in the control rats, while the plasma concentrations of corticosterone following 8-OH-DPAT were not influenced by either DMI or sertraline treatment. 6. The density (but not the affinity) of cerebral cortical binding of [3H]-dihydroalprenolol was significantly reduced by DMI treatment. 7. These results show that DMI treatment blunted the sensitivity of post-synaptic alpha 2-adrenoceptors, accompanied by complex interactions manifested as increased responsiveness of alpha 1-adrenoceptors and 5-HT1A receptors. Sertraline had no significant neurendocrine effects at a dose which significantly reduced gross activity. (+info)
The effect of selective serotonin re-uptake inhibitors on cytochrome P4502D6 (CYP2D6) activity in human liver microsomes.
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Inhibition of human cytochrome P4502D6 (CYP2D6)-catalysed metabolism can lead to clinically significant alterations in pharmacokinetics. Since there is evidence that the selective serotonin reuptake inhibitor (SSRI) class of antidepressant drugs might inhibit CYP2D6, the effects of five SSRIs on human liver microsomal CYP2D6 activity were compared with each other and with three tricyclic antidepressant drugs. On a molar basis, paroxetine was the most potent of the SSRIs at inhibiting the CYP2D6-catalysed oxidation of sparteine (Ki = 0.15 microM), although fluoxetine (0.60 microM) and sertaline (0.70 microM) had Ki values in the same range. Fluvoxamine (8.2 microM) and citalopram (5.1 microM) also inhibited CYP2D6 activity. The major circulating metabolites of paroxetine in man produced negligible inhibition. In contrast, norfluoxetine the active metabolite of fluoxetine, was a potent CYP2D6 inhibitor (0.43 microM). CYP2D6 activity was also diminished by the tricyclic antidepressant drugs clomipramine (2.2 microM), desipramine (2.3 microM) and amitriptyline (4.0 microM). These findings suggest that compounds with SSRI activity are likely to interact with human CYP2D6 in vivo with the potential of causing drug interactions. (+info)