Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Pegaptanib sodium (Macugen; Eyetech Pharmaceuticals/Pfizer) is an RNA aptamer directed against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for pathological ocular neovascularization and vascular permeability. After nearly a decade of preclinical development to optimize and characterize its biological effects, pegaptanib was shown in clinical trials to be effective in treating choroidal neovascularization associated with age-related macular degeneration. Pegaptanib therefore has the notable distinction of being the first aptamer therapeutic approved for use in humans, paving the way for future aptamer applications.

At a dose as low as 1 microgram per kilogram of body weight, lysergic acid diethylamide (LSD) significantly decreased the suppressive effect of electric shock on licking behavior of the rat. Attenuation of punishment was also obtained with mescaline, but neither dimethyltryptamine nor delta9-tetrahydrocannabinol was active in this test. Cyproheptadine and alpha-propyldopacetamide, drugs that interfere with the function of neurons that contain serotonin, have a behavioral effect similar to that of LSD and mescaline, which suggests that the attenuation of punishment produced by these hallucinogens may result from decreased activity of such neurons.

SummaryThe Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signaturetag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

We identified a quinolone resistance locus,flqB, linked to transposon insertion Q1108 andfjis on the SmaI D fragment of the Staphylococcus aureus NCTC 8325 chromosome, the same fragment that contains the norA gene. S. aureus norA cloned fromflqB andflqB+ strains in Escherichia coli differed only in a single nucleotide in the putative promoter region. There was no detectable change in the number of copies of norA on the chromosomes of flqB strains, but they had increased levels of norA transcripts. Cloned norA produced resistance to norfloxacin and other hydrophilic quinolones and reduced norfloxacin accumulation in intact cells that was energy dependent, suggesting active drug efflux as the mechanism of resistance. Drug efflux was studied by measurement of norfloxacin uptake into everted inner membrane vesicles prepared from norA-containing E. coli cells. Vesicles exhibited norfloxacin uptake after the addition of lactate or NADH, and this uptake was abolished by carbonyl cyanide m-chlorophenylhydrazone and nigericin but not valinomycin, indicating that it was linked to the pH gradient across the cell membrane. Norfloxacin uptake into vesicles was also saturable, with an apparent Km of 6 ,zM, a concentration between those that inhibit the growth offlqB and flqB+ S. aureus cells, indicating that drug uptake is mediated by a carrier with a high apparent affinity for norfloxacin. Ciprofloxacin and ofloxacin competitively inhibited norfloxacin uptake into vesicles. Reserpine, which inhibits the multidrug efflux mediated by the bmr gene of Bacilus subtilis, which is similar to norA, abolished norfloxacin uptake into vesicles as well as the norfloxacin resistance of anflqB mutant, suggesting a potential means for circumventing quinolone resistance as a result of drug elux in S. aureus. These fiidings indicate that the chromosomal flqB resistance locus is associated with increased levels of expression of norA and strongly suggest that the NorA protein itself functions as a drug transporter that is coupled to the proton gradient across the cell membrane.Fluoroquinolones such as norfloxacin, ciprofloxacin, and ofloxacin are broad-spectrum, synthetic antimicrobial agents that are widely used for the treatment of bacterial infections. Development of drug resistance in some species, particularly Pseudomonas aeruginosa and Staphylococcus aureus, has, however, limited the utilities of fluoroquinolones in some clinical settings. The genetics and mechanisms of bacterial fluoroquinolone resistance have been studied most extensively in Escherichia coli and P. aeruginosa (9). These drugs act on DNA gyrase, and in E. coli, mutations in the gyrA and gyrB genes encoding the DNA gyrase A and B subunits, respectively, have been shown to cause resistance (6,11,38,40). In addition, mutations in genes that affect the expression of porin outer membrane proteins have been shown to cause resistance and to be associated with reduced levels of drug accumulation in intact cells (10,11). Moreover, in these mutant gram-negative bacteria, reduced ...