bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Nonexercise Activity Thermogenesis in Resistance to Fat Gain in Humans
Authors: Levine James A. , Eberhardt Norman L. , Jensen Michael D.
Published: July 2002

Humans show considerable interindividual variation in susceptibility to weight gain in response to overeating. The physiological basis of this variation was investigated by measuring changes in energy storage and expenditure in 16 nonobese volunteers who were fed 1000 kilocalories per day in excess of weight-maintenance requirements for 8 weeks. Two-thirds of the increases in total daily energy expenditure was due to increased nonexercise activity thermogenesis (NEAT), which is associated with fidgeting, maintenance of posture, and other physical activities of daily life. Changes in NEAT accounted for the 10-fold differences in fat storage that occurred and directly predicted resistance to fat gain with overfeeding (correlation coefficient = 0.77, probability < 0.001). These results suggest that as humans overeat, activation of NEAT dissipates excess energy to preserve leanness and that failure to activate NEAT may result in ready fat gain.
STAT5 Interaction with the T Cell Receptor Complex and Stimulation of T Cell Proliferation
Authors: Welte Thomas , Leitenberg David , Dittel Bonnie N. , al-Ramadi Basel K. , Xie Bing , Chin Yue E. , Janeway Charles A. , Bothwell Alfred L. M. , Bottomly Kim , Fu Xin-Yuan
Published: July 2002

The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.
Unexpected Modes of PDZ Domain Scaffolding Revealed by Structure of nNOS-Syntrophin Complex
Authors: Hillier Brian J. , Christopherson Karen S. , Prehoda Kenneth E. , Bredt David S. , Lim Wendell A.
Published: July 2002

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces—one face has the canonical peptide binding groove, whereas the other has a β-hairpin “finger.” This nNOS β finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp β turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.
Roles of Phosphorylation Sites in Regulating Activity of the Transcription Factor Pho4
Authors: Komeili Arash , O'Shea Erin K.
Published: July 2002

Transcription factors are often phosphorylated at multiple sites. Here it is shown that multiple phosphorylation sites on the budding yeast transcription factor Pho4 play distinct and separable roles in regulating the factor's activity. Phosphorylation of Pho4 at two sites promotes the factor's nuclear export and phosphorylation at a third site inhibits its nuclear import. Phosphorylation of a fourth site blocks the interaction of Pho4 with the transcription factor Pho2. Multiple phosphorylation sites provide overlapping and partially redundant layers of regulation that function to efficiently control the activity of Pho4.
Self-Organization of Microtubule Asters Induced in
Xenopus
Egg Extracts by GTP-Bound Ran
Authors: Ohba T. , Nakamura M. , Nishitani H. , Nishimoto T.
Published: July 2002

The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in
Xenopus
egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network.
A Functional Assay for Centromere-Associated Sister Chromatid Cohesion
Authors: Megee Paul C. , Koshland Douglas
Published: July 2002

Cohesion of sister chromatids occurs along the entire length of chromosomes, including the centromere where it plays essential roles in chromosome segregation. Here, minichromosomes in the budding yeast
Saccharomyces cerevisiae
are exploited to generate a functional assay for DNA sequences involved in cohesion. The centromeric DNA element CDEIII was found to be necessary but not sufficient for cohesion. This element was shown previously to be required for assembly of the kinetochore, the centromere-associated protein complex that attaches chromosomes to the spindle. These observations establish a link between centromere-proximal cohesion and kinetochore assembly.
Pharmacogenomics: Translating Functional Genomics into Rational Therapeutics
Authors: Evans William E. , Relling Mary V.
Published: July 2002

Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual differences in the efficacy and toxicity of many medications. Pharmacogenomic studies are rapidly elucidating the inherited nature of these differences in drug disposition and effects, thereby enhancing drug discovery and providing a stronger scientific basis for optimizing drug therapy on the basis of each patient's genetic constitution.
Pharmacological Rescue of Mutant p53 Conformation and Function
Authors: Foster Barbara A. , Coffey Heather A. , Morin Michael J. , Rastinejad Farzan
Published: July 2002

Compounds that stabilize the DNA binding domain of p53 in the active conformation were identified. These small synthetic molecules not only promoted the stability of wild-type p53 but also allowed mutant p53 to maintain an active conformation. A prototype compound caused the accumulation of conformationally active p53 in cells with mutant p53, enabling it to activate transcription and to slow tumor growth in mice. With further work aimed at improving potency, this class of compounds may be developed into anticancer drugs of broad utility.
Natural Selection and Parallel Speciation in Sympatric Sticklebacks
Authors: Rundle Howard D. , Nagel Laura , Boughman Janette Wenrick , Schluter Dolph
Published: July 2002

Natural selection plays a fundamental role in most theories of speciation, but empirical evidence from the wild has been lacking. Here the post-Pleistocene radiation of threespine sticklebacks was used to infer natural selection in the origin of species. Populations of sticklebacks that evolved under different ecological conditions show strong reproductive isolation, whereas populations that evolved independently under similar ecological conditions lack isolation. Speciation has proceeded in this adaptive radiation in a repeatable fashion, ultimately as a consequence of adaptation to alternative environments.
Complementary Neural Mechanisms for Tracking Items in Human Working Memory
Authors: Jiang Yang , Haxby James V. , Martin Alex , Ungerleider Leslie G. , Parasuraman Raja
Published: July 2002

Recognition of a specific visual target among equally familiar distracters requires neural mechanisms for tracking items in working memory. Event-related functional magnetic resonance imaging revealed evidence for two such mechanisms: (i) Enhanced neural responses, primarily in the frontal cortex, were associated with the target and were maintained across repetitions of the target. (ii) Reduced responses, primarily in the extrastriate visual cortex, were associated with stimulus repetition, regardless of whether the stimulus was a target or a distracter. These complementary neural mechanisms track the status of familiar items in working memory, allowing for the efficient recognition of a currently relevant object and rejection of irrelevant distracters.
Wnt Signaling in Oncogenesis and Embryogenesis--a Look Outside the Nucleus
Authors: Peifer Mark , Polakis Paul
Published: July 2002

The Wnt cell-cell signaling pathway plays a critical and evolutionarily conserved role in directing cell fates during embryogenesis. In addition, inappropriate activation of the Wnt signal transduction pathway plays a role in a variety of human cancers. Many recent studies of Wnt signaling have provided mechanistic insight into these dual roles. Here we focus on two areas of rapid advance: (i) the machinery that regulates the stability of the key signal transducer, β-catenin, and (ii) the effect of Wnt signaling on cellular targets outside the nucleus, the actin and microtubule cytoskeletons.
Structure of the RNA Polymerase Domain of
E. coli
Primase
Authors: Keck James L. , Roche Daniel D. , Lynch A. Simon , Berger James M.
Published: July 2002

All cellular organisms use specialized RNA polymerases called “primases” to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the
Escherichia coli
DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the “toprim” fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.
Unfolding Pathways of Individual Bacteriorhodopsins
Authors: Oesterhelt F. , Oesterhelt D. , Pfeiffer M. , Engel A. , Gaub H. E. , Müller D. J.
Published: July 2002

Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from
Halobacterium salinarum
. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.
Promoter-Selective Properties of the TBP-Related Factor TRF1
Authors: Holmes Michael C. , Tjian Robert
Published: July 2002

The TATA-binding protein (TBP)–related factor 1 (TRF1) is expressed in a tissue-restricted fashion during
Drosophila
embryogenesis and may serve as a promoter-specific recognition factor that can replace TBP in regulating transcription. However, bona fide target promoters that would preferentially respond to TRF1 have remained elusive. Polytene chromosome staining, chromatin immunoprecipitation, direct messenger RNA analysis, and transient cotransfection assays identified the
Drosophila
gene
tudor
as containing a TRF1-responsive promoter. Reconstituted in vitro transcription reactions and deoxyribonuclease I footprinting assays confirmed the ability of TRF1 to bind preferentially and direct transcription of the
tudor
gene from an alternate promoter. Thus, metazoans appear to have evolved gene-selective and tissue-specific components of the core transcription machinery to regulate gene expression.
Io's Thermal Emission from the Galileo Photopolarimeter- Radiometer
Authors: Spencer John R. , Rathbun Julie A. , Travis Larry D. , Tamppari Leslie K. , Barnard Laura , Martin Terry Z. , McEwen Alfred S.
Published: July 2002

Galileo's photopolarimeter-radiometer instrument mapped Io's thermal emission during the I24, I25, and I27 flybys with a spatial resolution of 2.2 to 300 kilometers. Mapping of Loki in I24 shows uniform temperatures for most of Loki Patera and high temperatures in the southwest corner, probably resulting from an eruption that began 1 month before the observation. Most of Loki Patera was resurfaced before I27. Pele's caldera floor has a low temperature of 160 kelvin, whereas flows at Pillan and Zamama have temperatures of up to 200 kelvin. Global maps of nighttime temperatures provide a means for estimating global heat flow.
Pig Cloning by Microinjection of Fetal Fibroblast Nuclei
Authors: Onishi Akira , Iwamoto Masaki , Akita Tomiji , Mikawa Satoshi , Takeda Kumiko , Awata Takashi , Hanada Hirohumi , Perry Anthony C. F.
Published: July 2002

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (
Sus scrofa
) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.
A Specificity-Enhancing Factor for the ClpXP Degradation Machine
Authors: Levchenko Igor , Seidel Meredith , Sauer Robert T. , Baker Tania A.
Published: July 2002

Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an
sspB
mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.
Control of Viremia and Prevention of Clinical AIDS in Rhesus Monkeys by Cytokine-Augmented DNA Vaccination
Authors: Barouch Dan H. , Santra Sampa , Schmitz Jörn E. , Kuroda Marcelo J. , Fu Tong-Ming , Wagner Wendeline , Bilska Miroslawa , Craiu Abie , Zheng Xin Xiao , Krivulka Georgia R. , Beaudry Kristin , Lifton Michelle A. , Nickerson Christine E. , Trigona Wendy L. , Punt Kara , Freed Dan C. , Guan Liming , Dubey Sheri , Casimiro Danilo , Simon Adam , Davies Mary-Ellen , Chastain Michael , Strom Terry B. , Gelman Rebecca S. , Montefiori David C. , Lewis Mark G. , Emini Emilio A. , Shiver John W. , Letvin Norman L.
Published: July 2002

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus–1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4
+
T cells, no virus-specific CD4
+
T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4
+
T cell counts, preserved virus-specific CD4
+
T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.
Song Replay During Sleep and Computational Rules for Sensorimotor Vocal Learning
Authors: Dave Amish S. , Margoliash Daniel
Published: July 2002

Songbirds learn a correspondence between vocal-motor output and auditory feedback during development. For neurons in a motor cortex analog of adult zebra finches, we show that the timing and structure of activity elicited by the playback of song during sleep matches activity during daytime singing. The motor activity leads syllables, and the matching sensory response depends on a sequence of typically up to three of the preceding syllables. Thus, sensorimotor correspondence is reflected in temporally precise activity patterns of single neurons that use long sensory memories to predict syllable sequences. Additionally, “spontaneous” activity of these neurons during sleep matches their sensorimotor activity, a form of song “replay.” These data suggest a model whereby sensorimotor correspondences are stored during singing but do not modify behavior, and off-line comparison (e.g., during sleep) of rehearsed motor output and predicted sensory feedback is used to adaptively shape motor output.
Defect-Induced Phase Separation in Dipolar Fluids
Authors: Tlusty T. , Safran S. A.
Published: July 2002

A defect-induced, critical phase separation in dipolar fluids is predicted, which replaces the usual liquid-gas transition that is driven by the isotropic aggregation of particles and is absent in dipolar fluids due to strong chaining. The coexisting phases are a dilute gas of chain ends that coexists with a high-density liquid of chain branching points. Our model provides a unified explanation for the branched structures, the unusually low critical temperature and density, and the consequent two-phase coexistence “islands” that were recently observed in experiment and simulation.
"Fluorescent Timer": Protein That Changes Color with Time
Authors: Terskikh Alexey , Fradkov Arkady , Ermakova Galina , Zaraisky Andrey , Tan Patrick , Kajava Andrey V. , Zhao Xiaoning , Lukyanov Sergey , Matz Mikhail , Kim Stuart , Weissman Irving , Siebert Paul
Published: July 2002

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock–dependent promoter in
Caenorhabditis elegans
and from the
Otx-2
promoter in developing
Xenopus
embryos. Thus, E5 is a “fluorescent timer” that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.
Cholinergic Enhancement and Increased Selectivity of Perceptual Processing During Working Memory
Authors: Furey Maura L. , Pietrini Pietro , Haxby James V.
Published: July 2002

Using functional magnetic resonance imaging, we investigated the mechanism by which cholinergic enhancement improves working memory. We studied the effect of the cholinesterase inhibitor physostigmine on subcomponents of this complex function. Cholinergic enhancement increased the selectivity of neural responses in extrastriate cortices during visual working memory, particularly during encoding. It also increased the participation of ventral extrastriate cortex during memory maintenance and decreased the participation of anterior prefrontal cortex. These results indicate that cholinergic enhancement improves memory performance by augmenting the selectivity of perceptual processing during encoding, thereby simplifying processing demands during memory maintenance and reducing the need for prefrontal participation.
Machine Learning for Science: State of the Art and Future Prospects
Authors: Mjolsness Eric , DeCoste Dennis
Published: July 2002

Recent advances in machine learning methods, along with successful applications across a wide variety of fields such as planetary science and bioinformatics, promise powerful new tools for practicing scientists. This viewpoint highlights some useful characteristics of modern machine learning methods and their relevance to scientific applications. We conclude with some speculations on near-term progress and promising directions.
The Plasticity of Dendritic Cell Responses to Pathogens and Their Components
Authors: Huang Qian , Liu Dongyu , Majewski Paul , Schulte Leah C. , Korn Joshua M. , Young Richard A. , Lander Eric S. , Hacohen Nir
Published: July 2002

Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to
Escherichia coli
,
Candida albicans
, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses.
Biological Cost of AmpC Production for
Salmonella enterica
Serotype Typhimurium
Authors: Morosini M. I. , Ayala J. A. , Baquero F. , Martínez J. L. , Blázquez J.
Published: July 2002

Chromosomally mediated AmpC-type β-lactamases are frequently found among
Enterobacteriaceae
. Hyperproduction of AmpC β-lactamase results in high-level resistance to β-lactam antibiotics. One striking feature of
Salmonella
is the absence of the structural
ampC
gene, encoding AmpC β-lactamase, in contrast with other members in the
Enterobacteriaceae
family, such as
Escherichia
,
Citrobacter
, or
Enterobacter
. The horizontal acquisition of
ampC
genes is one of the causes of the increased resistance to extended-spectrum cephalosporins and β-lactamase inhibitors among gram-negative rods. Nevertheless, despite the high number of β-lactam-resistant
Salmonella
isolates so far described, only two strains expressing resistance to cephalosporin and β-lactamase inhibitors which is mediated by AmpC-type enzymes have been found. In this work, data are provided which support the possibility that the maintenance and expression of the
ampC
gene may represent an unbearable cost for
Salmonella
in terms of reduction of some of its lifestyle attributes, such as growth rate and invasiveness. The deleterious AmpC burden can be eliminated by decreasing the production of AmpC when both the regulatory gene,
ampR
, and
ampC
are present in
Salmonella
. Thus, it is suggested that the two genes have to be acquired together by
Salmonella
, leading to an inducible β-lactam resistance phenotype. AmpC synthesis did not produce major variations in the peptidoglycan composition of
Salmonella
.
Resistance to Methotrexate Due to AcrAB-Dependent Export from
Escherichia coli
Authors: Kopytek Stephan J. , Dyer John C. D. , Knapp Gwendowlyn S. , Hu James C.
Published: July 2002

Many laboratory strains of
Escherichia coli
are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate either
tolC
or
acrA
confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations in
acrA
or
tolC
have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.
A New SHV-Derived Extended-Spectrum β-Lactamase (SHV-24) That Hydrolyzes Ceftazidime through a Single-Amino-Acid Substitution (D179G) in the Ω-Loop
Authors: Kurokawa Hiroshi , Yagi Tetsuya , Shibata Naohiro , Shibayama Keigo , Kamachi Kazunari , Arakawa Yoshichika
Published: July 2002

A new SHV-derived extended-spectrum β-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan. This enzyme was encoded by a transferable 150-kb plasmid from an
Escherichia coli
clinical isolate. The pI and

K
m

for CAZ of this enzyme were 7.5 and 30 μM, respectively. SHV-24 was found to have a D179G substitution in the Ω-loop of the enzyme.
Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in
Pseudomonas aeruginosa
Authors: Masuda Nobuhisa , Sakagawa Eiko , Ohya Satoshi , Gotoh Naomasa , Tsujimoto Hideto , Nishino Takeshi
Published: July 2002

To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in
Pseudomonas aeruginosa
, we constructed a series of isogenic mutants lacking
mexXY
and/or
mexAB
and/or
oprM
from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of the
mexAB-oprM
-deficient-/
mexXY
-maintaining mutant, yet caused no change in the susceptibility of a
mexAB-oprM
- and
mexXY
-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.
Concentration-Dependent Selection of Small Phenotypic Differences in TEM β-Lactamase-Mediated Antibiotic Resistance
Authors: Negri Maria-Cristina , Lipsitch Marc , Blázquez Jesús , Levin Bruce R. , Baquero Fernando
Published: July 2002

In this paper, the first robust experimental evidence of in vitro and in vivo concentration-dependent selection of low-level antibiotic-resistant genetic variants is described. The work is based on the study of an asymmetric competition assay with pairs of isogenic
Escherichia coli
strains, differing only (apart from a neutral chromosomal marker) in a single amino acid replacement in a plasmid-mediated TEM-1 beta-lactamase enzyme, which results in the new TEM-12 beta-lactamase. The mixture was challenged by different antibiotic concentrations, both in vitro and in the animal model, and the selective process of the variant population was carefully monitored. A mathematical model was constructed to test the hypothesis that measured growth and killing rates of the individual TEM variants at different antibiotic concentrations could be used to predict quantitatively the strength of selection for TEM-12 observed in competition experiments at these different concentrations.
A Ribosomal ATPase Is a Target for Hygromycin B Inhibition on
Escherichia coli
Ribosomes
Authors: Ganoza M. Clelia , Kiel Michael C.
Published: July 2002

We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of
Escherichia coli
70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.
Rifampin Resistance in
Mycobacterium kansasii
Is Associated with
rpoB
Mutations
Authors: Klein John L. , Brown Timothy J. , French Gary L.
Published: July 2002

Rifampin is the most potent drug used in the treatment of disease due to
Mycobacterium kansasii
. A 69-bp fragment of
rpoB
, the gene that encodes the β subunit of the bacterial RNA polymerase, was sequenced and found to be identical in five rifampin-susceptible clinical isolates of
M. kansasii
. This sequence showed 87% homology with the
Mycobacterium tuberculosis
gene, with an identical deduced amino acid sequence. In contrast, missense mutations were detected in the same fragment amplified from five rifampin-resistant isolates. A rifampin-resistant strain generated in vitro also harbored an
rpoB
gene missense mutation that was not present in the parent isolate. All mutations detected (in codons 513, 526, and 531) have previously been described in rifampin-resistant
M. tuberculosis
isolates. Rifampin MICs determined by E-test were <1 mg/liter for all rifampin-susceptible isolates and >256 mg/liter for all rifampin-resistant ones. In addition, four of the five rifampin-resistant isolates were also resistant to rifabutin. We have thus shown a strong association between
rpoB
gene missense mutations and rifampin resistance in
M. kansasii
. Although our results are derived from a small number of isolates and confirmation with larger numbers would be useful, they strongly suggest that mutations within
rpoB
form the molecular basis of rifampin resistance in this species.
CTX-M-Type Extended-Spectrum β-Lactamase That Hydrolyzes Ceftazidime through a Single Amino Acid Substitution in the Omega Loop
Authors: Poirel Laurent , Naas Thierry , Le Thomas Isabelle , Karim Amal , Bingen Edouard , Nordmann Patrice
Published: July 2002

Escherichia coli
ILT-1,
Klebsiella pneumoniae
ILT-2, and
K. pneumoniae
ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for
E. coli
ILT-1 and
K. pneumoniae
ILT-2 than for
K. pneumoniae
ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that
E. coli
ILT-1 and
K. pneumoniae
ILT-2 produced the CTX-M-18 β-lactamase, while
K. pneumoniae
ILT-3 produced the CTX-M-19 β-lactamase. The amino acid sequence of the CTX-M-18 β-lactamase differed from that of the CTX-M-9 β-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The
bla
CTX-M-18
and
bla
CTX-M-19
genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.
Resistance-Nodulation-Cell Division-Type Efflux Pump Involved in Aminoglycoside Resistance in
Acinetobacter baumannii
Strain BM4454
Authors: Magnet Sophie , Courvalin Patrice , Lambert Thierry
Published: July 2002

Multidrug-resistant strain
Acinetobacter baumannii
BM4454 was isolated from a patient with a urinary tract infection. The
adeB
gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of
adeB
in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide
m
-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The
adeB
gene was part of a cluster that included
adeA
and
adeC
which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the
adeABC
gene cluster.
Thermoreversible Gel Formulations Containing Sodium Lauryl Sulfate or
n
-Lauroylsarcosine as Potential Topical Microbicides against Sexually Transmitted Diseases
Authors: Roy Sylvie , Gourde Pierrette , Piret Jocelyne , Désormeaux André , Lamontagne Julie , Haineault Caroline , Omar Rabeea F. , Bergeron Michel G.
Published: July 2002

The microbicidal efficacies of two anionic surfactants, sodium lauryl sulfate (SLS) and
n
-lauroylsarcosine (LS), were evaluated in cultured cells and in a murine model of herpes simplex type 2 (HSV-2) intravaginal infection. In vitro studies showed that SLS and LS were potent inhibitors of the infectivity of HSV-2 strain 333. The concentrations of SLS which inhibit viral infectivity by 50% (50% inhibitory dose) and 90% (90% inhibitory dose) were 32.67 and 46.53 μM, respectively, whereas the corresponding values for LS were 141.76 and 225.30 μM. In addition, intravaginal pretreatment of mice with thermoreversible gel formulations containing 2.5% SLS or 2.5% LS prior to the inoculation of HSV-2 strain 333 completely prevented the development of genital herpetic lesions and the lethality associated with infection. Of prime interest, no infectious virus could be detected in mouse vaginal mucosa. Both formulations still provided significant protection when viral challenge was delayed until 1 h after pretreatment. Finally, intravaginal application of gel formulations containing 2.5% SLS or 2.5% LS once daily for 14 days to rabbits did not induce significant irritations to the genital mucosa, as demonstrated from macroscopic and histopathologic examinations. These results suggest that thermoreversible gel formulations containing SLS or LS could represent potent and safe topical microbicides for the prevention of HSV-2 and possibly other sexually transmitted pathogens, including human immunodeficiency virus.
Desbutyl-Benflumetol, a Novel Antimalarial Compound: In Vitro Activity in Fresh Isolates of
Plasmodium falciparum
from Thailand
Authors: Noedl Harald , Allmendinger Thomas , Prajakwong Somsak , Wernsdorfer Gunther , Wernsdorfer Walther H.
Published: July 2002

Desbutyl-benflumetol (DBB) is a novel antimalarial compound closely related to benflumetol (lumefantrine), of which it is a putative metabolite. The in vitro response of
Plasmodium falciparum
to DBB was studied in Mae Hong Son and Mae Sot, in northwest Thailand, in 1997 and 1998. In total, 155 fresh isolates were successfully tested using the World Health Organization standard in vitro microtest system (Mark II). The mean 50% effective concentration (EC
50
) and 90% effective concentration of DBB were 6.36 and 31.09 nmol/liter, respectively. The comparison of the activity of DBB and benflumetol yielded a highly significant potency ratio of 4.52, corresponding to a more than four times higher efficacy of DBB. A considerable potency difference was found between isolates from Mae Hong Son and those from Mae Sot, reflecting lesser sensitivity in the area with marked resistance to mefloquine and quinine. This observation is also supported by a highly significant activity correlation with benflumetol (
P
< 0.001) and to a similar degree with mefloquine (
P
< 0.001), reflecting a close relationship of DBB with the class II aryl amino alcohol blood schizontocides. A less distinct association was also found with artemisinin, which was significant only at the EC
50
level, and there was no correlation at all with chloroquine. DBB is a promising antimalarial compound that merits further investigation in order to define its practical therapeutic potential.
Alkyl-Lysophospholipid Resistance in Multidrug-Resistant
Leishmania tropica
and Chemosensitization by a Novel P-Glycoprotein-Like Transporter Modulator
Authors: Pérez-Victoria José M. , Pérez-Victoria F. Javier , Parodi-Talice Adriana , Jiménez Ignacio A. , Ravelo Angel G. , Castanys Santiago , Gamarro Francisco
Published: July 2002

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in
Leishmania
spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the
Leishmania
P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR)
Leishmania tropica
line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C
5
-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the
Leishmania
P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.
Mutator Bacteria as a Risk Factor in Treatment of Infectious Diseases
Authors: Giraud Antoine , Matic Ivan , Radman Miroslav , Fons Michel , Taddei François
Published: July 2002

We show in a gnotobiotic mouse model that, in addition to direct selection of antibiotic-resistant bacteria, some antibiotic treatments also select for mutator alleles. Because of these mutator alleles' high mutation rates, the initial treatment failure increases the probability of failures in subsequent treatments with other drugs.
Crystal Structure of (−)-Mefloquine Hydrochloride Reveals Consistency of Configuration with Biological Activity
Authors: Karle Jean M. , Karle Isabella L.
Published: July 2002

The absolute configuration of (−)-mefloquine has been established as 11
R
,12
S
by X-ray crystallography of the hydrochloride salt, thus allowing comparison of the configuration of mefloquine's optical isomers to those of quinine and quinidine. (−)-Mefloquine has the same stereochemistry as quinine, and (+)-mefloquine has the same stereochemistry as quinidine. Since (+)-mefloquine is more potent than (−)-mefloquine in vitro against the D6 and W2 strains of
Plasmodium falciparum
and quinidine is more potent than quinine, a common stereochemical component for antimalarial activity is implicated. The crystal of (−)-mefloquine hydrochloride contained four different conformations which mainly differ in a small rotation of the piperidine ring. These conformations are essentially the same as the crystalline conformations of racemic mefloquine methylsulfonate monohydrate, mefloquine hydrochloride, and mefloquine free base. The crystallographic parameters for (−)-mefloquine hydrochloride hydrate were as follows: C
17
H
17
F
6
N
2
O
+
Cl
−
· 0.25 H
2
O;

M
r

, 419.3; symmetry of unit cell, orthorhombic; space group,

P2
1
2
1
2
1

; parameters of unit cell,
a
= 12.6890 ± 0.0006 Å (1 Å = 0.1 nm),
b
= 18.9720 ± 0.0009 Å,
c
= 32.189 ± 0.017 Å; volume of unit cell, 7,749 ± 4 Å
3
; number of molecules per unit cell, 16; calculated density, 1.44 g cm
−3
; source of radiation, Cu Kα (λ = 1.54178 Å); μ (absorption coefficient), 2.373 mm
−1
; room temperature was used; final

R
1

(residual index), 0.0874 for 3,692 reflections with intensities greater than 2σ. All of the hydroxyl and amine hydrogen atoms participate in intermolecular hydrogen bonds with chloride ions. The orientation of the amine and hydroxyl groups in (+)-mefloquine may define the optimal geometry for hydrogen bonding with cellular constituents.
Broad-Spectrum Bactericidal Activity of Ag
2
O-Doped Bioactive Glass
Authors: Bellantone Maria , Williams Huw D. , Hench Larry L.
Published: July 2002

Bioactive glass has found extensive application as an orthopedic and dental graft material and most recently also as a tissue engineering scaffold. Here we report an initial investigation of the in vitro antibacterial properties of AgBG, a novel bioactive glass composition doped with Ag
2
O. The bacteriostatic and bactericidal properties of this new material and of two other bioactive glass compositions, 45S5 Bioglass and BG, have been studied by using
Escherichia coli
,
Pseudomonas aeruginosa
, and
Staphylococcus aureus
as test microorganisms. Concentrations of AgBG in the range of 0.05 to 0.20 mg of AgBG per ml of culture medium were found to inhibit the growth of these bacteria. Not only was AgBG bacteriostatic, but it also elicited a rapid bactericidal action. A complete bactericidal effect was elicited within the first hours of incubation at AgBG concentrations of 10 mg ml
−1
. 45S5 Bioglass and BG had no effect on bacterial growth or viability. The antibacterial action of AgBG is attributed exclusively to the leaching of Ag
+
ions from the glass matrix. Analytical measurements rule out any contribution to AgBG-mediated bacterial killing by changes in pH or ionic strength or the dissolution of other ionic species from the biomaterials. Our observations of the dissolution profiles of Ag
+
from AgBG in the presence and absence of bacteria are consistent with silver accumulation by the bacteria.
Seasonal Population Dynamics and Interactions of Competing Bacteriophages and Their Host in the Rhizosphere
Authors: Ashelford Kevin E. , Norris Susan J. , Fry John C. , Bailey Mark J. , Day Martin J.
Published: July 2002

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated withSerratia liquefaciensCP6RS or its lysogen, CP6RS-ly-Φ1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. APodoviridaephage, identified as ΦCP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, theSiphoviridaephage ΦCP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some ΦCP6-1 phage up to day 137, yet hardly any ΦCP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.
Prevalence of Lyme Disease
Borrelia
spp. in Ticks from Migratory Birds on the Japanese Mainland
Authors: Ishiguro Fubito , Takada Nobuhiro , Masuzawa Toshiyuki , Fukui Takako
Published: July 2002

Borrelia
sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature
Haemaphysalis flava
(94.4%),
Haemaphysalis longicornis
,
Ixodes columnae
,
Ixodes persulcatus
,
Ixodes turdus
, and an unidentified
Ixodes
species. Of these, 27 juveniles of
H. flava
on
Turdus pallidus
,
Turdus cardis
, or
Emberiza spodocephala
, 2 juveniles of
I. persulcatus
on
T. pallidus
, and 1 female
H. flava
molted from a
T. pallidus
-derived nymph were positive for the presence of
Borrelia
by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as
Borrelia garinii
with pattern B/B′ based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to
B. garinii
strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing
B. garinii
may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of
B. garinii
from nymph to adult
H. flava
suggests that the infected
H. flava
may transmit
Borrelia
to large animals.