We have studied the plasma membrane protein phenotype of human cultureamplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytom-

The occurrence of active efflux and cell wall modifications were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciprofloxacin (MIC ‫؍‬ 0.125 g/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciprofloxacin (MICs ‫؍‬ 2 and 8 g/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efflux was provided by a classical fluorimetric method, which revealed a three-to fourfold decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annuled by addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In mutant BN18/71, a second fluorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efflux pump and suggested that decreased susceptibility to ciprofloxacin due to active efflux probably related to overproduction of this pump could occur before that due to gyrA mutations. Alterations were also found in the outer membrane protein and lipopolysaccharide profiles of the mutants, and these alterations were possibly responsible for the decrease in the permeability of the outer membrane that was observed in the mutants and that could act synergistically with active efflux to decrease the level of ciprofloxacin accumulation.Fluoroquinolones are often the treatment of choice in the cases of life-threatening salmonellosis due to multidrug-resistant strains (4, 27). Salmonella sp. strains that exhibit treatment-compromising resistance to fluoroquinolones are uncommon, but the increasing incidence of strains with decreased susceptibility is a matter of concern (12, 28). In other gramnegative bacteria, such as Escherichia coli, Neisseria gonorrhoeae, or Klebsiella pneumoniae, high-level fluoroquinolone resistance is always associated with the presence of multiple mutations in the quinolone resistance-determining regions (QRDRs) of the genes that code for the intracellular targets of these antibiotics, gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) (2, 7, 11). For Salmonella enterica serovar Typhimurium, however, we showed in a previous study (9) that fluoroquinolone resistance is not well correlated with the presence of such mutations: highly fluoroquinolone-resistant mutants selected in vitro had no mutations in the genes that code for topoisomerase IV, and some had only one mutation in the gyrA gene, whereas E. coli isolates that exhibited the same level of resistance harbored at leas...

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent di erentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA-and also Srcdependent manner. This activation is required for FSHinduced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic e ect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their di erentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite e ects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells. Oncogene (2001) 20, 4696 ± 4709.

A microlarval development test for the detection of anthelmintic resistance in nematodes is described. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis eggs were cultured to third stage larvae in the presence of Earle's balanced salt solution, yeast extract and bacteria in a total volume of 150 microliters. Good dose-response data were obtained with thiabendazole, levamisole, pyrantel tartrate and ivermectin allowing the determination of the 50 per cent lethal concentration and of resistance factors when resistant strains were available. The test was found to be accurate, sensitive, easy to carry out and applicable to the routine detection of resistance.

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