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.
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