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Buffer 50 mM NaH2PO4-300 mM NaCl-20 mM imidazole [pH 8.0] ; . Four elutions followed, two elutions with 250 mM imidazole and then two elutions with 325 mM imidazole 25 ; . All centrifugations were performed at 700 g for 2 min at 4C. Samples were dialyzed immediately in Slide-A-lyzer 3.5 K Pierce, Rockford, IL ; for 18 h at Tris-HCl pH 7.5 ; . After dialysis, 10% glycerol was added, and the samples were stored at 20C. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, and protein final concentration was determined by using a protein assay kit Bio-Rad ; . DNA gyrase assay. The DNA supercoiling assay was realized in the presence of the relaxed plasmid pBR322 Topogen, Marne la Vallee, France ; with purified E. coli DNA gyrase John Innes Enterprises, Norwich, United Kingdom ; , as recommended by the manufacturer. One unit of DNA gyrase was incubated with 0.5 g of relaxed pBR322 in a reaction volume of 30 l 37C for 30 min in incubation buffer Tris-HCl 35 mM [pH 7.5], 24 mM KCl, 4 mM MgCl2, 2 mM dithiothreitol, 1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, and 0.1 mg ml albumin ; . The different concentrations of ofloxacin tested 0, 0.25, 0.5, 1, and 10 g ml ; were incubated with the reaction mixture for 1 h at 25C and for 30 min at 37C. The resulting topoisomers of pBR322 were resolved by running a 1% agarose gel stained with ethidium bromide for 16 h at 3.5 V cm that was visualized under UV light. In experiments using QnrE. faecalis, the dialyzed protein extract was first preincubated with DNA gyrase for 1 h at 25C, before ofloxacin was added to the reaction mixture. Heterologous expression. To assess the effects of the qnrE. faecalis gene expression in other bacterial species, the recombinant plasmid pORI23: : qnr-cat was introduced into electrocompetent strains E. coli DH10B, S. aureus RN4220, and L. lactis IL-1419. The transformants were selected on medium containing erythromycin, 500 g ml, and chloramphenicol, 20 g ml, for the E. coli DH10B strain and on medium containing erythromycin, 5 g ml, and chloramphenicol, 5 g ml, for the S. aureus RN4220 and L. lactis IL-1419 strains. The MICs of ofloxacin, ciprofloxacin, sparfloxacin, levofloxacin, and moxifloxacin were determined as described above.

Moxifloxacin was clastogenic in the v79 chromosome aberration assay, but it did not induce unscheduled dna synthesis in cultured rat hepatocytes. Ably results from the fact that fluoroquinolones at their Cmax are quickly bactericidal against extracellular bacteria, so that there is not much to gain by the association in this context ; . Finally, like other investigators 13, 30, 31, ; , we suggest that fluoroquinolones may represent a valuable option for the treatment of listeriosis, especially even in difficult situations such as meningitis, because of their marked bactericidal activities. Although the concentrations of fluoroquinolones in cerebrospinal fluid are only 20 to 50% of the concentrations in serum 1 ; , our data show that this level already allows almost a maximal effect both extra- and intracellularly Fig. 5 ; . More data, however, are needed concerning the penetration of moxifloxacin into brain tissues under conditions of inflamed meninges. Nevertheless, the present data open interesting perspectives and set up the conditions for the performance of meaningful trials of moxifloxacin with animals.

Moxifloxacin versus clarithromycin in AECB requiring further antibiotic therapy ; , only applicable to day 14 and days 2835 assessments; and v ; indeterminate clinical evaluation not possible for any reason ; . urinalysis ; were performed on blood and urine samples. Any clinically significant abnormalities were followed up until normalized.
Resistance to moxifloxacin, and two- to eightfold increases in resistance to the other drugs. Thus, none of the drugs tested was a NorA-specific substrate, but moxifloxacin appeared to be a NorB-specific substrate. For norfloxacin the introduction of pQT8 into MT23142 produced a twofold increase in resistance, in contrast to the expected eightfold increase in resistance if the resistance phenotypes were additive. Similar nonadditivity was also seen for the other drugs that were substrates for both NorA and NorB. In contrast, pQT8 produced in both MT23142 and ISP794 the expected fourfold increase in resistance to moxifloxacin, the NorB-specific substrate, suggesting that norB was equivalently expressed from pQT8 in both genetic backgrounds. Thus, overexpression of norB appears to result in a limit in the extent of resistance to NorA substrates but not to the NorB-specific substrate. One possible explanation for this finding is that overexpression of norB limits the overexpression or causes the downregulation of norA, an in and mrv.

348 Effect of Propranolol on heart rate and arrhythmias in freely moving mice and isolated hearts of deltaKPQ-SCN5A LQT3 mice L. Fablitz I , P. Kirchhof 2, M. Emmelich 2, L. Fortm ller 2, G. M nnig 2, B. Riemann 3 , M. Sch fers 3 , G. Breithardt 2, E. Carmeliet 4, P. Carmeliet 4.

I. ABOUT THIS RESEARCH A Human Rights Watch team of three experienced researchers spent seven days in Jenin from April 19, 2002 to April 28, 2002 to research this report. The team interviewed over one hundred residents of Jenin refugee camp, gathering detailed accounts from victims and witnesses and carefully corroborating and cross-checking their accounts with those of others. Human Rights Watch investigators also collected information from other first-hand observers of the events in the Jenin refugee camp, including international aid workers, medical workers, and local officials. The research also included information from public sources, including Israeli governmental sources, about the incursion. However, the IDF has not agreed to Human Rights Watch's repeated requests for information about its military incursions into the West Bank and Gaza Strip. Although Human Rights Watch's research has been extensive, we do not pretend that it is comprehensive. Further inquiry is still in order, particularly as the excavation process proceeds, and if Israel ultimately decides to make its soldiers involved in the operation available for interview. II. SUMMARY On April 3, 2002, the Israeli Defense Forces IDF ; launched a major military operation in the Jenin refugee camp, home to some fourteen thousand Palestinians, the overwhelming majority of them civilians. The Israelis' expressed aim was to capture or kill Palestinian militants responsible for suicide bombings and other attacks that have killed more than seventy Israeli and other civilians since March 2002. The IDF military incursion into the Jenin refugee camp was carried out on an unprecedented scale compared to other military operations mounted by the IDF since the current Israeli-Palestinian conflict began in September 2000. The presence of armed Palestinian militants inside Jenin refugee camp, and the preparations made by those armed Palestinian militants in anticipation of the IDF incursion, does not detract from the IDF's obligation under international humanitarian law to take all feasible precautions to avoid harm to civilians. Israel also has a legal duty to ensure that its attacks on legitimate military targets did not cause disproportionate harm to civilians. Unfortunately, these obligations were not met. Human Rights Watch's research demonstrates that, during their incursion into the Jenin refugee camp, Israeli forces committed serious violations of international humanitarian law, some amounting prima facie to war crimes. Due to the dense urban setting of the refugee camp, fighters and civilians were never at great distances. Civilian residents of the camp described days of sustained missile fire from helicopters hitting their houses. Some residents were forced to flee from house to house seeking shelter, while others were trapped by the fighting, unable to escape to safety, and were threatened by a curfew that the IDF enforced with lethal force, using sniper fire. Human Rights Watch documented instances in which soldiers converted civilian houses into military positions, and confined the inhabitants to a single room. In other instances, civilians who attempted to flee were expressly told by IDF soldiers that they should return to their homes. Despite these close quarters, the IDF had a legal duty to distinguish civilians from military targets. At times, however, IDF military attacks were indiscriminate, failing to make this distinction. Firing was particularly indiscriminate on the morning of April 6, when missiles were launched from helicopters, catching many sleeping civilians unaware. One woman was killed by helicopter fire during that attack; a four-year-old child in another part of the town was injured when a missile hit the house where she was sleeping. Both were buildings housing only civilians, with no fighters in the immediate vicinity. The IDF used armored bulldozers to demolish residents' homes. The apparent purpose was to clear paths through Jenin's narrow and winding alleys to enable their tanks and other heavy weaponry to penetrate the camp interior, particularly since some of these had evidently been booby-trapped. However, particularly in the Hawashin district, the destruction extended well beyond any conceivable purpose of gaining access to fighters, and was vastly disproportionate to the military objectives pursued. The damage to Jenin camp by missile and tank Human Rights Watch 3 May 2002, Vol. 14, No. 3 E.

Fluoroquinolone resistance is beginning to appear among isolates of Streptococcus pneumoniae 4, 7, 8, ; . We have argued that resistance arises as a consequence of dosing that places tissue concentrations between the MIC and the mutant prevention concentration MPC ; , a new measure of activity related to the MIC of the least susceptible, single-step mutant 15, 16 ; . If this is true, MPC can be used to identify fluoroquinolones that are least likely to selectively enrich resistant subpopulations. We previously estimated MPC for several fluoroquinolones with about 100 clinical isolates of S. pneumoniae obtained from the Royal University Hospital, Saskatoon, Canada 2 ; . We now add gemifloxacin to the list of compounds compared and increase the number of isolates tested to 146 for all of the compounds. Table 1 lists MICs and MPCs for gemifloxacin, moxifloxacin, gatifloxacin, and levofloxacin determined as described previously 2 ; using the same set of isolates for each compound. Fluoroquinolone-resistant isolates were excluded. Gemifloxacin had the lowest modal MPC 0.25 g ml ; , followed by moxifloxacin 0.5 g ml ; , gatifloxacin 1 g ml ; , and levofloxacin 2 g ml ; The same rank order was observed when MPC was determined for 90% of the isolates. These data are consistent with gemifloxacin having more activity than the other compounds against resistant mutants 9, 14 ; . When the MIC at which 90% of the susceptible isolates are inhibited MIC90 ; was determined, gemifloxacin was also more active than moxifloxacin, gatifloxacin, and levofloxacin in these comparisons by 2, 3, and 4 dilutions, respectively. Since the effectiveness of an antibacterial agent is likely to be a function of both activity MIC and MPC ; and pathogen exposure 5, 11 ; , comparision of compounds requires consideration of drug pharmacokinetics in human tissues. From published values of concentrations in serum, we calculated the time above MPC for each compound when dosed as recommended by the manufacturer. Moxifloxacin is expected to have a concentration in serum above the MPC at which 90% of the isolates tested are prevented MPC90 ; for 18 h. For gemifloxacin, gatifloxacin, and levofloxacin, those times are 4, 1 to 2, and 0 h, respectively. This suggests that moxifloxacin may be the most effective at restricting the development of resistance, even though gemifloxacin has the lowest MIC and MPC. Table 1 also lists values of the area under the concentrationtime curve from 0 to 24 MIC and the maximum concentration of drug in serum Cmax ; MIC for recommended doses. For both parameters gemifloxacin exhibits higher values than moxifloxacin. If these two parameters are inversely related to the selection of resistant mutants 1, 6, 13 ; , resistance should develop less often from treatment with gemifloxacin than with moxifloxacin. But time above MPC Table 1 ; and low-concentration cycling 12 ; predict the opposite outcome. A clinical comparison of these two compounds may help distinguish between MPC-based ideas 15 ; and empirical pharmacodynamics 6, 13 ; for predicting the development of resistance. Such a comparison is important because neither method can be easily tested: MPC is an in vitro measure that does not take into account compartments in patients where drug concentrations and bacterial growth properties are poorly defined, and pharmacodynamic methods require examining very large numbers of patients to identify the point at which the overall prevalence of resistance does not increase.

Topical antibiotics are more commonly used. A combination of systemic and topical antibiotics is used to treat Neisseria infections. Eye drops are usually preferred over ointments for bacterial conjunctivitis. Some common eye drops are trimethropim-polymyxin B Polytrim ; and the fluoroquinolones ofloxacin Ocuflox, Allergan ; , ciprofloxacin Ciloxan ; , levofloxacin Quixin, Santen ; , moxifloxacin Vigamox, Alcon ; , and gatifloxacin Zymar, Allergan and mycostatin. MATERIALS AND METHODS Bacterial strains and growth conditions. Strains were routinely grown at 37C in tryptone soy TS ; broth or agar Bio-Rad, Marnes-la-Coquette, France ; , except when noted. TS agar medium was supplemented with 10% horse blood for Lactococcus lactis. Media for the selection of transformants contained kanamycin 20 and 500 g ml ; and or erythromycin 5 and 500 g ml ; and or chloramphenicol 5 and 20 g ml ; faecalis JH2-2, E. coli DH10B and BL21 DE3 ; , L. lactis IL-1419, and S. aureus RN4220 were used as recipient strains in transformation experiments. Antimicrobial susceptibility. MICs of fluoroquinolones for the different strains were determined in three independent experiments by the broth microdilution method in Mueller-Hinton broth Bio-Rad ; , as recommended by the CA-SFM : sfm.asso ; last release, January 2007 ; . For E. faecalis JH2-2, the dilutions tested were 1, 1.5, 2, and 128 g ml for ofloxacin and 0.5, 0.75, 1, and 64 g ml for ciprofloxacin. For heterologous expression experiments, MICs of ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, and moxifloxacin were determined by a standard twofold dilution method. PCR conditions. Oligonucleotide primers used in this study are listed in Table 1. The PCR consisted of 30 cycles of denaturation 94C, 30 s ; , annealing 50C, 30 s ; , and extension 72C, 30 s to 1 min ; . E. faecalis JH2-2 DNA was extracted by an Instagen Matrix kit Bio-Rad ; as recommended by the manufacturer. Identification of a homologue of the qnr gene in E. faecalis JH2-2. A BLAST analysis of the genomic sequence of E. faecalis V583 was performed : www .ncbi.nih.gov BLAST; accession number AE016830 ; . A homologue of the qnr gene was identified at the locus EF0905 and named E. faecalis qnr qnrE. faecalis ; . This gene was amplified with specific primers qnr-1F and qnr-1R Table 1 ; from E. faecalis JH2-2 DNA and sequenced Ceq 8000; Beckmann Coulter, Villepinte, France ; for comparison with the E. faecalis V583 qnrE. faecalis gene. Inactivation of qnrE. faecalis. A fragment internal to the qnrE. faecalis gene was amplified from E. faecalis JH2-2 DNA with specific primers qnr-2F and qnr-2R.

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