Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of moxifloxacin on its target enzymes was evaluated by three different approaches: (i) the MICs of moxifloxacin and nine other fluoroquinolones were determined for mutants of Escherichia coli (n = 13) and Staphylococcus aureus (n = 5) carrying different combinations of resistance mutations; (ii) the activity of moxifloxacin on isolated targets was determined as IC50 values for wild-type and mutant type II topoisomerases from E. coli; and (iii) the mutation frequencies were determined for two single-step mutants (MI with a Ser83-->Leu mutation in gyrA and WT-4 with a Ser80-->Ile mutation in parC) and their parent strain (WT). Of the quinolones tested, moxifloxacin was the only one showing an equivalent high activity against both targets. This is reflected by a comparable high susceptibility of the test strains of E. coli and S. aureus and by the IC50 values of moxifloxacin which were 50-90% lower than those of ciprofloxacin, norfloxacin and sparfloxacin for the wild-type and single mutant enzymes of gyrase and topoisomerase IV. However, double mutant GyrA was significantly more sensitive to moxifloxacin than to the other fluoroquinolones tested, while wild-type topoisomerase IV was two-fold more refractory. Mutation rates of WT, MI and WT-4 for ciprofloxacin and moxifloxacin were 5 x 10(-8) vs 4 x 10(-10); <6 x 10(-11) vs <6 x 10(-11); and 2 x 10(-6) vs 5 x 10(-7), respectively. These data indicate an equivalent high inhibitory activity of moxifloxacin on DNA gyrase and topoisomerase IV of E. coli.
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PMID:The effect of moxifloxacin on its target topoisomerases from Escherichia coli and Staphylococcus aureus. 1038 73

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.
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PMID:Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis. 1109 95

The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.
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PMID:Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. 1145 83

Most Aeromonas strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of Aeromonas caviae (n = 10), A. hydrophila (n = 5), and A. sobria (n = 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the gyrA, gyrB, parC, and parE genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of Aeromonas spp. were highly conserved (> or =96.1% identity), despite some genetic polymorphism; they were most closely related to those of Vibrio spp., Pseudomonas spp., and members of the family Enterobacteriaceae (72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-83-->Ile (10 strains) or Ser-83-->Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80-->Ile (three strains) or Ser-80-->Arg change, or at position 84, yielding a Glu-84-->Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double gyrA-parC missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single gyrA mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic Aeromonas spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.
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PMID:Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance. 1179 41

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.
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PMID:Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility. 1185 Feb 46

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 micro g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71-->Val in one, Thr82-->Ile in six, and Ala118-->Thr in one) or in gyrB (six mutations, amino acid changes Asp426-->Asn in five and Arg447-->Leu in one). These changes are similar to those already described in other species except for Asp71-->Val, which is novel, and Ala118-->Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 micro g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.
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PMID:gyrA and gyrB mutations are implicated in cross-resistance to Ciprofloxacin and moxifloxacin in Clostridium difficile. 1238 45

Significant levels of fluoroquinolone resistance were obtained in Campylobacterjejuni isolates after an unique step of selection using enrofloxacin. An Asp90-to-Asn and a Thr86-to-Ile change in the gyrase subunit GyrA were found associated with a low (MIC < or = 8 /microg/ml) or a high (MIC > or = 16 microg/ml) level of resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC > or = 128 microg/ml). Further steps of selection increased the MICs of fluoroquinolones but did not result in a multiple antibiotic resistance phenotype. The Thr86-to-Ile change was found to confer different levels of resistance, pointing out other mechanisms of resistance. However, sequencing revealed no mutation in gyrB, and several attempts did not enable any amplification of the parC gene coding for topoisomerase IV, suggesting an absence of this secondary target in C. jejuni. In addition, no difference in the major outer membrane protein expression was found among the isolates. Furthermore, the use of the recently identified efflux pump inhibitor Phe-Arg-beta-naphthylamide did not result in a significant decrease of fluoroquinolone MICs or change in the frequency of isolation of enrofloxacin-resistant mutants, and thus appears ineffective against fluoroquinolone-resistant C. jejuni isolates. Results obtained during ciprofloxacin accumulation studies confirmed that efflux probably plays a minor role in fluoroquinolone resistance of C. jejuni.
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PMID:Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin. 1252 31

The clinical use of bleomycin is limited by a dose-dependent pulmonary toxicity. Bleomycin is thought to be growth inhibitory by virtue of its ability to oxidatively damage DNA through its complex with iron. Our previous preclinical studies showed that bleomycin-induced pulmonary toxicity can be reduced by pretreatment with the doxorubicin cardioprotective agent dexrazoxane. Dexrazoxane is thought to protect against iron-based oxygen radical damage through the iron chelating ability of its hydrolyzed metabolite ADR-925, an analog of ethylenediaminetetraacetic acid (EDTA). ADR-925 quickly and effectively displaced either ferrous or ferric iron from its complex with bleomycin. This result suggests that dexrazoxane may have the potential to antagonize the iron-dependent growth inhibitory effects of bleomycin. A study was undertaken to determine if dexrazoxane could antagonize bleomycin-mediated cytotoxicity using a CHO-derived cell line (DZR) that was highly resistant to dexrazoxane through a threonine-48 to isoleucine mutation in topoisomerase IIalpha. Dexrazoxane is also a cell growth inhibitor that acts through its ability to inhibit the catalytic activity of topoisomerase II. Thus, the DZR cell line allowed us to examine the cell growth inhibitory effects of bleomycin in the presence of dexrazoxane without the confounding effect of dexrazoxane inhibiting cell growth. The cell growth inhibitory effects of bleomycin were unaffected by pretreating DZR cells with dexrazoxane. These results suggest that dexrazoxane may be clinically used in combination with bleomycin as a pulmonary protective agent without adversely affecting the antitumor activity of bleomycin.
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PMID:The iron chelating cardioprotective prodrug dexrazoxane does not affect the cell growth inhibitory effects of bleomycin. 1552 9

To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.
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PMID:Alterations in DNA gyrase and topoisomerase IV in resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones. 1567 22

The presence of fluoroquinolone resistance-associated alterations in topoisomerase II and IV were investigated for 103 nfxC-like type Pseudomonas aeruginosa isolates. The most nfxC-like type isolates (98.1%) possessed the substitution of Ile for Thr-83 in GyrA. A single alteration in GyrA (Thr-83-->Ile) was the most frequently detected and the next common alteration was two alterations with Thr-83-->Ile in GyrA and Ser-87-->Leu in ParC. A novel alteration at position Glin-106 of GyrA, which was suggested to be responsible for fluoroquinolone resistance, was identified. Our study revealed that the alterations in GyrB (Glu-468-->Asp) and in ParE (Asp-419-->Asn or Glu-459-->Asp) play a complementary role in the acquisition of resistance to fluoroquinolone. There was a correlation between the ciprofloxacin MIC and the number of resistance-associated alterations in GyrA, GyrB, ParC and ParE of P. aeruginosa isolates.
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PMID:Alterations in the GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subunits of topoisomerase IV in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa. 1578 7


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