bluuebunny/crossref_metadata_2025 huggingface dataset:
A Myosin I Isoform in the Nucleus
Authors: Pestic-Dragovich Lidija , Stojiljkovic Ljuba , Philimonenko Anatoly A. , Nowak Grzegorz , Ke Yunbo , Settlage Robert E. , Shabanowitz Jeffrey , Hunt Donald F. , Hozak Pavel , de Lanerolle Primal
Published: July 2002

A nuclear isoform of myosin I β that contains a unique 16–amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16–amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I β colocalized with RNA polymerase II in an α-amanitin– and actinomycin D–sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I β appears to be in a complex with RNA polymerase II and may affect transcription.
Structure of a Glycerol-Conducting Channel and the Basis for Its Selectivity
Authors: Fu Daxiong , Libson Andrew , Miercke Larry J. W. , Weitzman Cindy , Nollert Peter , Krucinski Jolanta , Stroud Robert M.
Published: July 2002

Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane. We report the crystal structure of the
Escherichia coli
glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution. Glycerol molecules line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms. Two conserved aspartic acid–proline–alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded.
Role of
BAX
in the Apoptotic Response to Anticancer Agents
Authors: Zhang Lin , Yu Jian , Park Ben Ho , Kinzler Kenneth W. , Vogelstein Bert
Published: July 2002

To assess the role of
BAX
in drug-induced apoptosis in human colorectal cancer cells, we generated cells that lack functional
BAX
genes. Such cells were partially resistant to the apoptotic effects of the chemotherapeutic agent 5-fluorouracil, but apoptosis was not abolished. In contrast, the absence of
BAX
completely abolished the apoptotic response to the chemopreventive agent sulindac and other nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibited the expression of the antiapoptotic protein Bcl-X
L
, resulting in an altered ratio of BAX to Bcl-X
L
and subsequent mitochondria-mediated cell death. These results establish an unambiguous role for
BAX
in apoptotic processes in human epithelial cancers and may have implications for cancer chemoprevention strategies.
The Formation of Chondrules at High Gas Pressures in the Solar Nebula
Authors: Galy Albert , Young Edward D. , Ash Richard D. , Keith O'Nions R.
Published: July 2002

High-precision magnesium isotope measurements of whole chondrules from the Allende carbonaceous chondrite meteorite show that some aluminum-rich Allende chondrules formed at or near the time of formation of calcium-aluminum–rich inclusions and that some others formed later and incorporated precursors previously enriched in magnesium-26. Chondrule magnesium-25/magnesium-24 correlates with [magnesium]/[aluminum] and size, the aluminum-rich, smaller chondrules being the most enriched in the heavy isotopes of magnesium. These relations imply that high gas pressures prevailed during chondrule formation in the solar nebula.
Teleconvection: Remotely Driven Thermal Convection in Rotating Stratified Spherical Layers
Authors: Zhang Keke , Schubert Gerald
Published: July 2002

We report the discovery of a convective phenomenon found to occur in a rotating spherical system in which an inner convectively unstable fluid layer is bounded by a corotating outer convectively stable fluid layer. Although convection is thermally driven in the unstable interior, the resulting convective motions concentrate primarily in the stable outer region. This phenomenon, which we term teleconvection, suggests that fluid motions observed at the “surface” of a planet (such as Jupiter's alternating zonal winds) may be driven by an energy source located deep inside the planet.
Strange Magnetism and the Anapole Structure of the Proton
Authors: Hasty R. , Hawthorne-Allen A. M. , Averett T. , Barkhuff D. , Beck D. H. , Beise E. J. , Blake A. , Breuer H. , Carr R. , Covrig S. , Danagoulian A. , Dodson G. , Dow K. , Farkhondeh M. , Filippone B. W. , Gao J. , Herda M. C. , Ito T. M. , Jones C. E. , Korsch W. , Kramer K. , Kowalski S. , Lee P. , McKeown R. D. , Mueller B. , Pitt M. , Ritter J. , Roche J. , Savu V. , Spayde D. T. , Tieulent R. , Tsentalovich E. , Wells S. P. , Yang B. , Zwart T.
Published: July 2002

The violation of mirror symmetry in the weak force provides a powerful tool to study the internal structure of the proton. Experimental results have been obtained that address the role of strange quarks in generating nuclear magnetism. The measurement reported here provides an unambiguous constraint on strange quark contributions to the proton's magnetic moment through the electron-proton weak interaction. We also report evidence for the existence of a parity-violating electromagnetic effect known as the anapole moment of the proton. The proton's anapole moment is not yet well understood theoretically, but it could have important implications for precision weak interaction studies in atomic systems such as cesium.
Electron Spin Resonance in Studies of Membranes and Proteins
Authors: Borbat P. P. , Costa-Filho A. J. , Earle K. A. , Moscicki J. K. , Freed J. H.
Published: July 2002

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.
Insulators and Boundaries: Versatile Regulatory Elements in the Eukaryotic Genome
Authors: Bell Adam C. , West Adam G. , Felsenfeld Gary
Published: July 2002

Insulators mark the boundaries of chromatin domains by limiting the range of action of enhancers and silencers. Although the properties of insulators have been well studied, their role in vivo has largely been a subject of speculation. Recent results make it possible to ascribe specific and essential functions to the insulators of
Drosophila
, yeast, and vertebrates. In some cases, insulator activity can be modulated by nearby regulatory elements, bound cofactors, or covalent modification of the DNA. Not simply passive barriers, insulators are active participants in eukaryotic gene regulation.
Coupling and Entangling of Quantum States in Quantum Dot Molecules
Authors: Bayer M. , Hawrylak P. , Hinzer K. , Fafard S. , Korkusinski M. , Wasilewski Z. R. , Stern O. , Forchel A.
Published: July 2002

We demonstrate coupling and entangling of quantum states in a pair of vertically aligned, self-assembled quantum dots by studying the emission of an interacting electron-hole pair (exciton) in a single dot molecule as a function of the separation between the dots. An interaction-induced energy splitting of the exciton is observed that exceeds 30 millielectron volts for a dot layer separation of 4 nanometers. The results are interpreted by mapping the tunneling of a particle in a double dot to the problem of a single spin. The electron-hole complex is shown to be equivalent to entangled states of two interacting spins.
Nucleotide-Dependent Single- to Double-Headed Binding of Kinesin
Authors: Kawaguchi Kenji , Ishiwata Shin'ichi
Published: July 2002

The motility of kinesin motors is explained by a “hand-over-hand” model in which two heads of kinesin alternately repeat single-headed and double-headed binding with a microtubule. To investigate the binding mode of kinesin at the key nucleotide states during adenosine 5′-triphosphate (ATP) hydrolysis, we measured the mechanical properties of a single kinesin-microtubule complex by applying an external load with optical tweezers. Both the unbinding force and the elastic modulus in solutions containing AMP-PNP (an ATP analog) were twice the value of those in nucleotide-free solution or in the presence of both AMP-PNP and adenosine 5′-diphosphate. Thus, kinesin binds through two heads in the former and one head in the latter two states, which supports a major prediction of the hand-over-hand model.
Inland Thinning of Pine Island Glacier, West Antarctica
Authors: Shepherd Andrew , Wingham Duncan J. , Mansley Justin A. D. , Corr Hugh F. J.
Published: July 2002

The Pine Island Glacier (PIG) transports 69 cubic kilometers of ice each year from ∼10% of the West Antarctic Ice Sheet (WAIS). It is possible that a retreat of the PIG may accelerate ice discharge from the WAIS interior. Satellite altimetry and interferometry show that the grounded PIG thinned by up to 1.6 meters per year between 1992 and 1999, affecting 150 kilometers of the inland glacier. The thinning cannot be explained by short-term variability in accumulation and must result from glacier dynamics.
Effects of Marine Reserves on Adjacent Fisheries
Authors: Roberts Callum M. , Bohnsack James A. , Gell Fiona , Hawkins Julie P. , Goodridge Renata
Published: July 2002

Marine reserves have been widely promoted as conservation and fishery management tools. There are robust demonstrations of conservation benefits, but fishery benefits remain controversial. We show that marine reserves in Florida (United States) and St. Lucia have enhanced adjacent fisheries. Within 5 years of creation, a network of five small reserves in St. Lucia increased adjacent catches of artisanal fishers by between 46 and 90%, depending on the type of gear the fishers used. In Florida, reserve zones in the Merritt Island National Wildlife Refuge have supplied increasing numbers of world record–sized fish to adjacent recreational fisheries since the 1970s. Our study confirms theoretical predictions that marine reserves can play a key role in supporting fisheries.
In Vivo Activities of Evernimicin (SCH 27899) against Vancomycin-Susceptible and Vancomycin-Resistant Enterococci in Experimental Endocarditis
Authors: Souli Maria , Thauvin-Eliopoulos Claudie , Eliopoulos George M.
Published: July 2002

To assess the potential efficacy of evernimicin (SCH 27899) against serious enterococcal infections, we used a rat model of aortic valve endocarditis established with either a vancomycin-susceptible
Enterococcus faecalis
or a vancomycin-resistant
Enterococcus faecium
strain. Animals infected with either one of the test strains were assigned to receive no treatment (controls) or 5-day therapy with one of the following regimens: evernimicin 60-mg/kg of body weight intravenous (i.v.) bolus once daily, 60-mg/kg i.v. bolus twice daily (b.i.d.), 60 mg/kg/day i.v. by continuous infusion, or 120 mg/kg/day i.v. by continuous infusion. These regimens were compared with vancomycin at 150 mg/kg/day. In animals infected with
E. faecalis
, evernimicin at 120 mg/kg/day by continuous infusion significantly reduced bacterial counts in vegetations (final density, 5.75 ± 3.38 log
10
CFU/g) compared with controls (8.51 ± 1.11 log
10
CFU/g). In animals infected with 0.5 ml of an 8 × 10
7
-CFU/ml inoculum of the vancomycin-resistant
E. faecium
, both 60-mg/kg bolus once a day and b.i.d. dose regimens of evernimicin were very effective (viable counts, 3.45 ± 1.44 and 3.81 ± 1.98 log
10
CFU/g, respectively). Vancomycin was unexpectedly active against infections induced with that inoculum. In animals infected with a 10
9
-CFU/ml inoculum of the vancomycin-resistant
E. faecium
, the evernimicin 60-mg/kg i.v. bolus b.i.d. reduced viable counts in vegetations compared with controls (6.27 ± 1.63 versus 8.34 ± 0.91 log
10
CFU/g;
P
< 0.05), whereas vancomycin was ineffective. Although resistant colonies could be selected in vitro, we were not able to identify evernimicin-resistant clones from cardiac vegetations. An unexplained observation from these experiments was the great variability in final bacterial densities within cardiac vegetations from animals in each of the evernimicin treatment groups.
Contribution of Dithiol Ligands to In Vitro and In Vivo Trypanocidal Activities of Dithiaarsanes and Investigation of Ligand Exchange in an Aqueous Solution
Authors: Loiseau Philippe M. , Lubert Patrick , Wolf Jean-Gerard
Published: July 2002

Twelve new dithiaarsanes were evaluated for their in vitro and in vivo trypanocidal properties in regard to their three parent molecules, 4-amino-phenylarsenoxide, melarsenoxide, and 4-dansylamino-phenylarsenoxide. The most potent dithiaarsane, compound 2b, had a minimum effective concentration of 1.5 nM after 48 h of incubation and at a dose of 0.39 μmol/kg of body weight (0.2 mg/kg) administered subcutaneously cured 100% of mice acutely infected with
Trypanosoma brucei brucei
CMP. With this model, the chemotherapeutic index of compound 2b was 512, compared to 256 for melarsamine dihydrochloride (Cymelarsan) under the same conditions. With a chronic infection produced by
T. brucei brucei
GVR, compound 2b cured 100% of mice after treatment at a dose of 25 μmol/kg (12.5 mg/kg) for 4 consecutive days, whereas melarsamine dihydrochloride and potassium melarsonyl (Trimelarsan) cured less than 50% mice at this dose. For both acute and late-stage infections, dithiaarsanes having a melaminophenyl ring exhibited the most-potent trypanocidal activity. Compound 2b is thus one of the most active organoarsenicals described in a mouse trypanosomiasis model. Considering that the main intracellular targets of organoarsenicals are thiol groups, we studied the possibility of ligand exchange between Cymelarsan and several dithiols. In aqueous solution, we observed a rapid exchange of cysteamine from melarsamine with free cysteamine and also with various dithiols always in favor of more stable cyclic derivatives. These ligand exchanges suggest the ability of trivalent organoarsenicals to react with targets such as trypanothione and dihydrolipoic acid. Among several ligands, a 1,3-dimercaptopropane moiety appeared the most suitable for trypanocidal activity.
Comparative Efficacies of Terbinafine and Fluconazole in Treatment of Experimental Coccidioidal Meningitis in a Rabbit Model
Authors: Sorensen Kevin N. , Sobel Raymond A. , Clemons Karl V. , Calderon Leilani , Howell Kimberley J. , Irani Plomarz R. , Pappagianis Demosthenes , Williams Paul L. , Stevens David A.
Published: July 2002

A rabbit model of coccidioidal meningitis was used to compare the therapeutic efficacies of terbinafine (TBF) and fluconazole (FCZ). Hydrocortisone acetate-treated New Zealand White male rabbits were infected intracisternally with either 2.2 × 10
4
or 6.4 × 10
4
Coccidioides immitis
arthroconidia. Oral treatment with polyethylene glycol 200 (PEG) twice daily (
n
= 8), TBF twice daily (
n
= 9; 200 mg/kg of body weight/day), or FCZ once daily (
n
= 8; 80 mg/kg/day) began on day 5 and continued for 21 days. Mean survival times were 20, 24, and 32 days for rabbits treated with PEG, TBF, and FCZ, respectively. All of the FCZ-treated animals (100%;
P
= 0.003), 56% of the TBF-treated animals (
P
= 0.4), and 25% of the PEG-treated animals survived the length of the study. Both FCZ and TBF were effective at reducing the incidence of paresis. Only FCZ was effective at reducing most neurological and systemic signs. FCZ treatments resulted in lower cerebrospinal fluid (CSF) protein concentrations and leukocyte counts and faster clearing of CSF fungal cultures compared with those for PEG-treated controls, but TBF treatments had no significant effect on these parameters. Neither drug affected CSF glucose levels. Mean serum TBF levels by bioassay were within the range of 3.5 to 6.2 μg/ml at 1, 2, and 4 h postdosing and 0.35 to 7.0 μg/ml at 14 h postdosing. No TBF was detected in CSF. Mean FCZ levels (24 to 25.5 h postdosing) by bioassay were 16.4 to 19.2 and 13.5 to 19.2 μg/ml in serum and CSF, respectively. The reduction in the numbers of CFU in the spinal cord and brain was over 100-fold (
P
= 0.0005) in FCZ-treated animals and 2-fold (
P
≤ 0.2) in TBF-treated animals compared with those in PEG-treated animals. Histopathologic severity (semiquantitative scoring system) was significantly attenuated by FCZ treatment (
P
= 0.05) and was slightly attenuated by TBF treatment compared with that for the controls. In conclusion, TBF appeared to have a slight effect on survival, histology, and reduction of the numbers of CFU in tissue; however, these effects were not significant. FCZ was effective at controlling coccidioidal meningitis.
Simultaneous Quantitation of Intracellular Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency Virus-Infected Individuals
Authors: Rodriguez Jose F. , Rodriguez Jorge L. , Santana Jorge , García Hermes , Rosario Osvaldo
Published: July 2002

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.
Early Bactericidal Activity of Paromomycin (Aminosidine) in Patients with Smear-Positive Pulmonary Tuberculosis
Authors: Donald P. R. , Sirgel F. A. , Kanyok T. P. , Danziger L. H. , Venter A. , Botha F. J. , Parkin D. P. , Seifart H. I. , Van de Wal B. W. , Maritz J. S. , Mitchison D. A.
Published: July 2002

The early bactericidal activity of the aminoglycoside paromomycin (aminosidine) in doses of 7.5 and 15 mg/kg of body weight was measured in 22 patients with previously untreated smear-positive pulmonary tuberculosis. The fall in log
10
CFU per milliliter of sputum per day during the first 2 days of treatment for 7 patients receiving a paromomycin dosage of 7.5 mg/kg/day was 0.066, with a standard deviation (SD) of 0.216 and confidence limits from −0.134 to 0.266, and that for 15 patients receiving 15 mg/kg/day was 0.0924, with an SD of 0.140 and confidence limits from 0.015 to 0.170. The difference between the mean and zero was not significant for the 7.5-mg/kg dose group but was significant for the 15-mg/kg dose group (
t
= 2.55,
P
= 0.023). Since paromomycin has no cross-resistance with streptomycin and has no greater toxicity than other aminoglycosides, these results suggest that it has the potential to substitute for streptomycin in antituberculosis regimens and may be a particularly valuable addition to the drug armamentarium for the management of multidrug-resistant tuberculosis.
In Vitro Development of Resistance to Telithromycin (HMR 3647), Four Macrolides, Clindamycin, and Pristinamycin in
Streptococcus pneumoniae
Authors: Davies Todd A. , Dewasse Bonifacio E. , Jacobs Michael R. , Appelbaum Peter C.
Published: July 2002

The ability of 50 sequential subcultures in subinhibitory concentrations of telithromycin (HMR 3647), azithromycin, clarithromycin, erythromycin A, roxithromycin, clindamycin, and pristinamycin to select for resistance was studied in five macrolide-susceptible and six macrolide-resistant pneumococci containing
mefE
or
ermB
. Telithromycin selected for resistance less often than the other drugs.
Pharmacokinetics and Safety of Ascending Single Doses of DZ-2640, a New Oral Carbapenem Antibiotic, Administered to Healthy Japanese Subjects
Authors: Tanaka Makoto , Kato Kinuyo , Hakusui Hideo , Murakami Yoichi , Sato Kenichi , Ito Yasushi , Kawamoto Keiji
Published: July 2002

DZ-2640 is the ester-type oral carbapenem prodrug of an active parent compound, DU-6681. The pharmacokinetics and safety of DU-6681 were investigated in six studies after oral administration of a single dose of DZ-2640 to healthy male Japanese volunteers at doses of 25, 50, 100, 200, and 400 mg (as the equivalents of DU-6681) in the fasted state. The same volunteers received the drug at a dose of 100 mg in the fasted and fed states to examine the effect of food intake on the bioavailability of DZ-2640. The concentrations of DU-6681 in plasma and urine were determined by a validated high-performance liquid chromatography method and a bioassay. A good correlation between both methods was seen, indicating an absence of major active metabolites. The mean maximum concentrations of DU-6681 in plasma (
C
max
) ranged from 0.263 μg/ml (25-mg dose) to 2.489 μg/ml (400-mg dose) and were reached within 1.5 h following drug administration. After reaching the
C
max
, plasma DU-6681 concentrations declined in a monophasic manner, with a half-life of 0.47 to 0.89 h. The area under the concentration-time curve (AUC) and
C
max
increased almost linearly with the dose up to the 200-mg dose. The AUC and
C
max
increased less than proportionally after administration of the 400-mg dose, suggesting a reduction in drug absorption. The plasma protein binding of DU-6681 was in the range of 23.3 to 25.6%. The cumulative urinary recoveries (0 to 24 h) were in the range of 31.9 to 44.9%. The AUC was slightly but statistically significantly reduced by food intake. However, the
C
max
, half-life, and recovery in urine were not affected by food intake. The renal clearance (402 to 510 ml/min) was much greater than the mean glomerular filtration rate (ca. 120 ml/min), which indicated active tubular secretion of the drug. A mild transient and moderate diarrhea was observed in two of six volunteers in the study with a single dose of 25 mg. Mild soft stools were observed in two of six volunteers who received a 400-mg dose of the drug.
SHV-13, a Novel Extended-Spectrum β-Lactamase, in
Klebsiella pneumoniae
Isolates from Patients in an Intensive Care Unit in Amsterdam
Authors: Yuan Meifang , Hall Lucinda M. C. , Savelkoul Paul H. M. , Vandenbroucke-Grauls Christina M. J. E. , Livermore David M.
Published: July 2002

Eleven clonally related
Klebsiella pneumoniae
isolates were examined. These had been isolated at an intensive care unit in Amsterdam in 1994. Their resistance was associated with a conjugative 170-kb plasmid which encoded a novel SHV β-lactamase designated SHV-13. The SHV-13 enzyme had two substitutions compared with SHV-1: Leu35Gln and Gly238Ala. It hydrolyzed cefotaxime much more rapidly than ceftazidime or aztreonam.
Effect of Fluconazole on the Pharmacokinetics of Doxorubicin in Nonhuman Primates
Authors: Warren Katherine E. , McCully Cynthia M. , Walsh Thomas J. , Balis Frank M.
Published: July 2002

Antifungal prophylaxis in cancer patients who are undergoing chemotherapy is associated with prolonged neutropenia. We measured the effect of fluconazole on doxorubicin pharmacokinetics in nonhuman primates to determine if neutropenia is related to a pharmacokinetic interaction that delays the clearance of the chemotherapeutic agent. Fluconazole pretreatment had no effect on doxorubicin pharmacokinetics.
Postantifungal Effects of Echinocandin, Azole, and Polyene Antifungal Agents against
Candida albicans
and
Cryptococcus neoformans
Authors: Ernst Erika J. , Klepser Michael E. , Pfaller Michael A.
Published: July 2002

The postantifungal effect (PAFE) of fluconazole, MK-0991, LY303366, and amphotericin B was determined against isolates of
Candida albicans
and
Cryptococcus neoformans
. Concentrations ranging from 0.125 to 4 times the MIC were tested following exposure to the antifungal for 0.25 to 1 h. Combinations of azole and echinocandin antifungals (MK-0991 and LY303366) were tested against
C. neoformans
. Fluconazole displayed no measurable PAFE against
Candida albicans
or
Cryptococcus neoformans
, either alone or in combination with either echinocandin antifungal. MK-0991, LY303366, and amphotericin B displayed a prolonged PAFE of greater than 12 h against
Candida
spp. when tested at concentrations above the MIC for the organism and 0 to 2 h when tested at concentrations below the MIC for the organism.
A Bacteriophage Lambda-Based Genetic Screen for Characterization of the Activity and Phenotype of the Human Immunodeficiency Virus Type 1 Protease
Authors: Martínez Miguel-Angel , Cabana Marta , Parera Mariona , Gutierrez Arantxa , Esté José A. , Clotet Bonaventura
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor
c
I is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.
Evaluation of the Invader Assay, a Linear Signal Amplification Method, for Identification of Mutations Associated with Resistance to Rifampin and Isoniazid in
Mycobacterium tuberculosis
Authors: Cooksey Robert C. , Holloway Brian P. , Oldenburg Mary C. , Listenbee Sonja , Miller Carolyn W.
Published: July 2002

We evaluated a recently described linear signal amplification method for sensitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) in
Mycobacterium tuberculosis
. The assay utilizes the thermostable flap endonuclease Cleavase VIII, derived from
Archaeoglobus fulgidus
, which cleaves a structure formed by the hybridization of two overlapping oligonucleotide probes to a target nucleic acid strand. This method, termed the Invader assay, can discriminate single-base differences. Nine pairs of probes, encompassing five mutations in
rpoB
and
katG
that are associated with resistance to either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using amplified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucleotides in length, depending on the genotype of the test sample, were separated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiated on the basis of product size. Multiple mutations at a specific
rpoB
nucleotide in target PCR products could be identified, as could mutants that were present at ≥0.5% of the total population of target sequences. The Invader assay is a sensitive screen for some mutations associated with antituberculosis drug resistance in amplified gene regions.
Comparative In Vitro Activities of Clinafloxacin and Trovafloxacin against 1,000 Isolates of
Bacteroides fragilis
Group: Effect of the Medium on Test Results
Authors: Snydman David R. , Jacobus Nilda V. , McDermott Laura A. , Supran Stacey E.
Published: July 2002

The in vitro antibacterial activities of clinafloxacin, trovafloxacin, ciprofloxacin, and cefoxitin against 1,000 clinical isolates of
Bacteroides fragilis
group were compared by agar dilution in brucella blood agar (BBA) and Wilkins Chalgren agar (WCA). Significantly higher geometric mean MICs for the three quinolones and cefoxitin (
P
< 0.001) were obtained in BBA than in WCA. Regardless of medium, clinafloxacin was slightly more active than trovafloxacin. The activity of clinafloxacin and trovafloxacin was greater than that of cefoxitin against
B. distasonis
,
B. ovatus
, and
B. thetaiotaomicron
but lower against
B. vulgatus
. High cross resistance between trovafloxacin and clinafloxacin was observed.
In Vitro Activities of Voriconazole, Itraconazole, and Amphotericin B against
Blastomyces dermatitidis
,
Coccidioides immitis
, and
Histoplasma capsulatum
Authors: Li Ren-Kai , Ciblak Meral A. , Nordoff Nicole , Pasarell Lester , Warnock David W. , McGinnis Michael R.
Published: July 2002

The in vitro activity of voriconazole was compared to those of itraconazole and amphotericin B against the mold forms of 304 isolates of three dimorphic fungi,
Blastomyces dermatitidis
,
Coccidioides immitis
, and
Histoplasma capsulatum
. MICs were determined by a broth microdilution adaptation of the National Committee for Clinical Laboratory Standards M27-A procedure. RPMI 1640 medium was used for tests with voriconazole and itraconazole, whereas Antibiotic Medium 3 with 2% glucose was used for amphotericin B. Minimum fungicidal concentrations (MFCs) were also determined. Amphotericin B was active against all three dimorphic fungi, with MICs at which 90% of the isolates tested are inhibited (MIC
90
s) of 0.5 to 1 μg/ml. Itraconazole had MIC
90
s of 0.06 μg/ml for
H. capsulatum
, 0.125 μg/ml for
B. dermatitidis
, and 1 μg/ml for
C. immitis
. The MIC
90
s of voriconazole were 0.25 μg/ml for all three fungi. Amphotericin B was fungicidal for
B. dermatitidis
and
H. capsulatum
with MFCs at which 90% of strains tested are killed (MFC
90
s) of 0.5 and 2 μg/ml, respectively. It was less active against
C. immitis
, with MFCs ranging from 0.5 to >16 μg/ml. Voriconazole and itraconazole were lethal for most isolates of
B. dermatitidis
, with MFC
50
s and MFC
90
s of 0.125 and 4 μg/ml, respectively. Both azoles were fungicidal for some isolates of
H. capsulatum
, with MFC
50
s of 2 and 8 μg/ml for itraconazole and voriconazole, respectively; neither had a lethal effect upon
C. immitis
. Our results suggest that voriconazole possesses promising activity against these important human pathogens.
Antimicrobial Susceptibilities and Clinical Sources of
Facklamia
Species
Authors: LaClaire L. , Facklam R.
Published: July 2002

Facklamia
spp. are gram-positive cocci, arranged in short chains or diplos, and resemble viridans streptococci on 5% sheep blood agar. Eighteen strains representing four species of
Facklamia
were isolated from blood cultures, an abscess, bone, cerebrospinal fluid, gall bladder, vaginal swab, and one unknown source. Cultures were tested against 15 antimicrobial agents by using the broth microdilution MIC method. Reduced susceptibilities to the beta lactams, erythromycin, clindamycin, trimethoprim-sulfamethoxazole, and tetracycline were found. These results indicate that the susceptibilities of the
Facklamia
species are varied and that some strains have resistance patterns which may present difficulty in managing systemic infections in patients.
Contribution of a Thickened Cell Wall and Its Glutamine Nonamidated Component to the Vancomycin Resistance Expressed by
Staphylococcus aureus
Mu50
Authors: Cui Longzhu , Murakami Hiroko , Kuwahara-Arai Kyoko , Hanaki Hideaki , Hiramatsu Keiichi
Published: July 2002

Staphylococcus aureus
Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase and
l
-glutamine
d
-fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of
14
C-labeled
d
-glucose into the cell wall was observed in Mu50. Unlike a
femC
mutant
S. aureus
strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50. The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell wall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.
Accumulation of Norfloxacin by
Bacteroides fragilis
Authors: Ricci Vito , Piddock Laura J. V.
Published: July 2002

The accumulation of norfloxacin by
Bacteroides fragilis
NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing
B. fragilis
to accumulate norfloxacin in an aerobic or an anaerobic environment was ∼2 min; the SSC achieved in air was 90.28 ± 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 ± 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 μg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per μg of exogenous norfloxacin per ml. At concentrations above 10 μg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a

K
m

of 5.03 μg/ml and a
V
max
of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30°C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/°C. At low concentrations of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 μM carbonyl cyanide
m
-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 ± 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 ± 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by
B. fragilis
is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium.
Trovafloxacin Treatment of Viridans Group Streptococcus Experimental Endocarditis
Authors: Piper Kerryl E. , Rouse Mark S. , Ronningen Karen L. , Steckelberg James M. , Wilson Walter R. , Patel Robin
Published: July 2002

The activity of trovafloxacin was compared with those of vancomycin and penicillin in a model of
Streptococcus sanguis
species group (trovafloxacin MIC, 0.125 μg/ml) and
Streptococcus mitis
species group (trovafloxacin MIC, 0.125 μg/ml) experimental endocarditis. Rabbits with catheter-induced aortic valve vegetations were given no treatment, trovafloxacin at 15 mg/kg of body weight three times a day (t.i.d.), vancomycin at 15 mg/kg twice a day, or penicillin at 1.2 × 10
6
IU t.i.d. After 3 days of treatment, the animals were sacrificed; cardiac valve vegetations were aseptically removed and cultured quantitatively. Penicillin was as active as vancomycin as measured by in vivo clearance of bacteria. Trovafloxacin was less active (
P
< 0.05) than vancomycin or penicillin against
S. sanguis
species group infection but had similar efficacy against
S. mitis
species group infection. Quinolones, despite MICs in the susceptible range, may not be active for serious infections caused by some viridans group streptococci.
Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent
Escherichia coli
in the Neutropenic Mouse Thigh Model of Infection
Authors: Rocchetta H. L. , Boylan C. J. , Foley J. W. , Iversen P. W. , LeTourneau D. L. , McMillian C. L. , Contag P. R. , Jenkins D. E. , Parr T. R.
Published: July 2002

A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The
lux
gene cluster of
Photorhabdus luminescens
was 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.
Identification of an
erm
(A) Erythromycin Resistance Methylase Gene in
Streptococcus pneumoniae
Isolated in Greece
Authors: Syrogiannopoulos George A. , Grivea Ioanna N. , Tait-Kamradt Amelia , Katopodis George D. , Beratis Nicholas G. , Sutcliffe Joyce , Appelbaum Peter C. , Davies Todd A.
Published: July 2002

In a serotype 11A clone of erythromycin-resistant pneumococci isolated from young Greek carriers, we identified the nucleotide sequence of
erm
(A), a methylase gene previously described as
erm
(TR) in
Streptococcus pyogenes
. The
erm
(A) pneumococci were resistant to 14- and 15-member macrolides, inducibly resistant to clindamycin, and susceptible to streptogramin B. To our knowledge, this is the first identification of resistance to erythromycin in
S. pneumoniae
attributed solely to the carriage of the
erm
(A) gene.
Cellular Uptake of Two Fluoroketolides, HMR 3562 and HMR 3787, by Human Polymorphonuclear Neutrophils In Vitro
Authors: Abdelghaffar H. , Vazifeh D. , Labro M. T.
Published: July 2002

We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (
V
max
, about 2,300 ng/2.5 × 10
6
PMN/5 min;

K
m

, about 50 μg/ml); (iii) the inhibitory effects of Ni
2+
(a blocker of the Na
+
-Ca
2+
exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system.
Transfer of Tn
916
-Like Elements in Microcosm Dental Plaques
Authors: Roberts Adam P. , Cheah Grace , Ready Derren , Pratten Jonathan , Wilson Michael , Mullany Peter
Published: July 2002

Microcosm dental plaques were grown from an inoculum of human saliva in a constant-depth film fermentor. The inoculum contained four tetracycline-resistant streptococcal species, each of which contained a Tn
916
-like element. This element was shown to transfer to other streptococci both in filter-mating experiments and within the biofilms in the fermentor.
High Incidence of Erythromycin Resistance among Clinical Isolates of
Streptococcus agalactiae
in Taiwan
Authors: Hsueh Po-Ren , Teng Lee-Jene , Lee Li-Na , Ho Shen-Wu , Yang Pan-Chyr , Luh Kwen-Tay
Published: July 2002

The in vitro susceptibilities of 266 isolates of
Streptococcus agalactiae
determined by the agar dilution method showed that 6% of isolates were nonsusceptible to penicillin and 46% was resistant to erythromycin. Of the erythromycin-resistant isolates, 86.3% had the macrolide-lincosamide-streptogramin (MLS) resistance phenotype (constitutive MLS, 85.5%; inducible MLS, 0.8%) and 13.7% had the M phenotype.
Fractional Maximal Effect Method for In Vitro Synergy between Amoxicillin and Ceftriaxone and between Vancomycin and Ceftriaxone against
Enterococcus faecalis
and Penicillin-Resistant
Streptococcus pneumoniae
Authors: Desbiolles Norbert , Piroth Lionel , Lequeu Catherine , Neuwirth Catherine , Portier Henri , Chavanet Pascal
Published: July 2002

In the present study we assessed the use of a new in vitro testing method and graphical representation of the results to investigate the potential effectiveness of combinations of amoxicillin (AMZ) plus ceftriaxone (CRO) and of CRO plus vancomycin (VAN) against strains of
Streptococcus pneumoniae
highly resistant to penicillin and cephalosporins (PRP strains). We used the fractional maximal effect (FME) method of time-kill curves to calculate adequate concentrations of the drugs to be tested rather than relying on arbitrary choices. The concentrations obtained, each of which corresponded to a fraction of the maximal effect, were tested alone and in combination with the bacterial strains in a broth medium. Synergy was defined as a ratio of observed effect/theoretical effect, called FME, of greater than 1, additivity was defined as an FME equal to 1, and antagonism was defined as an observed effect lower than the best effect of one of the antibiotics used alone. The area between antagonism and additivity is the indifference zone. The well-known synergy between amoxicillin and gentamicin against a reference strain of
Enterococcus faecalis
was confirmed, with a best FME equal to 1.07. Two strains of PRP, strains PRP-1 and PRP-2, were studied. The MICs for PRP-1 and PRP-2 were as follows: penicillin, 4 and 16 μg/ml, respectively; AMZ, 2 and 8 μg/ml, respectively, CRO, 1 and 4 μg/ml, respectively; and VAN, 0.5 and 0.25 μg/ml, respectively. For PRP-1 the best FME for the combination AMZ-CRO was 1.22 with drug concentrations of 1.68 mg/liter for AMZ and 0.17 mg/liter for CRO; the best FME for the combination VAN-CRO was 1.75 with VAN at 0.57 mg/liter and CRO at 0.17 mg/liter. For PRP-2 the best FME obtained for the combination AMZ-CRO was 1.05 with drug concentrations of 11.28 mg/liter for AMZ and 0.64 mg/liter for CRO; the best FME obtained for the combination VAN-CRO was 1.35 with VAN at 0.25 mg/liter and CRO at 1.49 mg/liter. These results demonstrated the synergy of both combinations, AMZ-CRO and VAN-CRO, against PRP strains at drug concentrations achievable in humans. Consequently, either of the combinations can be proposed for use for the treatment of PRP infections.
Pharmacokinetic Interactions between Nelfinavir and 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Atorvastatin and Simvastatin
Authors: Hsyu Poe-Hirr , Schultz-Smith Melissa D. , Lillibridge James H. , Lewis Ronald H. , Kerr Bradley M.
Published: July 2002

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are effective agents in lowering cholesterol and triglycerides and are being used by human immunodeficiency virus-positive patients to treat the lipid elevation that may be associated with antiretroviral therapy. Many HMG-CoA reductase inhibitors and protease inhibitors are metabolized by the same cytochrome P450 enzyme 3A4 (CYP3A4). In addition, many protease inhibitors are potent inhibitors of CYP3A4. Therefore, coadministration of these two classes of drugs may cause significant drug interactions. This open-label, multiple-dose study was performed to determine the interactions between nelfinavir, a protease inhibitor, and two HMG-CoA reductase inhibitors, atorvastatin and simvastatin, in healthy volunteers. Thirty-two healthy subjects received either atorvastatin calcium (10 mg once a day) or simvastatin (20 mg once a day) for the first 14 days of the study. Nelfinavir (1,250 mg twice a day) was added on days 15 to 28. Pharmacokinetic assessment was performed on days 14 and 28. The study drugs were well tolerated. Nelfinavir increased the steady-state area under the plasma concentration-time curve during one dosing period (AUC
τ
) of atorvastatin 74% and the maximum concentration (
C
max
) of atorvastatin 122% and increased the AUC
τ
of simvastatin 505% and the
C
max
of simvastatin 517%. Neither atorvastatin nor simvastatin appeared to alter the pharmacokinetics of nelfinavir. It is recommended that coadministration of simvastatin with nelfinavir should be avoided, whereas atorvastatin should be used with nelfinavir with caution.