) 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...

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.

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Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gelsDeposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5-mm-thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2-mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris-glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer-assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm3 in 10 %-20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R-250 staining of protein (10 nanograms), this method for silver staining is at least 370-fold more sensitive.

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High-level resistance to quinolones has previously been shown to occur in Campylobacter spp. both in vitro and in patients treated with quinolones. We have selected isolates that are resistant to quinolones by plating cells from a susceptible C. jejuni strain, UA535, on medium containing nalidixic acid at 32 ,ug/ml. Fluctuation analysis indicated that resistance occurred by mutation at a frequency of 5 x 10-8 per cell plated. Unlike what is observed with other gram-negative organisms, the nalidixic acid-resistant mutants demonstrated high-level cross-resistance (MIC, .4 ,ug/ml) to newer quinolones, including ciprofloxacin, norfloxacin, and temafloxacin, yet remained susceptible to coumermycin Al and several other unrelated antibiotics. Mutants with an identical resistance phenotype could also be selected from UA535 with ciprofloxacin and norfloxacin at a similar frequency. To study the mechanism of quinolone resistance, DNA gyrases were purified from C. jejuni UA535 and two resistant mutants by heparin-agarose and novobiocin-Sepharose chromatography. After the respective enzyme concentrations were adjusted to equivalent units of activity in the DNA supercoiling reaction, the DNA gyrases from the resistant mutants were found to be 100-fold less susceptible than the wild-type enzyme to inhibition by quinolones. Subunit switching experiments with purified A and B subunits from the wild type and one of the quinolone-resistant mutants indicated that an alteration in the A subunit was responsible for resistance. These results show that a single-step mutation can occur in vitro in the gene encoding DNA gyrase in C. jejuni, producing clinically relevant levels of resistance to the newer quinolones.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel ␥-secretase inhibitor that reduces amyloid-␤ (A␤) production with an in vitro IC 50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T-and B-cell maturation in an in vitro thymocyte assay with an EC 50 of 2.1 M. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma A␤ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma A␤. To further characterize A␤ dynamics in brain, CSF, and plasma in relation to drug exposure and Notchrelated toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in A␤, and the magnitude and duration of A␤ lowering exceeded those of the reductions in B-cell endpoints. Other ␥-secretase inhibitors have shown high potency at elevating A␤ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in A␤11-40 and A␤1-43 at doses that potently inhibited A␤1-40 and A␤1-42. PF-3084014, like previously described ␥-secretase inhibitors, preferentially reduced A␤1-40 relative to A␤1-42. Potency at A␤ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.Amyloid-␤ (A␤) peptide is the primary component of senile plaques (Glenner and Wong, 1984) and is the protein product of a gene [amyloid precursor protein (APP)] whose mutation can result in early-onset Alzheimer's disease. The intersection of this genetic and pathologic evidence has led to a strong focus on A␤ as a major culprit in the etiology of Alzheimer's disease. A number of compounds have advanced to the clinic with the goal of either reducing production of this peptide (e.g., ␤-or ␥-secretase inhibitors) or increasing its clearance from the brain (e.g., A␤ vaccines or monoclonal antibodies). Of these approaches, ␥-secretase has yielded the greatest diversity of chemical tools that enable the study of A␤ pharmacodynamics in animal models and humans. Bioavailable small-molecule inhibitors of ␥-secretase from various chemical series have been shown to rapidly reduce A␤ levels in brain, cerebrospinal fluid (CSF), and plasma from wild-type mice (Yohrling et al., 2007), rats (Best et al., 2005;El Mouedden et al., 2006;Lanz and Schachter, 2006), guinea pigs (Anderson et al., 2005;, and multiple muArticle, publication date, and citation information can be found at