Nalidixic acid, enoxacin, and other antibacterial 4-quinolones inhibit DNA gyrase activity by interrupting DNA breakage and reunion by A subunits of the A2B2 gyrase complex. Despite their clinical importance, the mode of quinolone action and mechanisms of resistance are poorly understood at the molecular level. Using a DNA fragment enrichment procedure, we isolated the gyrA gene from a uropathogenic Escherichia coli strain that encodes a gyrase A protein cross-resistant to a variety of quinolones. When complemented with gyrase B subunit, the purified A protein reconstituted DNA supercoiling activity 100-fold more resistant to inhibition by enoxacin than the susceptible enzyme and failed to mediate quinolone-dependent DNA cleavage. Nucleotide sequence analysis revealed that the gene differed at 58 nucleotide positions compared with the K-12 gyrA sequence. The 875-amino-acid residue-resistant gyrase A protein differed at three positions from its wild-type E. coli K-12 counterpart: tryptophan, glutamate, and serine replaced serine, aspartate, and alanine residues at positions 83, 678, and 828, respectively. By genetic analysis of chimeric gyrA genes in a gyrA(Ts) background, we showed that the Ser-83-*Trp mutation in the gyrase A protein was solely responsible for high-level bacterial resistance to nalidixic acid and fluoroquinolones.Bacterial DNA is maintained in a negatively supercoiled state by DNA gyrase, an ATP-dependent type II DNA topoisomerase (10). Gyrase is essential for cell viability, being implicated in a range of DNA transactions, including DNA replication and recombination, and in the control of gene expression (8,35). Gyrase catalyzes DNA supercoiling by an interesting and unusual mechanism. It passes a duplex DNA segment through a transient double-stranded DNA break made within a 120-to 150-base-pair (bp) loop of DNA wrapped on the surface of the tetrameric A2B2 gyrase complex. DNA strand passage is the salient mechanistic feature that allows DNA supercoiling and ATP-independent DNA relaxation by gyrase and also accounts for its catenation or decatenation and DNA unknotting activities (3,16,18).Antibacterial quinolones such as oxolinic acid inhibit DNA supercoiling by gyrase in vitro and rapidly arrest DNA replication in vivo (2, 9, 12, 28). Addition of detergent to gyrase-DNA complexes formed in the presence of oxolinic acid results in site-specific double-stranded DNA breakage and covalent attachment of the gyrase A subunits to each 5'-phosphate end via tyrosine 7,9,15,22,31). Thus, the A subunits appear to promote DNA breakage and reunion during catalysis, a process interrupted by quinolone inhibitors. In contrast, the B subunits bind ATP and are the locus of action of coumarin antibiotics such as novobiocin (11,20,30). The A and B subunits can be individually purified but must be combined to generate the topoisomerase activities of gyrase (14, 19

Background-Combination therapy consisting of mechanical unloading using a left ventricular assist device (LVAD) and pharmacological intervention can promote recovery from end-stage heart failure, but the mechanism is unknown. Preliminary microarray analysis revealed a significant and unexpected decrease in myocardial arginine:glycine amidinotransferase (AGAT) gene expression during recovery in these patients. The aim of this study was to evaluate the expression and role of AGAT expression in heart failure and recovery. Methods and Results-We used quantitative real time (TaqMan) polymerase chain reaction to examine myocardial AGAT mRNA expression in implant and explant samples from recovering patients after combination therapy (nϭ12), end-stage heart failure (ESHF) samples from stable patients undergoing transplantation without LVAD support (nϭ10), and donor hearts with normal hemodynamic function (nϭ8). AGAT mRNA expression was significantly elevated in all heart failure patients relative to donors (4.3-fold [PϽ0.001] and 2.7-fold [PϽ0.005] in LVAD and ESHF relative to donors, respectively) and returned to normal levels after recovery. AGAT enzyme activity was detectable in both human and rat myocardia and was elevated in heart failure. Conclusions-Our data highlight local and potentially regulated expression of AGAT activity in the myocardium and suggest a specific response to heart failure involving elevated local creatine synthesis. These findings have implications both for the management of recovery patients undergoing combination therapy and for heart failure in general.

This data set represents the first description of signalling pathways associated with the functional recovery of end-stage human heart failure and the identification of new targets in the human heart that are modified by this combination therapy.

DNA gyrase catalyses DNA supercoiling by making a transient double‐stranded DNA break within its 120‐150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme‐DNA intermediate inducing sequence‐specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site‐directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.

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Compared with studies worldwide, the relative proportions of different organisms causing otitis externa and the patterns of antimicrobial resistance differ. Increasing resistance of P aeruginosa to aminoglycosides demonstrates a changing pattern of antimicrobial resistance that has not been previously reported. Reassuringly, quinolone antibiotics remain highly effective when treating P aeruginosa.

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The β(2)-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.