) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase II. To determine the contribution of the C-8 fluorine to drug potency, we compared the effects of -(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of Drosophila melanogaster topoisomerase II with those of 953 (the 6,955). Removal of the C-8 fluoro group decreased the ability of the quinolone to enhance enzyme-mediated DNA cleavage -2.5-fold. Like its difluorinated counterpart, CP-115,955 increased the levels of cleavage intermediates without impairing the DNA religation reaction of the enzyme. Removal of the C-8 fluorine reduced the ability of the quinolone to inhibit topoisomerase II-catalyzed DNA relaxation. In addition, the cytotoxicity of CP-115,955 towards Chinese hamster ovary cells was decreased compared with that of CP-115,953. These results demonstrate that the C-8 fluorine increases the potency of quinolone derivatives against eukaryotic topoisomerase II and mammalian cells. Further comparisons of 804 (the N-1 ethyl-substituted derivative of the difluoro parent compound) indicate that the two intrinsic activities of quinolone-based drugs towards topoisomerase II (i.e., enhancement of DNA cleavage and inhibition of catalytic strand passage) can be differentially influenced by alteration of ring substituents. Finally, correlations between the biochemical and cytological activities of these drugs suggest that the ability to inhibit catalytic strand passage enhances the cytotoxic potential of quinolones towards eukaryotic cells.Topoisomerase II is an essential enzyme (9,21,23,53) that is required for chromosome structure (5,13,14,16,17), condensation (1, 36, 52, 56), and segregation (9,23,47,54). It also appears to play roles in DNA replication, transcription, and recombination in eukaryotic cells (3,6,8,30,39,43,47,51,55).In addition to its cellular functions, topoisomerase II is the primary target for several classes of antineoplastic drugs (32,48,59). These agents are widely used for the treatment of human cancers (32,48,59) and their clinical efficacies correlate with their abilities to stabilize covalent enzymecleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme (31,32,43,48,59). Previous studies with etoposide (40, 46) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (45, 46) demonstrated that these topoisomerase 1I-targeted drugs stabilize cleavage complexes primarily by inhibiting the ability of the enzyme to religate cleaved DNA.Recent work indicates that the DNA cleavage complex of eukaryotic topoisomerase II is also a target for novel 6,8-difluoroquinolone derivatives (4, 44). While quinolone-based drugs have been developed extensively as antimicrobial agents (targeted to DNA gyrase, the prokaryotic counterpart of topoisomerase II) (12,24,58), these studies provided evidence that quinolones may have potential as antineoplastic drugs. One of the difluoro compounds examined, 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopro...

No abstract

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in woundsonly during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10 , most macrophages in the wound were negative for iNOS. These cells , however , acquired iNOS antigen and activity in culture. Wound fluids , but not normal rat serum , suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound. (Am J Pathol 1999, 154:1097-1104)Previous work described a temporally restricted pattern of activity for two distinct enzymes of L-arginine metabolism, arginase and the inducible form of nitric oxide synthase (iNOS), in healing wounds. 1,2 Arguing from the accumulation of specific L-arginine catabolites in extracellular fluid obtained from experimental wounds and in cultures of whole-wound explants, it was proposed from this laboratory that the expression of iNOS in acute wounds was restricted to the period of polymorphonuclear leukocyte (PMN) infiltration, which encompasses the initial 24 to 72 hours after injury. Results also indicated that macrophage-derived arginase was the preponderant, and probably the only, high-flux enzyme of arginine catabolism in the wounds thereafter. Additional support to the latter conclusion and an enzymatic basis for the virtual disappearance of L-arginine from the extracellular space of late wounds were given by the accumulation of arginase in extracellular fluids obtained from wounds of increasing maturity.1,2 Additional studies identified other wound-associated microenvironmental factors, such as hypoxia, as decidedly preferential in inducing L-arginine metabolism in macrophages through arginase rather than iNOS. 3,4The relevance of the product of iNOS, NO, and its putative derivatives to inflammatory processes in general and more specifically to wound healing has recently been highlighted. 5 It has been proposed, in this regard, that the sustained expression of NOS in healing wounds is critical to the accumulation of collagen and the acquisition of mechanical strength in wounds.5 These results were, interestingly, obtained in part usi...

During a recent clinical trial of ciprofloxacin in the therapy of acute diarrhea, two subjects infected with Campylobacter jejuni who received ciprofloxacin failed microbiologically and one also failed clinically. Although both pretreatment isolates were susceptible to ciprofloxacin, the posttreatment isolates were resistant to ciprofloxacin (MIC = 32 micrograms/ml) and to other quinolones. The posttreatment isolates remained susceptible to nonquinolone antimicrobials. DNA gyrase holoenzyme was isolated from one of the resistant posttreatment isolates and was 8- to 16-fold less sensitive to inhibition by ciprofloxacin than was the gyrase from the paired pretreatment susceptible isolate. Ciprofloxacin accumulation was diminished in the two resistant posttreatment isolates. These results show that mutation in C. jejuni can occur in vivo and is associated with clinically significant resistance to the newer quinolones.