bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of Outer Membrane Protein A in
Escherichia coli
Killing by Neutrophil Elastase
Authors: Belaaouaj Abderr azzaq , Kim Kwang Sik , Shapiro Steven D.
Published: July 2002

In determining the mechanism of neutrophil elastase (NE)–mediated killing of
Escherichia coli
, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient,
E. coli
. Also, whereas NE-deficient mice had impaired survival in response to
E. coli
sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient
E. coli
. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.
Hydrological Impact of Heinrich Events in the Subtropical Northeast Atlantic
Authors: Bard Edouard , Rostek Frauke , Turon Jean-Louis , Gendreau Sandra
Published: July 2002

Reconstructing the impact of Heinrich events outside the main belt of ice rafting is crucial to understanding the underlying causes of these abrupt climatic events. A high-resolution study of a marine sediment core from the Iberian margin demonstrates that this midlatitude area was strongly affected both by cooling and advection of low-salinity arctic water masses during the last three Heinrich events. These paleoclimatic time series reveal the internal complexity of each of the last three Heinrich events and illustrate the value of parallel studies of the organic and inorganic fractions of the sediments.
The Osteoblast: A Sophisticated Fibroblast under Central Surveillance
Authors: Ducy Patricia , Schinke Thorsten , Karsenty Gerard
Published: July 2002

The study of the biology of osteoblasts, or bone-forming cells, illustrates how mammalian genetics has profoundly modified our understanding of cell differentiation and physiologic processes. Indeed, genetic-based studies over the past 5 years have revealed how osteoblast differentiation is controlled through growth and transcription factors. Likewise, the recent identification, using mutant mouse models, of a central component in the regulation of bone formation expands our understanding of the control of bone remodeling. This regulatory loop, which involves the hormone leptin, may help to explain the protective effect of obesity on bone mass in humans. In addition, it provides a novel physiologic concept that may shed light on the etiology of osteoporosis and help to identify new therapeutic targets.
Fairness Versus Reason in the Ultimatum Game
Authors: Nowak Martin A. , Page Karen M. , Sigmund Karl
Published: July 2002

In the Ultimatum Game, two players are offered a chance to win a certain sum of money. All they must do is divide it. The proposer suggests how to split the sum. The responder can accept or reject the deal. If the deal is rejected, neither player gets anything. The rational solution, suggested by game theory, is for the proposer to offer the smallest possible share and for the responder to accept it. If humans play the game, however, the most frequent outcome is a fair share. In this paper, we develop an evolutionary approach to the Ultimatum Game. We show that fairness will evolve if the proposer can obtain some information on what deals the responder has accepted in the past. Hence, the evolution of fairness, similarly to the evolution of cooperation, is linked to reputation.
The 100,000-Year Ice-Age Cycle Identified and Found to Lag Temperature, Carbon Dioxide, and Orbital Eccentricity
Authors: Shackleton Nicholas J.
Published: July 2002

The deep-sea sediment oxygen isotopic composition (δ
18
O) record is dominated by a 100,000-year cyclicity that is universally interpreted as the main ice-age rhythm. Here, the ice volume component of this δ
18
O signal was extracted by using the record of δ
18
O in atmospheric oxygen trapped in Antarctic ice at Vostok, precisely orbitally tuned. The benthic marine δ
18
O record is heavily contaminated by the effect of deep-water temperature variability, but by using the Vostok record, the δ
18
O signals of ice volume, deep-water temperature, and additional processes affecting air δ
18
O (that is, a varying Dole effect) were separated. At the 100,000-year period, atmospheric carbon dioxide, Vostok air temperature, and deep-water temperature are in phase with orbital eccentricity, whereas ice volume lags these three variables. Hence, the 100,000-year cycle does not arise from ice sheet dynamics; instead, it is probably the response of the global carbon cycle that generates the eccentricity signal by causing changes in atmospheric carbon dioxide concentration.
Greenhouse Gases in Intensive Agriculture: Contributions of Individual Gases to the Radiative Forcing of the Atmosphere
Authors: Robertson G. Philip , Paul Eldor A. , Harwood Richard R.
Published: July 2002

Agriculture plays a major role in the global fluxes of the greenhouse gases carbon dioxide, nitrous oxide, and methane. From 1991 to 1999, we measured gas fluxes and other sources of global warming potential (GWP) in cropped and nearby unmanaged ecosystems. Net GWP (grams of carbon dioxide equivalents per square meter per year) ranged from 110 in our conventional tillage systems to −211 in early successional communities. None of the annual cropping systems provided net mitigation, although soil carbon accumulation in no-till systems came closest to mitigating all other sources of GWP. In all but one ecosystem, nitrous oxide production was the single greatest source of GWP. In the late successional system, GWP was neutral because of significant methane oxidation. These results suggest additional opportunities for lessening the GWP of agronomic systems.
Technique for Enhanced Rare Earth Separation
Authors: Uda Tetsuya , Jacob K. Thomas , Hirasawa Masahiro
Published: July 2002

A process is demonstrated for the efficient separation of rare earth elements, using a combination of selective reduction and vacuum distillation of halides. The large differences in the redox chemistry of the rare earth elements and in the vapor pressures of rare earth di- and trihalides are exploited for separation. Experimental proof of concept is provided for the binary systems praseodymium-neodymium and neodymium-samarium. This process enhances the separation factor for the isolation of samarium and neodymium from their mixture by more than an order of magnitude.
Imaging the Electron Wave Function in Self-Assembled Quantum Dots
Authors: Vdovin E. E. , Levin A. , Patanè A. , Eaves L. , Main P. C. , Khanin Yu. N. , Dubrovskii Yu. V. , Henini M. , Hill G.
Published: July 2002

Magnetotunneling spectroscopy is used as a noninvasive and nondestructive probe to produce two-dimensional spatial images of the probability density of an electron confined in a self-assembled semiconductor quantum dot. The technique exploits the effect of the classical Lorentz force on the motion of a tunneling electron and can be regarded as the momentum (
k
) space analog of scanning tunneling microscopy imaging. The images reveal the elliptical symmetry of the ground state and the characteristic lobes of the higher energy states.
Molecular Analysis of
FRIGIDA
, a Major Determinant of Natural Variation in
Arabidopsis
Flowering Time
Authors: Johanson Urban , West Joanne , Lister Clare , Michaels Scott , Amasino Richard , Dean Caroline
Published: July 2002

Vernalization, the acceleration of flowering by a long period of cold temperature, ensures that many plants overwinter vegetatively and flower in spring. In
Arabidopsis
, allelic variation at the
FRIGIDA
(
FRI
) locus is a major determinant of natural variation in flowering time. Dominant alleles of
FRI
confer late flowering, which is reversed to earliness by vernalization. We cloned
FRI
and analyzed the molecular basis of the allelic variation. Most of the early-flowering ecotypes analyzed carry
FRI
alleles containing one of two different deletions that disrupt the open reading frame. Loss-of-function mutations at
FRI
have thus provided the basis for the evolution of many early-flowering ecotypes.
Emissions of Methyl Halides and Methane from Rice Paddies
Authors: Redeker K. R. , Wang N.-Y. , Low J. C. , McMillan A. , Tyler S. C. , Cicerone R. J.
Published: July 2002

Methyl halide gases are important sources of atmospheric inorganic halogen compounds, which in turn are central reactants in many stratospheric and tropospheric chemical processes. By observing emissions of methyl chloride, methyl bromide, and methyl iodide from flooded California rice fields, we estimate the impact of rice agriculture on the atmospheric budgets of these gases. Factors influencing methyl halide emissions are stage of rice growth, soil organic content, halide concentrations, and field-water management. Extrapolating our data implies that about 1 percent of atmospheric methyl bromide and 5 percent of methyl iodide arise from rice fields worldwide. Unplanted flooded fields emit as much methyl chloride as planted, flooded rice fields.
Protection Against Cutaneous Leishmaniasis Resulting from Bites of Uninfected Sand Flies
Authors: Kamhawi Shaden , Belkaid Yasmine , Modi Govind , Rowton Edgar , Sacks David
Published: July 2002

Despite the fact that
Leishmania
are transmitted exclusively by sand flies, none of the experimental models of leishmaniasis have established infection via sand fly bites. Here we describe a reproducible murine model of
Leishmania major
infection transmitted by
Phlebotomus papatasi
. Prior exposure of mice to bites of uninfected sand flies conferred powerful protection against
Leishmania major
that was associated with a strong delayed-type hypersensitivity response and with interferon-γ production at the site of parasite delivery. These results have important implications for the epidemiology of cutaneous leishmaniasis and suggest a vaccination strategy against this and possibly other vector-borne diseases.
Biochemical Basis of Oxidative Protein Folding in the Endoplasmic Reticulum
Authors: Tu Benjamin P. , Ho-Schleyer Siew C. , Travers Kevin J. , Weissman Jonathan S.
Published: July 2002

The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1. In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein. We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p. Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds.
How Cells Handle Cholesterol
Authors: Simons Kai , Ikonen Elina
Published: July 2002

Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Recent advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts in a functional state. How rafts contribute to cholesterol metabolism and transport in the cell is still an open issue. It has long been known that cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells, and influx of lipoprotein cholesterol into cells. The regulation of cholesterol homeostasis is now receiving a new focus, and this changed perspective may throw light on diseases caused by cholesterol excess, the prime example being atherosclerosis.
Support for the Lunar Cataclysm Hypothesis from Lunar Meteorite Impact Melt Ages
Authors: Cohen B. A. , Swindle T. D. , Kring D. A.
Published: July 2002

Lunar meteorites represent a more random sampling of lunar material than the Apollo or Luna collections and, as such, lunar meteorite impact melt ages are the most important data in nearly 30 years with which to reexamine the lunar cataclysm hypothesis. Within the lunar meteorite breccias MAC 88105, QUE 93069, DaG 262, and DaG 400, seven to nine different impact events are represented with
40
Ar-
39
Ar ages between 2.76 and 3.92 billion years ago (Ga). The lack of impact melt older than 3.92 Ga supports the concept of a short, intense period of bombardment in the Earth-moon system at ∼3.9 Ga. This was an anomalous spike of impact activity on the otherwise declining impact- frequency curve.
Intersubband Electroluminescence from Silicon-Based Quantum Cascade Structures
Authors: Dehlinger G. , Diehl L. , Gennser U. , Sigg H. , Faist J. , Ensslin K. , Grützmacher D. , Müller E.
Published: July 2002

The quantum cascade laser, which uses electronic transitions within a single band of a semiconductor, constitutes a possible way to integrate active optical components into silicon-based technology. This concept necessitates a transition with a narrow linewidth and an upper state with a sufficiently long lifetime. We report the observation of intersubband electroluminescence from a p-type silicon/silicon-germanium quantum cascade structure, centered at 130 millielectron volts with a width of 22 millielectron volts, with the expected polarization, and discernible up to 180 kelvin. The nonradiative lifetime is found to depend strongly on the design of the quantum well structure, and is shown to reach values comparable to that of an equivalent GaInAs/AlInAs laser structure.
Millennial-Scale Dynamics of Southern Amazonian Rain Forests
Authors: Mayle Francis E. , Burbridge Rachel , Killeen Timothy J.
Published: July 2002

Amazonian rain forest–savanna boundaries are highly sensitive to climatic change and may also play an important role in rain forest speciation. However, their dynamics over millennial time scales are poorly understood. Here, we present late Quaternary pollen records from the southern margin of Amazonia, which show that the humid evergreen rain forests of eastern Bolivia have been expanding southward over the past 3000 years and that their present-day limit represents the southernmost extent of Amazonian rain forest over at least the past 50,000 years. This rain forest expansion is attributed to increased seasonal latitudinal migration of the Intertropical Convergence Zone, which can in turn be explained by Milankovitch astronomic forcing.
Identification of
HE1
as the Second Gene of Niemann-Pick C Disease
Authors: Naureckiene Saule , Sleat David. E. , Lackland Henry , Fensom Anthony , Vanier Marie T. , Wattiaux Robert , Jadot Michel , Lobel Peter
Published: July 2002

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the
HE1
gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein–derived cholesterol.
Inhibition of Eukaryotic DNA Replication by Geminin Binding to Cdt1
Authors: Wohlschlegel James A. , Dwyer Brian T. , Dhar Suman K. , Cvetic Christin , Walter Johannes C. , Dutta Anindya
Published: July 2002

In all eukaryotic organisms, inappropriate firing of replication origins during the G
2
phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G
1
and S, whereas geminin is present in S and G
2
phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.
Direct Imaging of Transient Molecular Structures with Ultrafast Diffraction
Authors: Ihee Hyotcherl , Lobastov Vladimir A. , Gomez Udo M. , Goodson Boyd M. , Srinivasan Ramesh , Ruan Chong-Yu , Zewail Ahmed H.
Published: July 2002

Ultrafast electron diffraction (UED) has been developed to study transient structures in complex chemical reactions initiated with femtosecond laser pulses. This direct imaging of reactions was achieved using our third-generation apparatus equipped with an electron pulse (1.07 ± 0.27 picoseconds) source, a charge-coupled device camera, and a mass spectrometer. Two prototypical gas-phase reactions were studied: the nonconcerted elimination reaction of a haloethane, wherein the structure of the intermediate was determined, and the ring opening of a cyclic hydrocarbon containing no heavy atoms. These results demonstrate the vastly improved sensitivity, resolution, and versatility of UED for studying ultrafast structural dynamics in complex molecular systems.
Fast Drop Movements Resulting from the Phase Change on a Gradient Surface
Authors: Daniel Susan , Chaudhury Manoj K. , Chen John C.
Published: July 2002

The movement of liquid drops on a surface with a radial surface tension gradient is described here. When saturated steam passes over a colder hydrophobic substrate, numerous water droplets nucleate and grow by coalescence with the surrounding drops. The merging droplets exhibit two-dimensional random motion somewhat like the Brownian movements of colloidal particles. When a surface tension gradient is designed into the substrate surface, the random movements of droplets are biased toward the more wettable side of the surface. Powered by the energies of coalescence and collimated by the forces of the chemical gradient, small drops (0.1 to 0.3 millimeter) display speeds that are hundreds to thousands of times faster than those of typical Marangoni flows. This effect has implications for passively enhancing heat transfer in heat exchangers and heat pipes.
Simultaneous Binding of PtdIns(4,5)P
2
and Clathrin by AP180 in the Nucleation of Clathrin Lattices on Membranes
Authors: Ford Marijn G. J. , Pearse Barbara M. F. , Higgins Matthew K. , Vallis Yvonne , Owen David J. , Gibson Adele , Hopkins Colin R. , Evans Philip R. , McMahon Harvey T.
Published: July 2002

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH
2
-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P
2
] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH
2
-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P
2
-binding site. Because AP180 could bind to PtdIns(4,5)P
2
and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.
Impairment of the Ubiquitin-Proteasome System by Protein Aggregation
Authors: Bence Neil F. , Sampat Roopal M. , Kopito Ron R.
Published: July 2002

Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.
Postantibiotic Leukocyte Enhancement of Meropenem against Gram-Positive and Gram-Negative Strains
Authors: Novelli Andrea , Fallani Stefania , Cassetta Maria Iris , Conti Silvia , Mazzei Teresita
Published: July 2002

The postantibiotic leukocyte enhancement (PALE) of meropenem in vitro in comparison with that of imipenem was evaluated with 24 recently isolated gram-positive and gram-negative strains. In general, pre-exposure to carbapenems (at four times the MIC for 2 h) led to increased polymorphonuclear cell phagocytic killing. The PALE of imipenem was generally significantly less than that observed with meropenem.
Effect of Subinhibitory Antibiotic Concentrations on Polysaccharide Intercellular Adhesin Expression in Biofilm-Forming
Staphylococcus epidermidis
Authors: Rachid Shwan , Ohlsen Knut , Witte Wolfgang , Hacker Jörg , Ziebuhr Wilma
Published: July 2002

Biofilm production is an important step in the pathogenesis of
Staphylococcus epidermidis
polymer-associated infections and depends on the expression of the
ica
ADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the
ica
promoter and the beta-galactosidase gene
lac
Z from
Escherichia coli
was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on
ica
expression in
S. epidermidis
. It was shown that
S. epidermidis
biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance
ica
expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on
ica
expression. A weak (i.e., 2.5-fold) induction of
ica
expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of
ica
transcription and quantitative analyses of biofilm formation in a colorimetric assay.
New Gene Cassettes for Trimethoprim Resistance,
dfr13
, and Streptomycin-Spectinomycin Resistance,
aadA4
, Inserted on a Class 1 Integron
Authors: Adrian Peter V. , Thomson Christopher J. , Klugman Keith P. , Amyes Sebastian G. B.
Published: July 2002

In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene,
dfr13
, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with
dfr12
and exhibited a trimethoprim inhibition profile similar to that of
dfr12
. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of
dfr13
, a new cassette, an aminoglycoside resistance gene of the class AADA [ANT(3")(9)-I], which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus
aadA1
(
ant(3")-Ia
) and has been called
aadA4
(
ant(3")-Id). The 3′ end of the aadA4
cassette was truncated by IS
26
, which was contiguous with a truncated form of Tn
3
. On the same plasmid, pUK2381, a second copy of IS
26
was associated with
sul2
, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes
dfr13
,
aadA4
,
bla
TEM-1
, and
sul2
.
Mechanisms of Resistance to Quinupristin-Dalfopristin among Isolates of
Enterococcus faecium
from Animals, Raw Meat, and Hospital Patients in Western Europe
Authors: Soltani Mehnam , Beighton David , Philpott-Howard John , Woodford Neil
Published: July 2002

Twenty-eight quinupristin-dalfopristin-resistant isolates of
Enterococcus faecium
from hospital patients and nonhuman sources in European countries were studied. High-level resistance (MICs, ≥32 μg/ml) was associated with the presence of
vat
(E) (
satG
) (14 isolates [50%]) or
vat
(D) (
satA
) (6 isolates [21%]). These genes were not detected in eight (29%) isolates with lower levels of quinupristin-dalfopristin resistance (MICs, 4 to 16 μg/ml). This suggests the presence of further mechanisms of resistance to quinupristin-dalfopristin in
E. faecium
.
Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of
Staphylococcus aureus
and
Micrococcus luteus
Authors: Novo David J. , Perlmutter Nancy G. , Hunt Richard H. , Shapiro Howard M.
Published: July 2002

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated
Staphylococcus aureus
and
Micrococcus luteus
cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC
2
(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC
2
(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.
A Rapid Phenotypic Assay for Detection of Acyclovir-Resistant Varicella-Zoster Virus with Mutations in the Thymidine Kinase Open Reading Frame
Authors: Sahli Roland , Andrei Graciela , Estrade Christine , Snoeck Robert , Meylan Pascal R. A.
Published: July 2002

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdkmutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transformtdkmutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl β-d-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.
Activities of Trovafloxacin and Ampicillin-Sulbactam Alone or in Combination versus Three Strains of Vancomycin- Intermediate
Staphylococcus aureus
in an In Vitro Pharmacodynamic Infection Model
Authors: Aeschlimann Jeffrey R. , Hershberger Ellie , Rybak Michael J.
Published: July 2002

The recent isolation of clinical strains of methicillin-resistant
Staphylococcus aureus
(MRSA) with intermediate susceptibility (MICs, 8 μg/ml) to vancomycin (vancomycin-intermediate
S. aureus
[VISA]) emphasizes the importance of developing novel antimicrobial regimens and/or agents for future treatment. We studied the activities of ampicillin-sulbactam and trovafloxacin alone or in combination against three unique strains of VISA in an in vitro infection model. Two VISA strains were trovafloxacin susceptible (MICs, ≤2 μg/ml); one VISA strain was trovafloxacin resistant (MIC, 4 μg/ml). Trovafloxacin was administered to simulate a dose of 200 or 400 mg every 24 h. Ampicillin-sulbactam was administered to simulate a dose of 3 g every 6 h. Samples were removed from the infection models over 48 h, and reductions in colony counts were compared between regimens. Trovafloxacin (200 mg) produced rapid killing of a control MRSA strain over the 48-h experiment but produced only slight killing of all three VISA strains. The higher dose of trovafloxacin improved killing but did not produce bactericidal activity at 48 h. Ampicillin-sulbactam produced rapid bactericidal activity against all four strains tested, and colony counts at 8 h were at the limits of detection. However, regrowth occurred by 48 h for each strain. The combination of ampicillin-sulbactam and trovafloxacin provided additive activity against two of the three VISA strains. In conclusion, trovafloxacin or ampicillin-sulbactam alone did not provide adequate activity against the VISA strains for the 48-h evaluation period, but the combination could help improve activity against some strains of VISA.
Pharmacokinetics and Safety of AMD-3100, a Novel Antagonist of the CXCR-4 Chemokine Receptor, in Human Volunteers
Authors: Hendrix Craig W. , Flexner Charles , MacFarland Ronald T. , Giandomenico Christen , Fuchs Edward J. , Redpath Ella , Bridger Gary , Henson Geoffrey W.
Published: July 2002

AMD-3100, a bicyclam, is a novel agent that uniquely inhibits the entry of human immunodeficiency virus type 1 (HIV-1) into CD4
+
T cells via selective blockade of the chemokine CXCR-4 receptor. Twelve healthy volunteers were given AMD-3100 as a single 15-min intravenous infusion at 10, 20, 40, or 80 μg/kg. Five subjects also received a single subcutaneous injection of AMD-3100 (40 or 80 μg/kg). Three subjects received two escalating oral doses each (80 and 160 μg/kg). All subjects tolerated their dose(s) well without any grade 2 toxicity or dose adjustment. Six subjects experienced mild, transient symptoms, primarily gastrointestinal in nature and not dose related. All subjects experienced a dose-related elevation of the white blood cell count, from 1.5 to 3.1 times the baseline, which returned to the baseline 24 h after dosing. AMD-3100 demonstrated dose proportionality for the maximum drug concentration in serum (
C
max
) and the area under the concentration-time curve from 0 h to ∞ (AUC
0–∞
) over the entire dose range. At the highest intravenous dose (80 μg/kg), the median
C
max
was 515 (range, 470 to 521) ng/ml and the AUC
0–∞
was 1,044 (range, 980 to 1,403) ng-h/ml. The median systemic absorption after subcutaneous dosing was 87% (range, 67 to 106%). No drug was detectable in the blood following oral dosing. Using a two-compartment model, the median pharmacokinetic parameter estimates (ranges) were as follows: volume of distribution, 0.34 (0.27 to 0.36) liter/kg; clearance, 1.30 (0.97 to 1.34) liters/h; elimination half-life, 3.6 (3.5 to 4.9) h. After a single, well-tolerated intravenous dose of AMD-3100, concentrations were sustained for 12 h above the in vitro antiretroviral 90% inhibitory concentrations and for 8 h above antiviral concentrations identified in the SCID-hu Thy/Liv mouse model of HIV infection.
Prevalence of β-Lactamases among 1,072 Clinical Strains of
Proteus mirabilis
: a 2-Year Survey in a French Hospital
Authors: Chanal C. , Bonnet R. , De Champs C. , Sirot D. , Labia R. , Sirot J.
Published: July 2002

β-Lactam resistance was studied in 1,072 consecutive
P. mirabilis
clinical strains isolated at the Clermont-Ferrand teaching hospital between April 1996 and March 1998. The frequency of amoxicillin resistance was 48.5%. Among the 520 amoxicillin-resistant isolates, three resistance phenotypes were detected: penicillinase (407 strains [78.3%]), extended-spectrum β-lactamase (74 strains [14.2%]), and inhibitor resistance (39 strains [7.5%]). The penicillinase phenotype isolates were divided into three groups according to the level of resistance to β-lactams, which was shown to be related to the strength of the promoter. The characterization of the different β-lactamases showed that amoxicillin resistance in
P. mirabilis
was almost always (97%) associated with TEM or TEM-derived β-lactamases, most of which evolved via TEM-2.
In Vitro and In Vivo Activities of Nitazoxanide against
Clostridium difficile
Authors: McVay Catherine S. , Rolfe Rial D.
Published: July 2002

We have used the hamster model of antibiotic-induced
Clostridium difficile
intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against
C. difficile
, (ii) toxicity, (iii) ability to prevent
C. difficile
-associated ileocecitis, and (iv) propensity to induce
C. difficile
-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of
C. difficile
were comparable to those of vancomycin or metronidazole.
C. difficile
-associated ileocecitis was induced with oral clindamycin and toxigenic
C. difficile
in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-
C. difficile
challenge. During the treatment period, NTZ (≥7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced
C. difficile
intestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of
C. difficile
disease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic
C. difficile
. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or
C. difficile
intestinal disease, nor was
C. difficile
detected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal
C. difficile
intestinal disease in 20 and 70% of recipients, respectively.
Characterization of the Extended-Spectrum β-Lactamase Reference Strain,
Klebsiella pneumoniae
K6 (ATCC 700603), Which Produces the Novel Enzyme SHV-18
Authors: Rasheed J. Kamile , Anderson Gregory J. , Yigit Hesna , Queenan Anne Marie , Doménech-Sánchez Antonio , Swenson Jana M. , Biddle James W. , Ferraro Mary Jane , Jacoby George A. , Tenover Fred C.
Published: July 2002

Klebsiella pneumoniae
K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of
K. pneumoniae
K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a
bla
SHV
gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a
bla
SHV
-specific probe indicated that a
bla
SHV
gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with
Escherichia coli
HB101. DNA sequencing of this
bla
SHV
-related gene revealed that it differs from
bla
SHV-1
at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.
Pharmacokinetics of Levofloxacin and Ciprofloxacin during Continuous Renal Replacement Therapy in Critically Ill Patients
Authors: Malone Rebecca S. , Fish Douglas N. , Abraham Edward , Teitelbaum Isaac
Published: July 2002

The pharmacokinetics of intravenously administered levofloxacin and ciprofloxacin were studied in intensive care unit patients during continuous venovenous hemofiltration (CVVH; four patients received levofloxacin, and five received ciprofloxacin) or hemodiafiltration (CVVHDF; six patients received levofloxacin, and five received ciprofloxacin). Levofloxacin clearance was substantially increased during both CVVH and CVVHDF, while ciprofloxacin clearance was affected less. The results of this study suggest that doses of levofloxacin of 250 mg/day and ciprofloxacin of 400 mg/day are sufficient to maintain effective drug concentrations in the plasma of patients undergoing CVVH or CVVHDF.
Rapamycin and Less Immunosuppressive Analogs Are Toxic to
Candida albicans
and
Cryptococcus neoformans
via FKBP12-Dependent Inhibition of TOR
Authors: Cruz M. Cristina , Goldstein Alan L. , Blankenship Jill , Del Poeta Maurizio , Perfect John R. , McCusker John H. , Bennani Youssef L. , Cardenas Maria E. , Heitman Joseph
Published: July 2002

Candida albicans
and
Cryptococcus neoformans
cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity.
C. albicans rbp1/rbp1
mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in
C. albicans
,
C. neoformans
, and
Saccharomyces cerevisiae
. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.
Sensitive Bioassay for Determination of Fluconazole Concentrations in Plasma Using a
Candida albicans
Mutant Hypersusceptible to Azoles
Authors: Marchetti Oscar , Majcherczyk Paul A. , Glauser Michel P. , Bille Jacques , Moreillon Philippe , Sanglard Dominique
Published: July 2002

The antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay using
Candida pseudotropicalis
, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using
C. albicans
DSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes
CDR1, CDR2, CaMDR1
, and
FLU1
. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient > 0.997). The absolute sensitivity was 0.026 μg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required ±15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% ± 15.31% and 2.66% ± 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.
In Vitro Pharmacodynamic Properties of Colistin and Colistin Methanesulfonate against
Pseudomonas aeruginosa
Isolates from Patients with Cystic Fibrosis
Authors: Li Jian , Turnidge John , Milne Robert , Nation Roger L. , Coulthard Kingsley
Published: July 2002

The in vitro pharmacodynamic properties of colistin and colistin methanesulfonate were investigated by studying the MICs, time-kill kinetics, and postantibiotic effect (PAE) against mucoid and nonmucoid strains of
Pseudomonas aeruginosa
isolated from patients with cystic fibrosis. Twenty-three clinical strains, including multiresistant strains, and one type strain were selected for MIC determination. Eleven strains were resistant; MICs for these strains were >128 mg/liter. For the susceptible strains, MICs of colistin ranged from 1 to 4 mg/liter, while the MICs of colistin methanesulfonate were significantly higher and ranged from 4 to 16 mg/liter. The time-kill kinetics were investigated with three strains at drug concentrations ranging from 0.5 to 64 times the MIC. Colistin showed extremely rapid killing, resulting in complete elimination at the highest concentrations within 5 min, while colistin methanesulfonate killed more slowly, requiring a concentration of 16 times the MIC to achieve complete killing within 24 h. Colistin exhibited a significant PAE of 2 to 3 h at 16 times the MIC against the three strains after 15 min of exposure. For colistin methanesulfonate, PAEs were shorter at the concentrations tested. Colistin methanesulfonate had lower overall bactericidal and postantibiotic activities than colistin, even when adjusted for differences in MICs. Our data suggest that doses of colistin methanesulfonate higher than the recommended 2 to 3 mg/kg of body weight every 12 h may be required for the effective treatment of
P. aeruginosa
infections in cystic fibrosis patients.
Real-Time PCR and Melting Curve Analysis for Reliable and Rapid Detection of SHV Extended-Spectrum β-Lactamases
Authors: Randegger Corinne C. , Hächler Herbert
Published: July 2002

Extended-spectrum β-lactamases (ESBLs), e.g., ESBLs of the TEM or SHV type, compromise the efficacies of expanded-spectrum cephalosporins. An SHV non-ESBL that hydrolyzes only narrow-spectrum cephalosporins can be converted into an SHV ESBL through substitutions at three amino acid positions, 179, 238, or 238–240. In order to improve detection of SHV ESBLs, a novel method, based on real-time PCR monitored with fluorescently labeled hybridization probes and followed by melting curve analysis, was developed. It is able to (i) detect
bla
SHV
genes with high degrees of sensitivity and specificity, (ii) discriminate between
bla
SHV non-ESBL
and
bla
SHV ESBL
, and (iii) categorize the SHV ESBL producers into three phenotypically relevant subgroups. This method, termed the SHV melting curve mutation detection method, represents a powerful tool for epidemiological studies with SHV ESBLs. It even has the potential to be used in the diagnostic microbiology laboratory, because up to 32 clinical isolates can be processed in less than 1 h by starting with just a few bacterial colonies.
Antimicrobial Activity of Intraurethrally Administered ProbioticLactobacillus caseiin a Murine Model ofEscherichia coliUrinary Tract Infection
Authors: Asahara Takashi , Nomoto Koji , Watanuki Masaaki , Yokokura Teruo
Published: July 2002

The antimicrobial activity of the intraurethrally administered probioticLactobacillus caseistrain Shirota againstEscherichia coliin a murine urinary tract infection (UTI) model was examined. UTI was induced by intraurethral administration ofEscherichia colistrain HU-1 (a clinical isolate from a UTI patient, positive for type 1 and P fimbriae), at a dose of 1 × 106to 2 × 106CFU in 20 μl of saline, into a C3H/HeN mouse bladder which had been traumatized with 0.1 N HCl followed immediately by neutralization with 0.1 N NaOH 24 h before the challenge infection. Chronic infection with the pathogen at 106CFU in the urinary tract (bladder and kidneys) was maintained for more than 3 weeks after the challenge, and the number of polymorphonuclear leukocytes and myeloperoxidase activity in the urine were markedly elevated during the infection period. A single administration ofL. caseiShirota at a dose of 108CFU 24 h before the challenge infection dramatically inhibitedE. coligrowth and inflammatory responses in the urinary tract. Multiple daily treatments withL. caseiShirota during the postinfection period also showed antimicrobial activity in this UTI model. A heat-killed preparation ofL. caseiShirota exerted significant antimicrobial effects not only with a single pretreatment (100 μg/mouse) but also with multiple daily treatments during the postinfection period. The otherLactobacillusstrains tested, i.e.,L. fermentumATCC 14931T,L. jenseniiATCC 25258T,L. plantarumATCC 14917T, andL. reuteriJCM 1112T, had no significant antimicrobial activity. Taken together, these results suggest that the probioticL. caseistrain Shirota is a potent therapeutic agent for UTI.