scholarly journals Use of a Rapid Throughput In Vivo Screen To Investigate Inhibitors of Eukaryotic Topoisomerase II Enzymes

1998 ◽  
Vol 42 (4) ◽  
pp. 889-894 ◽  
Author(s):  
Timothy R. Hammonds ◽  
Anthony Maxwell ◽  
John R. Jenkins
Keyword(s):  
Topoisomerase Ii ◽  
Antitumor Agents ◽  
Viable Cell ◽  
Cytotoxic Agents ◽  
New Method ◽  
Temperature Sensitive ◽  
Cell Counts ◽  
Similar Drug ◽  
In Vivo Screen

ABSTRACT Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIα or IIβ or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

scholarly journals Efficacies of piperacillin-tazobactam and cefepime in rats with experimental intra-abdominal abscesses due to an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae.

1997 ◽  
Vol 41 (5) ◽  
pp. 1053-1057 ◽  
Author(s):  
C Thauvin-Eliopoulos ◽  
M F Tripodi ◽  
R C Moellering ◽  
G M Eliopoulos
Keyword(s):  
Klebsiella Pneumoniae ◽  
Antimicrobial Agents ◽  
Total Duration ◽  
Viable Cell ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Abdominal Abscesses ◽  
Cell Counts ◽  
Extended Spectrum

The in vivo activities of piperacillin-tazobactam and cefepime were compared with those of ticarcillin-clavulanate, ceftazidime, cefotaxime, and imipenem in a rat model of intra-abdominal abscess with a strain of Klebsiella pneumoniae elaborating an extended-spectrum beta-lactamase (TEM-26). With the exception of ceftazidime, all of the antimicrobial agents significantly reduced bacterial counts within abscesses at the end of therapy compared with those in untreated controls. Residual viable cell counts (mean +/- standard deviation in log10 CFU/gram) were as follows: control, 8.76 +/- 0.97; ceftazidime, 8.00 +/- 0.76; piperacillin-tazobactam, 3.87 +/- 1.72; ticarcillin-clavulanate, 3.74 +/- 1.34; cefepime, 3.15 +/- 1.19; cefotaxime, 2.61 +/- 0.77; imipenem, 2.41 +/- 0.93. Imipenem was more effective than either of the inhibitor combinations (P < 0.05). Cefotaxime was unexpectedly effective given its poor in vivo activity against this organism in our earlier studies, which used a different dose and total duration of therapy (L. B. Rice, J. D. C. Yao, K. Klimm, G. M. Eliopoulos, and R. C. Moellering, Jr., Antimicrob. Agents Chemother. 35:1243-1244, 1991). These observations suggest that the effectiveness of cephalosporins in the treatment of experimental infections caused by extended-spectrum beta-lactamase-producing K. pneumoniae may be highly dependent on dosing regimens, even for a specific organism and site of infection.

scholarly journals In Vivo Bypass of Chaperone by Extended Coiled-Coil Motif in T4 Tail Fiber

2004 ◽  
Vol 186 (24) ◽  
pp. 8363-8369 ◽  
Author(s):  
Yun Qu ◽  
Paul Hyman ◽  
Timothy Harrah ◽  
Edward Goldberg
Keyword(s):  
Bacteriophage T4 ◽  
Coiled Coil ◽  
Critical Factor ◽  
Coiled Coils ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Type Gene ◽  
In Vivo Screen

ABSTRACT The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30°C but not at 42°C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50°C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for “clamps” to initiate the folding of trimeric β-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.

scholarly journals Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent Escherichia coli in the Neutropenic Mouse Thigh Model of Infection

2001 ◽  
Vol 45 (1) ◽  
pp. 129-137 ◽  
Author(s):  
H. L. Rocchetta ◽  
C. J. Boylan ◽  
J. W. Foley ◽  
P. W. Iversen ◽  
D. L. LeTourneau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Antimicrobial Agents ◽  
Viable Cell ◽  
Multicopy Plasmid ◽  
In Vivo Evaluation ◽  
Therapeutic Success ◽  
Cell Counts

ABSTRACT A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. Thelux gene cluster of Photorhabdus luminescenswas introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

scholarly journals Vancomycin Acts Synergistically with Gentamicin against Penicillin-Resistant Pneumococci by Increasing the Intracellular Penetration of Gentamicin

2003 ◽  
Vol 47 (1) ◽  
pp. 144-147 ◽  
Author(s):  
P. Cottagnoud ◽  
M. Cottagnoud ◽  
M. G. Täuber
Keyword(s):  
Viable Cell ◽  
Underlying Mechanism ◽  
Intracellular Concentration ◽  
Combination Regimen ◽  
Cell Counts ◽  
Short Period ◽  
Before And After ◽  
Gentamicin Concentration

ABSTRACT Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.

scholarly journals Cellular Responses to Postsegregational Killing by Restriction-Modification Genes

2000 ◽  
Vol 182 (8) ◽  
pp. 2218-2229 ◽  
Author(s):  
Naofumi Handa ◽  
Asao Ichige ◽  
Kohji Kusano ◽  
Ichizo Kobayashi
Keyword(s):  
Viable Cell ◽  
Underlying Mechanism ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Gene Complex ◽  
Cellular Processes ◽  
Cell Counts ◽  
Gene Complexes ◽  
Restriction Modification

ABSTRACT Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in anEscherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC,recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted “nonself” DNA and repair restricted “self” DNA.

scholarly journals Antifungal Activities of R-135853, a Sordarin Derivative, in Experimental Candidiasis in Mice

2005 ◽  
Vol 49 (1) ◽  
pp. 52-56 ◽  
Author(s):  
Yasuki Kamai ◽  
Masayo Kakuta ◽  
Takahiro Shibayama ◽  
Takashi Fukuoka ◽  
Shogo Kuwahara
Keyword(s):  
Subcutaneous Administration ◽  
Viable Cell ◽  
Cell Counts ◽  
Single Oral Administration ◽  
Fluconazole Resistant ◽  
Resistant Strains ◽  
Mucosal Candidiasis ◽  
Dose Dependent

ABSTRACT The activities of R-135853, a novel sordarin derivative that possesses a 1,4-oxazepane ring moiety, were evaluated in vitro and in vivo. R-135853 exhibited potent in vitro activities against Candida albicans (fluconazole-susceptible strains), Candida glabrata, Candida tropicalis, and Cryptococcus neoformans, with MICs at which 90% of isolates were inhibited of 0.03, 1, 0.5, and 0.5 μg/ml, respectively. R-135853 also exhibited potent activities against fluconazole-susceptible dose-dependent and fluconazole-resistant strains of C. albicans, with MICs ranging from 0.03 to 0.06 μg/ml. However, R-135853 exhibited weak or no activity against Candida parapsilosis, Candida krusei, and Aspergillus spp. R-135853 exhibited dose-dependent efficacy against experimental murine hematogenous candidiasis induced by C. albicans when it was administered by both the subcutaneous and the oral routes and reduced viable cell counts in the kidneys significantly when it was administered at 50 mg/kg of body weight/dose (administration three times a day). In this model, R-135853 also exhibited dose-dependent efficacy by single oral administration. Subcutaneous administration of R-135853 exhibited dose-dependent efficacy against experimental murine esophageal candidiasis induced by fluconazole-resistant C. albicans, against which fluconazole at 50 mg/kg/dose was ineffective, and reduced viable cell counts in the esophagus significantly when it was administered at 10 and 50 mg/kg/dose. R-135853 eradicated C. albicans from the esophagi of one and four of five mice when it was administered at 10 and 50 mg/kg/dose, respectively. These results suggest that R-135853 is promising for the treatment of disseminated or mucosal candidiasis, including fluconazole-refractory infections.

scholarly journals Antineoplastic and Cytotoxic Activities of Nickel(II) Complexes of Thiosemicarbazones

1997 ◽  
Vol 4 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Iris H. Hall ◽  
Merrill C. Miller ◽  
Douglas X. West
Keyword(s):  
Topoisomerase Ii ◽  
Ehrlich Ascites Carcinoma ◽  
Cytotoxic Agents ◽  
Cytotoxic Activities ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Ehrlich Ascites ◽  
Dna Strand ◽  
Cultured Tumor Cells

Nickel(II) complexes of thiosemicarbazons were observed to be potent cytotoxic agents in human and rodent tissue cultured tumor cells. Each compound demonstrated a slightly different profile in the various histological types of tumors. The nickel complex of Appip demonstrated the most potent in vivo activity in the Ehrlich ascites carcinoma. This agent selectively inhibited L1210 DNA and purine syntheses, and DNA polymerase α, PRPP-amido transferase, IMP-dehydrogenase, dihydrofolate reductase, TMP-kinase and thymidylate synthetase activities. L1210 DNA strand scission was evident and DNA viscosity was reduced after 24 hr incubation. The nickel complexes were not L1210 DNA topoisomerase II inhibitors.

scholarly journals Indolo[2,3-b]quinoline derivatives as novel promising antitumor agents

2016 ◽  
Author(s):  
Lukasz Kaczmarek ◽  
Katarzyna Badowska-Rosłonek ◽  
Anna Jaromin ◽  
Wojciech Łuniewski ◽  
Wanda Peczyńska-Czoch ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Topoisomerase Ii ◽  
Antitumor Agents ◽  
Aqueous Media ◽  
Human Tumors ◽  
Quinoline Derivatives ◽  
Dna Topoisomerase ◽  
Cryptolepis Sanguinolenta

In the search for novel antineoplastic compounds we have found that four-membered, linear 5,11-dimethyl -5H- indolo[2,3-b]quinoline (DIMIQ) reveals cytostatic activity in vitro and moderate antitumor activity in vivo in mice melanoma B16 as well as leukemias L1210 and P388. Preliminary studies showed that DIMIQ stabilizes DNA-topoisomerase II complex. Some years later indolo[2,3-b]quinoline was found as an alkaloid neocryptolepine in the West African shrub Cryptolepis sanguinolenta. Unfortunately, DIMIQ’s high toxicity, lack of selectivity and very low solubility in aqueous media seriously limit the practical use of this compound as an anticancer drug. Extensive research on the structure-activity relationships of different indolo[2,3-b]quinoline derivatives showed that the position and type of the substituent is conclusive both to the antitumor activity and general toxicity of the compound. These studies led us to discover derivatives of DIMIQ which exhibit very low toxicity against normal cells and are highly toxic against selected human tumors. These derivatives are soluble in water and some of them are able to overcome multidrug resistance in human tumors cells.

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