bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Streptomyces peucetius genes for doxorubicin resistance and aklavinone 11-hydroxylase in Streptomyces galilaeus ATCC 31133 and production of a hybrid aclacinomycin
Authors: Hwang C K , Kim H S , Hong Y S , Kim Y H , Hong S K , Kim S J , Lee J J
Published: June 2012

The aklavinone 11-hydroxylase gene and two doxorubicin resistance genes cloned from Streptomyces peucetius subsp. caesius ATCC 27952 were introduced into doxorubicin-sensitive Streptomyces galilaeus ATCC 31133, an aclacinomycin producer. The doxorubicin resistance genes drrA and drrB endowed S. galilaeus with high-level resistance to doxorubicin, indicating that the resistance mechanism for doxorubicin might be different from that for aclacinomycin A. Transformation of S. galilaeus ATCC 31133 with plasmid pMC213 containing the aklavinone 11-hydroxylase gene (dnrF) resulted in the production of many red pigments. A new metabolite was purified, and the position of the newly introduced hydroxyl group was determined. This result indicated that the aklavinone 11-hydroxylase gene was stably expressed in S. galilaeus ATCC 31133 and that it gave rise to a hybrid aclacinomycin A which showed highly specific in vitro cytotoxicity against leukemia and melanoma cell lines.
Analysis of mutations at position 184 in reverse transcriptase of human immunodeficiency virus type 1
Authors: Boyer P L , Hughes S H
Published: June 2012

We have analyzed recombinant human immunodeficiency virus type 1 reverse transcriptases that contain mutations at position 184. These variants retain high levels of RNA-dependent DNA polymerase activity and show resistance to ddITP. However, the mutants varied in their ability to polymerize processively. The variants Met184Ile and Met184Val showed slight reductions in processivity relative to that of the wild-type enzyme; the variants Met184Ala and Met184Leu showed considerable reductions in their processivity.
Prospective randomized trial of efficacy of ganciclovir versus that of anti-cytomegalovirus (CMV) immunoglobulin to prevent CMV disease in CMV-seropositive heart transplant recipients treated with OKT3
Authors: Aguado J M , Gomez-Sanchez M A , Lumbreras C , Delgado J , Lizasoain M , Otero J R , Rufilanchas J J , Noriega A R
Published: June 2012

We compared the efficacy of ganciclovir versus that of cytomegalovirus (CMV) immunoglobulin for the prevention of CMV disease in 31 CMV-seropositive heart transplant recipients who had received early immunoprophylaxis with OKT3 monoclonal antibodies. The incidence of CMV disease and visceral involvement was much higher in the CMV immunoglobulin group than in the ganciclovir group (40 versus 6%, respectively; P = 0.03). No adverse effects were found in the CMV immunoglobulin group, but 19% of the patients in the ganciclovir group developed mild leukopenia or a mild increase in their serum creatinine levels.
Effects of an acidic beverage (Coca-Cola) on absorption of ketoconazole
Authors: Chin T W , Loeb M , Fong I W
Published: June 2012

Absorption of ketoconazole is impaired in patients with achlorhydria. The purpose of this study was to determine the effectiveness of a palatable acidic beverage (Coca-Cola Classic, pH 2.5) in improving the absorption of ketoconazole in the presence of drug-induced achlorhydria. A prospective, randomized, three-way crossover design with a 1-week wash-out period between each treatment was employed. Nine healthy nonsmoking, nonobese volunteers between 22 and 41 years old were studied. Each subject was randomized to receive three treatments: (A) ketoconazole 200-mg tablet with water (control), (B) omeprazole (60 mg) followed by ketoconazole (200 mg) taken with water, and (C) omeprazole (60 mg) followed by ketoconazole (200 mg) taken with 240 ml of Coca-Cola Classic. The pH values of gastric aspirates were checked after omeprazole was administered to confirm attainment of a pH of > 6. Multiple serum samples were obtained for measurements of ketoconazole concentrations by high-pressure liquid chromatography. The mean area under the ketoconazole concentration-time curve from zero to infinity for the control treatment (17.9 +/- 13.1 mg.h/liter) was significantly greater than that for treatment B (3.5 +/- 5.1 mg.h/liter; 16.6% +/- 15.0% of control). The mean peak concentration was highest for the control treatment (4.1 +/- 1.9 micrograms/ml), for which the mean peak concentration showed a significant increase over that for treatment B. The absorption of ketoconazole was reduced in the presence of omeprazole-induced achlorhydria. However, drug absorption was significantly increased, to approximately 65% of the mean for the control treatment, when the drug was taken with an acidic beverage, such as Coca-Cola.
Role of extracellular calcium in in vitro uptake and intraphagocytic location of macrolides
Authors: Mtairag E M , Abdelghaffar H , Douhet C , Labro M T
Published: June 2012

We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism.
Effect of clarithromycin on sputum production and its rheological properties in chronic respiratory tract infections
Authors: Tamaoki J , Takeyama K , Tagaya E , Konno K
Published: June 2012

Macrolide antibiotics possess a variety of actions other than antimicrobial activities. To determine the effects of long-term administration of clarithromycin (CAM) on the amount and physical properties of sputum in patients with clinical conditions associated with excessive airway secretions, we conducted the present study in a parallel, double-blind, placebo-controlled fashion. Patients were divided into two groups: the first group (n = 16) received CAM (100 mg, twice a day) for 8 weeks, and the second group (n = 15) received placebo. In evaluating airway secretion, the daily amount of expectorated sputum, solid composition, viscoelastic properties (including elastic modulus and dynamic viscosity), and sputum microbiology were assessed. CAM decreased sputum production from 51 +/- 6 to 24 +/- 3 g/day after treatment, whereas placebo had no effect. The bacterial density and sputum flora were unaltered. In the group receiving CAM, the percent solid composition and elastic modulus increased from 2.44% +/- 0.29% to 3.01% +/- 0.20% and 66 +/- 7 to 87 +/- 8 dyne/cm2 (P < 0.05), respectively, but the dynamic viscosity remained unchanged. These results suggest that long-term treatment with CAM reduces the amount of sputum production, probably by inhibiting airway secretions, and increases sputum elasticity.
Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors
Authors: Tisdale M , Myers R E , Maschera B , Parry N R , Oliver N M , Blair E D
Published: June 2012

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.
Partial characterization and effect of omeprazole on ATPase activity in Helicobacter pylori by using permeabilized cells
Authors: Belli W A , Fryklund J
Published: June 2012

ATPase activity in permeabilized cells of Helicobacter pylori as well as those of Helicobacter felis and Campylobacter jejuni was analyzed. The ATPase activities in these cells were most susceptible to sodium azide, fluoroaluminate, and dicyclohexylcarbodiimide, which are typical inhibitors of F ATPases. Optimal values for maximal activity were found to be at approximately pH 6.4, 6.0, and 6.0 for C. jejuni, H. pylori, and H. felis, respectively. The substituted benzimidazole compounds omeprazole, lansoprazole, and Eisai 3810 were found to have no effect on the F ATPase activity of H. pylori at concentrations which are inhibitory for cell growth (MICs). In addition, an extracellular, vanadate-susceptible ATPase activity was detected in H. pylori, which was also relatively insusceptible to the benzimidazole compounds. Thus, the mechanism of killing mediated by omeprazole and related compounds in Helicobacter pylori does not appear to be due to diminished ATPase activity.
Antagonistic interactions between azoles and amphotericin B with yeasts depend on azole lipophilia for special test conditions in vitro
Authors: Scheven M , Schwegler F
Published: June 2012

The interactions of the azole antifungal agents fluconazole, itraconazole, ketoconazole, or miconazole with amphotericin B (AmB) in their effect on Candida albicans were investigated. These four azoles antagonized the fungistatic activity of AmB at sub-MICs if both substances acted simultaneously. This coincubation test was primarily developed to observe the azole-mediated demethylase inhibition quantitatively by bioassay. Interestingly, the occurrence of azole-AmB antagonism depended on azole lipophilia if specially selected test conditions were applied. By a consecutive incubation regimen, preincubation at high azole concentrations (1 to 50 micrograms/ml) and then subsequent incubation with AmB (1 microgram/ml), only preincubation with the three lipophilic azoles decreased the fungicidal activity of AmB but not that of FCZ. It was shown that yeasts absorb only lipophilic azoles to a remarkable extent. This fact might be responsible for the absence of antagonism of FCZ to AmB when yeasts were incubated consecutively. It can be concluded with caution that consecutive treatment of candidiasis with FCZ and AmB does not necessarily result in a clinically relevant antagonism.
Aerosol and parenteral pneumocandins are effective in a rat model of pulmonary aspergillosis
Authors: Kurtz M B , Bernard E M , Edwards F F , Marrinan J A , Dropinski J , Douglas C M , Armstrong D
Published: June 2012

The pneumocandins are semisynthetic analogs of echinocandin-like compounds that have shown efficacy in animal models of systemic candidiasis, disseminated aspergillosis, and pneumocystis pneumonia. However, the most common form of Aspergillus infection in susceptible patients is pulmonary aspergillosis, which was not directly tested in the mouse models used in the past. We have evaluated three pneumocandins, L-693,989, L-731,373, and L-733,560, in a rat model of pulmonary aspergillosis. Male Sprague-Dawley rats were treated for 2 weeks with cortisone and tetracycline and fed a low-protein diet before being inoculated via the trachea with 10(6) conidia of Aspergillus fumigatus H11-20. In the absence of drug treatment, the animals developed a progressive, rapidly fatal bronchopneumonia. All three pneumocandins at doses of 5 mg/kg (intraperitoneally [i.p.] every 12 h [q12h]) were effective in delaying mortality in this model. Survival at day 7 postinfection was 20% among controls (n = 10 for all groups), while it was 60, 80, and 90% in groups that were treated with L-693,989, L-731,373, and L-733,560, respectively. In another trial, survival at day 7 postinfection was 25% among controls (n = 8 for all groups); it was 87.5% in a group treated with amphotericin B (0.5 mg/kg i.p. q12h) and was 100% in a group treated with L-733,560 (0.625 mg/kg i.p. q12h). In a separate trial, aerosol L-693,989 administered 2 h before infection (5 mg/kg) delayed mortality. Eight of the 10 animals treated with aerosol L-693,989 survived for 7 days, whereas only 2 of 10 control animals survived. We conclude that the pneumocandins we tested were highly effective in an animal model of pulmonary aspergillosis.
Pharmacodynamics of ceftazidime administered as continuous infusion or intermittent bolus alone and in combination with single daily-dose amikacin against Pseudomonas aeruginosa in an in vitro infection model
Authors: Cappelletty D M , Kang S L , Palmer S M , Rybak M J
Published: June 2012

We compared the pharmacodynamics and killing activity of ceftazidime, administered by continuous infusion and intermittent bolus, against Pseudomonas aeruginosa ATCC 27853 and ceftazidime-resistant P. aeruginosa 27853CR with and without a single daily dose of amikacin in an in vitro infection model over a 48-h period. Resistance to ceftazidime was selected for by serial passage of P. aeruginosa onto agar containing increasing concentrations of ceftazidime. Human pharmacokinetics and dosages were simulated as follows: half-life, 2 h; intermittent-bolus ceftazidime, 2 g every 8 h (q8h) and q12h; continuous infusion, 2-g loading dose and maintenance infusions of 5, 10, and 20 micrograms/ml; amikacin, 15 mg/kg q24h. There was no significant difference in time to 99.9% killing between any of the monotherapy regimens or between any combination regimen against ceftazidime-susceptible P. aeruginosa. Continuous infusions of 10 and 20 micrograms/ml killed as effectively as an intermittent bolus of 2 g q12h and q8h, respectively. Continuous infusion of 20 micrograms/ml and an intermittent bolus of 2 g q8h were the only regimens which prevented organism regrowth at 48 h, while a continuous infusion of 5 micrograms/ml resulted in the most regrowth. All of the combination regimens exhibited a synergistic response, with rapid killing of ceftazidime-susceptible P. aeruginosa and no regrowth. Against ceftazidime-resistant P. aeruginosa, none of the ceftazidime monotherapy regimens achieved 99.9% killing. The combination regimens exhibited the same rapid killing of the resistant strain as occurred with the susceptible strain; however, regrowth occurred with all regimens. The combination regimens of continuous infusion of 20 micrograms/ml plus amikacin and intermittent bolus q8h or q12h plus amikacin continued to be synergistic. Overall, continuous infusion monotherapy with ceftazidime at concentrations 4 to 5 and 10 to 15 times the MIC was as effective as an intermittent bolus of 2 g q12h (10 to 15 times the MIC) and q8h (25 to 35 times the MIC), respectively, against ceftazidime-susceptible P. aeruginosa. Combination therapy with amikacin plus ceftazidime, either intermittently q8h or by continuous infusion of 20 micrograms/ml, appeared to be effective and exhibited synergism against ceftazidime-resistant P. aeruginosa.
Protein binding and serum bactericidal activities of vancomycin and teicoplanin
Authors: Dykhuizen R S , Harvey G , Stephenson N , Nathwani D , Gould I M
Published: June 2012

In a randomized crossover study, the protein binding and serum bactericidal activities (SBAs) of vancomycin and teicoplanin against Staphylococcus aureus and Streptococcus pyogenes were investigated in six healthy volunteers. Total concentrations in serum 1 h postadministration of vancomycin and teicoplanin were 25.5 +/- 2.7 and 10.8 +/- 8.9 mg/liter, respectively; mean free concentrations were 14.6 +/- 2.0 and 0.6 +/- 0.9 mg/liter, respectively. Protein binding for vancomycin was 36.9% +/- 2.87%, and that for teicoplanin was 97.4% +/- 2.6%. SBA determined in pooled human serum at 1 h against S. aureus ranged from 1:8 to 1:32 for both vancomycin and teicoplanin. Against S. pyogenes SBA at 1 h ranged from 1:16 to 1:128 for vancomycin and 1:256 to 1:2,048 for teicoplanin. In vitro kill curve studies showed that vancomycin is slowly bactericidal and that teicoplanin is bacteriostatic. Despite having less in vitro cidal activity against the study isolates and having low or unrecordable levels of free drug in serum, teicoplanin demonstrated a similar or better SBA than vancomycin. SBA was more closely related to the total drug level (r = 0.77 for S. aureus and r = 0.79 for S. pyogenes) than the free level of teicoplanin (r = 0.59 for S. aureus and r = 0.56 for S. pyogenes). The high level of protein binding of teicoplanin did not seem to impair its antibacterial activity as measured by its SBA.
Interactions of Yersinia pestis penicillin-binding proteins with beta-lactam antibiotics
Authors: Ferreira R C , Park J T , Camelo D , De Almeida D F , Ferreira L C
Published: June 2012

The affinities of six major penicillin-binding proteins (PBPs) of Yersinia pestis EV76 to different beta-lactam antibiotics were determined. The results indicate that, similar to their counterparts in Escherichia coli, PBP2 and PBP3 are the lethal targets of amdinocillin and furazlocillin, respectively. The PBP contents of four additional Y. pestis strains and the morphological effects produced by some beta-lactam antibiotics are also reported.
Early detection of rifampin in human nerve tissue after an oral dose of 600 milligrams
Authors: Guebre-Xabier M , Shannon E J , Kazen R , Kebret Z , Frommel D
Published: June 2012

Rifampin in picogram quantities inhibited the ability of Mycobacterium bovis 44 BCG P3 to release 14CO2 from the oxidation of [14C]palmitic acid. By using these mycobacteria in a bioassay, samples of serum and posterior tibial nerve were assayed for inhibitory concentrations of rifampin. Within 8 to 12 h after ingestion of 600 mg of rifampin, the drug was detected in eight patients in concentrations ranging from 0.52 to 4.1 micrograms/ml in serum and in concentrations ranging from 0.6 to 6.3 ng/mg in posterior tibial nerve fiber tissue.
Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams
Authors: Vakulenko S B , Tóth M , Taibi P , Mobashery S , Lerner S A
Published: June 2012

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime.
Liposomal amphotericin B therapy of murine histoplasmosis
Authors: Graybill J R , Bocanegra R
Published: June 2012

Liposomal amphotericin B (AmBisome) was compared with amphotericin B deoxycholate for the treatment of disseminated murine histoplasmosis. Liposomal amphotericin B was well tolerated and, milligram for milligram, was as potent as amphotericin B deoxycholate.
Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae
Authors: Lustigman S , Brotman B , Huima T , Castelhano A L , Singh R N , Mehta K , Prince A M
Published: June 2012

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.
Role of mexA-mexB-oprM in antibiotic efflux in Pseudomonas aeruginosa
Authors: Li X Z , Nikaido H , Poole K
Published: June 2012

We have earlier described mexA-mexB-oprK, an operon involved in pyoverdine export in Pseudomonas aeruginosa, and suggested that the products of these genes also contribute to the active efflux of several antibiotics (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993). Recently the outer membrane component of this efflux system was shown to be OprM, rather than OprK (N. Gotoh and K. Poole, unpublished results). In the present study, the conclusion concerning the efflux activity of this system was confirmed and extended by the measurement of drug accumulation in intact cells. Thus, the steady-state accumulation levels of tetracycline and norfloxacin were increased in mexA and oprM null mutants. mexA and oprM null mutants also showed an increase in susceptibility to a wide variety of beta-lactam antibiotics and an increase in the steady-state accumulation level of benzylpenicillin, indicating that the MexA-MexB-OprM pump also effluxes beta-lactams. Furthermore, deenergization of the cytoplasmic membrane with a proton conductor always produced a strong increase in the accumulation level. Finally, a single-step mutant over-producing MexAB-OprM accumulated less tetracycline and chloramphenicol than the parent strain and was more resistant to a wide range of antimicrobial compounds, including beta-lactams. These results support the notion that these proteins contribute to the intrinsic resistance of P. aeruginosa through the multidrug active efflux process.
Relevance of Chlamydia pneumoniae murine pneumonitis model to evaluation of antimicrobial agents
Authors: Masson N D , Toseland C D , Beale A S
Published: June 2012

A mouse model of Chlamydia pneumoniae pneumonitis was established in outbred MF1 mice immunosuppressed with cyclophosphamide. Following intranasal inoculation with 2.2 x 10(3) inclusion-forming units of C. pneumoniae TW-183 per mouse, chlamydiae were culturable from the lungs for at least 29 days. Progressive subacute pneumonitis with perivascular and peribronchial lymphoid cell hyperplasia was observed, and C. pneumoniae organisms were located in consolidated areas of tissue by immunocytochemistry. Mice were treated orally, commencing at 8 days after infection, with clinically achievable concentrations of amoxicillin-clavulanate or ciprofloxacin (three times daily for 7 days), ofloxacin, doxycycline, or erythromycin (twice daily for 7 days), or azithromycin (once daily for 4 days). Despite disparate antichlamydial activity in cell culture and different pharmacokinetic properties in infected animals, all treatments reduced the chlamydial load in the lungs (P < 0.05) when the loads were evaluated by culture at 1 and 10 days after the cessation of dosing, and this was reflected in the histopathological and immunocytochemistry scores. There was no significant difference between these treatments, and C. pneumoniae TW-183 was eradicated from the majority but not from all mice. These results confirm the limited clinical data available to date. In conclusion, a range of oral antimicrobial agents commonly used for the treatment of community-acquired respiratory infection was found to be efficacious in this experimental model of C. pneumoniae pneumonitis, which may therefore be of utility in chemotherapy and follow-up studies.
DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa
Authors: Yonezawa M , Takahata M , Matsubara N , Watanabe Y , Narita H
Published: June 2012

The mutations in the quinolone resistance-determining region of the gyrA gene from clinical isolates of Pseudomonas aeruginosa were determined by DNA sequencing. The strains were isolated in 1989 and 1993. No mutations were detected in the clinical isolates in 1989, while five types of mutations were identified in the isolates in 1993. These mutations were as follows: group 1, a Thr residue to an Ile residue at position 83 (Thr-83-Ile); group 2, Asp-87-Asn; group 3, Thr-83-Ile and Asp-87-Gly; group 4, Thr-83-Ile and Asp-87-Asn; group 5, Thr-83-Ile and Asp-87-His. Three types of double mutations (groups 3, 4, and 5) have not been described previously. These mutations were homologous to the Ser-83-Leu, Asp-87-Asn, and Asp-87-Gly changes observed in Escherichia coli. Thus, DNA gyrase A subunit mutations are implicated in resistance to quinolones in P. aeruginosa as well as E. coli.
Liposomal encapsulation of foscarnet protects against hypocalcemia induced by free foscarnet
Authors: Omar R F , Dusserre N , Désormeaux A , Poulin L , Tremblay M , Beauchamp D , Bergeron M G
Published: June 2012

Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the potential protective role of liposome-encapsulated foscarnet (LE-PFA) on these metabolic abnormalities. PFA administered as one bolus injection (0.5 or 1.0 g/kg) caused significant rapid decreases (approximately 20%) in the levels of calcium and phosphorus in serum within a few minutes and up to 30 min after injection. LE-PFA did not induce any of these changes, while peak levels in serum and the half-life of this formulation were much higher than those of the free drug. PFA administered for 2 weeks (340 or 500 mg/kg/day) resulted in no changes in creatinine or blood urea nitrogen levels in serum at the low-dosage level, but at the higher-dosage level, the creatinine level in serum increased by day 5 posttreatment. Furthermore, there was no increase in the creatinine or blood urea nitrogen level after 2 weeks of treatment with LE-PFA at a dosage of 35 mg/kg/day. When the pharmacokinetics of both free PFA and LE-PFA were compared, the plasma half-life of the encapsulated drug was approximately four times longer than that of the free drug. In addition, the systemic clearance of LE-PFA was approximately one-fifth of that of the free drug. In conclusion, free PFA causes hypocalcemia and hypophosphatemia and increases the creatinine level in serum, whereas the LE form of this drug seems to protect against the abnormal changes in calcium and phosphorus levels caused by the free drug. By preventing hypocalcemia and increasing its half-life, LE-PFA can be used at lower doses and at longer intervals. Clinical investigations of these formulations may be worthwhile.
Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol
Authors: Nair V , St Clair M H , Reardon J E , Krasny H C , Hazen R J , Paff M T , Boone L R , Tisdale M , Najera I , Dornsife R E
Published: June 2012

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Once-daily versus twice-daily administration of ceftazidime in the preterm infant
Authors: van den Anker J N , Schoemaker R C , van der Heijden B J , Broerse H M , Neijens H J , de Groot R
Published: June 2012

Ceftazidime pharmacokinetics in 28 preterm infants (gestational ages, 25.6 to 31.9 weeks) were studied on day 3 of life. Patients with suspected septicemia were randomized on day 1 of life in two groups. One group (n = 13) was administered 25 mg of ceftazidime per kg of body weight once daily, and the other (n = 15) was given 25 mg of ceftazidime per kg twice daily. Both groups also received 25 mg of amoxicillin per kg twice daily. Blood samples were collected on day 3 of life with an arterial catheter at 0, 0.5, 1, 2, 4, 8, and 12 h after an intravenous bolus injection. An additional blood sample was taken at 24 h from the group dosed once a day. High-performance liquid chromatography was used to determine serum ceftazidime concentrations. The pharmacokinetics of ceftazidime were best described by using a one-compartment model. The half-life for the elimination of the drug from serum, apparent volume of distribution, total body clearance of ceftazidime, and inulin clearance were not significantly different for both groups. The ceftazidime/inulin clearance ratio was 0.72 for both groups. However, trough concentrations in serum for the twice-daily group were significantly (P < 0.001) higher (42.0 +/- 13.4 mg/liter) than those for the once-daily group (13.1 +/- 4.7 mg/liter). The latter concentrations were all still substantially higher than the MIC of ceftazidime for major neonatal pathogens. We conclude that the currently recommended dosage of 25 mg of ceftazidime per kg twice daily for preterm infants with gestational ages below 32 weeks may be adjusted during the first days of life to one daily dose at 25 mg/kg, provided that for the empirical treatment of septicemia, amoxicillin at 25 mg/kg is also given twice daily.
Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation
Authors: Odds F C , Vranckx L , Woestenborghs F
Published: June 2012

The technical parameters for antifungal susceptibility testing with Candida species were reexamined to determine the optimal conditions for testing with semiautomated preparations of broth microdilution cultures, automated spectrophotometric readings of the cultures, and dose-response and endpoint determinations by means of a computer spreadsheet. Tests were based on proposed standard method M27P of the National Committee for Clinical Laboratory Standards for antifungal agents. RPMI 1640 broth with extra glucose to a final concentration of 2% gave higher and more reproducible drug-free control readings without affecting susceptibility endpoint readings. An inoculum of 8 x 10(4) yeasts per ml prepared from a carbon-limiting broth culture without further standardization was found to give optimal control readings after 48 h of incubation at 37 degrees C. For flucytosine, fluconazole, itraconazole, and ketoconazole, endpoints based on 50% growth inhibition (50% inhibitory concentration) gave the minimum variation with inoculum size and the fewest endpoint differences with RPMI 1640 medium obtained from two different suppliers. The 50% inhibitory concentration was also the optimal endpoint for fluconazole and ketoconazole susceptibilities in comparison with broth macrodilution MICs determined by the method of the National Committee for Clinical Laboratory Standards. Intralaboratory reproducibility was determined by retrospective analysis of replicate results for isolates retested at random over a 2-year period. This approach showed less favorable reproducibility than has been reported from purpose-designed, prospective antifungal susceptibility studies, but it may better reflect real-life test reproducibility. Susceptibility data for 616 clinical isolates of yeasts, representing 16 Candida and Saccharomyces spp., confirmed the tendency of Candida lusitaniae isolates to show relatively low susceptibilities to amphotericin B, the tendency of Candida krusei isolates to show low flucytosine and fluconazole susceptibilities, and the presence of some isolates in the species Candida albicans, Candida glabrata, and Candida tropicalis with low susceptibilities to azole derivative antifungal agents. The study demonstrates the value of automation and standardization in all stages of yeast susceptibility testing, from plate preparation to data analysis.
Distribution kinetics of enoxacin and its metabolite oxoenoxacin in excretory fluids of healthy volunteers
Authors: Jaehde U , Sörgel F , Naber K G , Zürcher J , Schunack W
Published: June 2012

The distribution kinetics of enoxacin and its main metabolite oxoenoxacin in excretory fluids was investigated in 11 healthy volunteers. A single intravenous dose of 428 mg of enoxacin was given as a 1-h infusion. Serial samples of plasma, urine, saliva, nasal secretions, tears, and sweat were drawn and analyzed for enoxacin and oxoenoxacin by reversed-phase high-pressure liquid chromatography. Large differences in the concentration-time profiles of the excretory fluids analyzed were observed. Nasal secretions exhibited the highest enoxacin exposure, as assessed by the area under the concentration-time curve. Excretory fluid/plasma area under the concentration-time curve ratios were found to be 1.67 +/- 0.36 for nasal secretions, 0.76 +/- 0.28 for saliva, 0.25 +/- 0.07 for sweat, and 0.23 +/- 0.11 for tears. The elimination half-life of enoxacin from sweat (8.27 +/- 2.63 h) was significantly longer than that for plasma (5.10 +/- 0.46 h). Oxoenoxacin was detected in urine and saliva and exhibited a higher renal clearance and a lower saliva exposure than the parent compound. In contrast to that of the metabolite, distribution of enoxacin in saliva was found to be time and pH dependent. In conclusion, our study revealed considerable differences in the distribution kinetics of enoxacin among various excretory sites. Because of distinct acidic and basic properties, the anionic oxometabolite significantly differs from the zwitterionic parent compound in its distribution characteristics.
Characterization of antimicrobial resistance in enterococci of animal origin
Authors: Thal L A , Chow J W , Mahayni R , Bonilla H , Perri M B , Donabedian S A , Silverman J , Taber S , Zervos M J
Published: June 2012

Among 97 enterococci cultured from animals, gentamicin MICs were > or = 2,000 micrograms/ml for 9 isolates and between 250 and 1,024 micrograms/ml for 6 isolates. For two isolates tested (gentamicin MICs, 256 and 512 micrograms/ml, respectively), there was no in vitro synergy with penicillin plus gentamicin, resistance was transferable, and there was no hybridization with a probe specific for 6'-aminoglycoside acetyltransferase-2"-aminoglycoside phosphotransferase. The results of the study indicate the presence of a unique gentamicin resistance genotype in enterococci of animal origin.
In vitro activity of levofloxacin, singly and in combination with rifamycin analogs, against Mycobacterium leprae
Authors: Dhople A M , Ibanez M A
Published: June 2012

The in vitro susceptibility of Mycobacterium leprae to levofloxacin was studied by using two biochemical parameters to measure the metabolic activity of the organism. Levofloxacin consistently exhibited twofold greater bactericidal activity than ofloxacin, with the MIC being 0.75 microgram/ml. When combined with one of the three rifamycin analogs, synergism was obtained with KRM-1648 and rifabutin but not with rifampin.
Influence of a newly developed quinolone, T-3761, on pharmacokinetics of theophylline in rats
Authors: Hasegawa T , Nadai M , Haghgoo S , Yamaki K , Takagi K , Nabeshima T
Published: June 2012

The effect of a new quinolone, T-3761, on the pharmacokinetics and metabolism of theophylline was investigated with rats. T-3761 at a high dose (20 mg/kg of body weight) was injected intravenously 10 min before an intravenous administration of theophylline (10 mg/kg). The presence of T-3761 slightly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and T-3761 were estimated by noncompartmental methods. A significant decrease (approximately 25%) in the systemic clearance of theophylline was observed in the presence of T-3761. However, no significant changes between the control group and the T-3761-treated groups in the volume of distribution at a steady state were observed. Pretreatment with T-3761 increased the urinary excretion of unchanged theophylline (by approximately 25%) and decreased the nonrenal clearances (by approximately 30%), indicating that T-3761 inhibits the metabolism of theophylline. These findings suggest that T-3761 at the dose used in this study affects the pharmacokinetics and metabolism of theophylline.
Development and Assay of RNA Transcripts of Enterovirus Species A to D, Rhinovirus Species A to C, and Human Parechovirus: Assessment of Assay Sensitivity and Specificity of Real-Time Screening and Typing Methods
Authors: McLeish Nigel J. , Witteveldt Jeroen , Clasper Lucy , McIntyre Chloe , McWilliam Leitch E. Carol , Hardie Alison , Bennett Susan , Gunson Rory , Carman William F. , Feeney Susan A. , Coyle Peter V. , Vipond Barry , Muir Peter , Benschop Kimberley , Wolthers Katja , Waris Matti , Osterback Riikka , Johannessen Ingo , Templeton Kate , Harvala Heli , Simmonds Peter
Published: June 2012

Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and −20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.
Distinct Roles for Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 in the Structure and Production of a Primate Gammaherpesvirus
Authors: Woodson Evonne N. , Kedes Dean H.
Published: June 2012

During their progression from intranuclear capsids to mature trilaminar virions, herpesviruses incorporate an extensive array of viral as well as a smaller subset of cellular proteins. Our laboratory previously reported that rhesus monkey rhadinovirus (RRV), a close homolog of the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is comprised of at least 33 different virally encoded proteins. In the current study, we found that RRV infection activated the extracellular signal-regulated kinase (ERK) pathway and nascent virions preferentially incorporated the activated form of ERK2 (pERK2) into the tegument. This was evident even in the face of greatly diminished stores of intracellular ERK2, suggesting a clear bias toward the incorporation of pERK2 into the RRV particle. Similar to earlier findings with KSHV, activation of ERK was essential for the production of lytic viral proteins and virions. Knockdown of intracellular ERK, however, failed to inhibit virus production, likely due to maintenance of residual pools of intracellular pERK2. Paradoxically, selective knockdown of ERK1 enhanced virion production nearly 5-fold and viral titers more than 10-fold. These data are the first to implicate ERK1 as a negative regulator of lytic replication in a herpesvirus and the first to demonstrate the incorporation of an activated signaling molecule within a herpesvirus. Together, the results further our understanding of how herpesviruses interact with host cells during infection and demonstrate how this family of viruses can exploit cellular signal transduction pathways to modulate their own replication.
Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus
Authors: Zhang Ying , Zhang Qianyi , Gao Yuwei , He Xijun , Kong Huihui , Jiang Yongping , Guan Yuntao , Xia Xianzhu , Shu Yuelong , Kawaoka Yoshihiro , Bu Zhigao , Chen Hualan
Published: June 2012

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.
Negative Elongation Factor-Mediated Suppression of RNA Polymerase II Elongation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression
Authors: Toth Zsolt , Brulois Kevin F. , Wong Lai-Yee , Lee Hye-Ra , Chung Brian , Jung Jae U.
Published: June 2012

Genome-wide chromatin immunoprecipitation assays indicate that the promoter-proximal pausing of RNA polymerase II (RNAPII) is an important postinitiation step for gene regulation. During latent infection, the majority of Kaposi's sarcoma-associated herpesvirus (KSHV) genes is silenced via repressive histone marks on their promoters. Despite the absence of their expression during latency, however, several lytic promoters are enriched with activating histone marks, suggesting that mechanisms other than heterochromatin-mediated suppression contribute to preventing lytic gene expression. Here, we show that the RNAPII-mediated transcription of the KSHV OriLytL, K5, K6, and K7 (OriLytL-K7) lytic genes is paused at the elongation step during latency. Specifically, the RNAPII-mediated transcription is stalled by the host's negative elongation factor (NELF) at the promoter regions of OriLytL-K7 lytic genes during latency, leading to the hyperphosphorylation of the serine 5 residue and the hypophosphorylation of the serine 2 of the C-terminal domain of the RNAPII large subunit, a hallmark of stalled RNAPII. Consequently, depletion of NELF expression induced transition of stalled RNAPII into a productive transcription elongation at the promoter-proximal regions of OriLytL-K7 lytic genes, leading to their RTA-independent expression. Using an RTA-deficient recombinant KSHV, we also showed that expression of the K5, K6, and K7 lytic genes was highly inducible upon external stimuli compared to other lytic genes that lack RNAPII on their promoters during latency. These results indicate that the transcription elongation of KSHV OriLytL-K7 lytic genes is inhibited by NELF during latency, but can also be promptly reactivated in an RTA-independent manner upon external stimuli.