bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Defensins: Linking Innate and Adaptive Immunity Through Dendritic and T Cell CCR6
Authors: Yang D. , Chertov O. , Bykovskaia S. N. , Chen Q. , Buffo M. J. , Shogan J. , Anderson M. , Schröder J. M. , Wang J. M. , Howard O. M. Z. , Oppenheim J. J.
Published: July 2002

Defensins contribute to host defense by disrupting the cytoplasmic membrane of microorganisms. This report shows that human β-defensins are also chemotactic for immature dendritic cells and memory T cells. Human β-defensin was selectively chemotactic for cells stably transfected to express human CCR6, a chemokine receptor preferentially expressed by immature dendritic cells and memory T cells. The β-defensin–induced chemotaxis was sensitive to pertussis toxin and inhibited by antibodies to CCR6. The binding of iodinated LARC, the chemokine ligand for CCR6, to CCR6-transfected cells was competitively displaced by β-defensin. Thus, β-defensins may promote adaptive immune responses by recruiting dendritic and T cells to the site of microbial invasion through interaction with CCR6.
Phosphorylation and Regulation of Raf by Akt (Protein Kinase B)
Authors: Zimmermann Sven , Moelling Karin
Published: July 2002

Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.
One Polypeptide with Two Aminoacyl-tRNA Synthetase Activities
Authors: Stathopoulos Constantinos , Li Tong , Longman Randy , Vothknecht Ute C. , Becker Hubert D. , Ibba Michael , Söll Dieter
Published: July 2002

The genome sequences of certain archaea do not contain recognizable cysteinyl–transfer RNA (tRNA) synthetases, which are essential for messenger RNA–encoded protein synthesis. However, a single cysteinyl–tRNA synthetase activity was detected and purified from one such organism,
Methanococcus jannaschii
. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl–tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl–tRNA synthetase can synthesize both cysteinyl-tRNA
Cys
and prolyl-tRNA
Pro
. The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.
Strength and Breaking Mechanism of Multiwalled Carbon Nanotubes Under Tensile Load
Authors: Yu Min-Feng , Lourie Oleg , Dyer Mark J. , Moloni Katerina , Kelly Thomas F. , Ruoff Rodney S.
Published: July 2002

The tensile strengths of individual multiwalled carbon nanotubes (MWCNTs) were measured with a “nanostressing stage” located within a scanning electron microscope. The tensile-loading experiment was prepared and observed entirely within the microscope and was recorded on video. The MWCNTs broke in the outermost layer (“sword-in-sheath” failure), and the tensile strength of this layer ranged from 11 to 63 gigapascals for the set of 19 MWCNTs that were loaded. Analysis of the stress-strain curves for individual MWCNTs indicated that the Young's modulusEof the outermost layer varied from 270 to 950 gigapascals. Transmission electron microscopic examination of the broken nanotube fragments revealed a variety of structures, such as a nanotube ribbon, a wave pattern, and partial radial collapse.
Inhibitors of Strand Transfer That Prevent Integration and Inhibit HIV-1 Replication in Cells
Authors: Hazuda Daria J. , Felock Peter , Witmer Marc , Wolfe Abigail , Stillmock Kara , Grobler Jay A. , Espeseth Amy , Gabryelski Lori , Schleif William , Blau Carol , Miller Michael D.
Published: July 2002

Integrase is essential for human immunodeficiency virus–type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.
High-Speed Electrically Actuated Elastomers with Strain Greater Than 100%
Authors: Pelrine Ron , Kornbluh Roy , Pei Qibing , Joseph Jose
Published: July 2002

Electrical actuators were made from films of dielectric elastomers (such as silicones) coated on both sides with compliant electrode material. When voltage was applied, the resulting electrostatic forces compressed the film in thickness and expanded it in area, producing strains up to 30 to 40%. It is now shown that prestraining the film further improves the performance of these devices. Actuated strains up to 117% were demonstrated with silicone elastomers, and up to 215% with acrylic elastomers using biaxially and uniaxially prestrained films. The strain, pressure, and response time of silicone exceeded those of natural muscle; specific energy densities greatly exceeded those of other field-actuated materials. Because the actuation mechanism is faster than in other high-strain electroactive polymers, this technology may be suitable for diverse applications.
Shaped Ceramics with Tunable Magnetic Properties from Metal-Containing Polymers
Authors: MacLachlan Mark J. , Ginzburg Madlen , Coombs Neil , Coyle Thomas W. , Raju Nandyala P. , Greedan John E. , Ozin Geoffrey A. , Manners Ian
Published: July 2002

A shaped, magnetic ceramic was obtained from a metal-containing polymer network, which was synthesized by thermal polymerization of a metal-containing organosilicon monomer. Pyrolysis of a cylinder, shape, or film of the metal-containing polymer precursor produced a low-density magnetic ceramic replica in high yield. The magnetic properties of the shaped ceramic could be tuned between a superparamagnetic and ferromagnetic state by controlling the pyrolysis conditions, with the particular state dependent on the size of iron nanoclusters homogeneously dispersed throughout the carbosilane-graphitic–silicon nitride matrix. These results indicate that cross-linked metal-containing polymers may be useful precursors to ceramic monoliths with tailorable magnetic properties.
Global Biodiversity Scenarios for the Year 2100
Authors: Sala Osvaldo E. , Stuart Chapin F. , III  , Armesto Juan J. , Berlow Eric , Bloomfield Janine , Dirzo Rodolfo , Huber-Sanwald Elisabeth , Huenneke Laura F. , Jackson Robert B. , Kinzig Ann , Leemans Rik , Lodge David M. , Mooney Harold A. , Oesterheld Martı́n , Poff N. LeRoy , Sykes Martin T. , Walker Brian H. , Walker Marilyn , Wall Diana H.
Published: July 2002

Scenarios of changes in biodiversity for the year 2100 can now be developed based on scenarios of changes in atmospheric carbon dioxide, climate, vegetation, and land use and the known sensitivity of biodiversity to these changes. This study identified a ranking of the importance of drivers of change, a ranking of the biomes with respect to expected changes, and the major sources of uncertainties. For terrestrial ecosystems, land-use change probably will have the largest effect, followed by climate change, nitrogen deposition, biotic exchange, and elevated carbon dioxide concentration. For freshwater ecosystems, biotic exchange is much more important. Mediterranean climate and grassland ecosystems likely will experience the greatest proportional change in biodiversity because of the substantial influence of all drivers of biodiversity change. Northern temperate ecosystems are estimated to experience the least biodiversity change because major land-use change has already occurred. Plausible changes in biodiversity in other biomes depend on interactions among the causes of biodiversity change. These interactions represent one of the largest uncertainties in projections of future biodiversity change.
Identification of Vaccine Candidates Against Serogroup B Meningococcus by Whole-Genome Sequencing
Authors: Pizza Mariagrazia , Scarlato Vincenzo , Masignani Vega , Giuliani Marzia Monica , Aricò Beatrice , Comanducci Maurizio , Jennings Gary T. , Baldi Lucia , Bartolini Erika , Capecchi Barbara , Galeotti Cesira L. , Luzzi Enrico , Manetti Roberto , Marchetti Elisa , Mora Marirosa , Nuti Sandra , Ratti Giulio , Santini Laura , Savino Silvana , Scarselli Maria , Storni Elisa , Zuo Peijun , Broeker Michael , Hundt Erika , Knapp Bernard , Blair Eric , Mason Tanya , Tettelin Hervé , Hood Derek W. , Jeffries Alex C. , Saunders Nigel J. , Granoff Dan M. , Venter J. Craig , Moxon E. Richard , Grandi Guido , Rappuoli Rino
Published: July 2002

Neisseria meningitidis
is a major cause of bacterial septicemia and meningitis
.
Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in
Escherichia coli
, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.
Heightened Odds of Large Earthquakes Near Istanbul: An Interaction-Based Probability Calculation
Authors: Parsons Tom , Toda Shinji , Stein Ross S. , Barka Aykut , Dieterich James H.
Published: July 2002

We calculate the probability of strong shaking in Istanbul, an urban center of 10 million people, from the description of earthquakes on the North Anatolian fault system in the Marmara Sea during the past 500 years and test the resulting catalog against the frequency of damage in Istanbul during the preceding millennium. Departing from current practice, we include the time-dependent effect of stress transferred by the 1999 moment magnitude
M
= 7.4 Izmit earthquake to faults nearer to Istanbul. We find a 62 ± 15% probability (one standard deviation) of strong shaking during the next 30 years and 32 ± 12% during the next decade.
Locally Resonant Sonic Materials
Authors: Liu Zhengyou , Zhang Xixiang , Mao Yiwei , Zhu Y. Y. , Yang Zhiyu , Chan C. T. , Sheng Ping
Published: July 2002

We have fabricated sonic crystals, based on the idea of localized resonant structures, that exhibit spectral gaps with a lattice constant two orders of magnitude smaller than the relevant wavelength. Disordered composites made from such localized resonant structures behave as a material with effective negative elastic constants and a total wave reflector within certain tunable sonic frequency ranges. A 2-centimeter slab of this composite material is shown to break the conventional mass-density law of sound transmission by one or more orders of magnitude at 400 hertz.
NF-κB-Induced Loss of
MyoD
Messenger RNA: Possible Role in Muscle Decay and Cachexia
Authors: Guttridge Denis C. , Mayo Marty W. , Madrid Lee V. , Wang Cun-Yu , Baldwin Jr. Albert S.
Published: July 2002

MyoD regulates skeletal muscle differentiation (SMD) and is essential for repair of damaged tissue. The transcription factor nuclear factor kappa B (NF-κB) is activated by the cytokine tumor necrosis factor (TNF), a mediator of skeletal muscle wasting in cachexia. Here, the role of NF-κB in cytokine-induced muscle degeneration was explored. In differentiating C2C12 myocytes, TNF-induced activation of NF-κB inhibited SMD by suppressing MyoD mRNA at the posttranscriptional level. In contrast, in differentiated myotubes, TNF plus interferon-γ (IFN-γ) signaling was required for NF-κB–dependent down-regulation of MyoD and dysfunction of skeletal myofibers. MyoD mRNA was also down-regulated by TNF and IFN-γ expression in mouse muscle in vivo. These data elucidate a possible mechanism that may underlie the skeletal muscle decay in cachexia.
High-Resolution Inkjet Printing of All-Polymer Transistor Circuits
Authors: Sirringhaus H. , Kawase T. , Friend R. H. , Shimoda T. , Inbasekaran M. , Wu W. , Woo E. P.
Published: July 2002

Direct printing of functional electronic materials may provide a new route to low-cost fabrication of integrated circuits. However, to be useful it must allow continuous manufacturing of all circuit components by successive solution deposition and printing steps in the same environment. We demonstrate direct inkjet printing of complete transistor circuits, including via-hole interconnections based on solution-processed polymer conductors, insulators, and self-organizing semiconductors. We show that the use of substrate surface energy patterning to direct the flow of water-based conducting polymer inkjet droplets enables high-resolution definition of practical channel lengths of 5 micrometers. High mobilities of 0.02 square centimeters per volt second and on-off current switching ratios of 10
5
were achieved.
Nonlinear Dimensionality Reduction by Locally Linear Embedding
Authors: Roweis Sam T. , Saul Lawrence K.
Published: July 2002

Many areas of science depend on exploratory data analysis and visualization. The need to analyze large amounts of multivariate data raises the fundamental problem of dimensionality reduction: how to discover compact representations of high-dimensional data. Here, we introduce locally linear embedding (LLE), an unsupervised learning algorithm that computes low-dimensional, neighborhood-preserving embeddings of high-dimensional inputs. Unlike clustering methods for local dimensionality reduction, LLE maps its inputs into a single global coordinate system of lower dimensionality, and its optimizations do not involve local minima. By exploiting the local symmetries of linear reconstructions, LLE is able to learn the global structure of nonlinear manifolds, such as those generated by images of faces or documents of text.
Sequencing of the
rpoB
Gene in
Legionella pneumophila
and Characterization of Mutations Associated with Rifampin Resistance in the
Legionellaceae
Authors: Nielsen K. , Hindersson P. , Høiby N. , Bangsborg J. M.
Published: July 2002

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the
rpoB
gene are known to cause rifampin resistance in
Escherichia coli
and
Mycobacterium tuberculosis
, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family
Legionellaceae
. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the
Legionella pneumophila rpoB
gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the
rpoB
gene (4,104 bp) and an ORF of 384 bp representing part of the
rpoC
gene was obtained. A 316-bp DNA fragment in the center of the
L. pneumophila rpoB
gene, corresponding to a previously described site for mutations leading to rifampin resistance in
M. tuberculosis
, was sequenced from 18 rifampin-resistant
Legionella
isolates representing four species (
L. bozemanii
,
L. longbeachae
,
L. micdadei
, and
L. pneumophila
), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family
Legionellaceae
.
Anti-Human Immunodeficiency Virus Activities of Nucleosides and Nucleotides: Correlation with Molecular Electrostatic Potential Data
Authors: Mickle Travis , Nair Vasu
Published: July 2002

Examination of the anti-human immunodeficiency virus (HIV) data of some normal and isomeric dideoxynucleosides (ddNs and isoddNs), their three-dimensional (3-D) electron density patterns, their electrostatic potential surfaces (EPS), and their conformational maps reveals some interesting correlations. For example, the EPS of (
S,S
)-isoddA shows regions of high and low electrostatic potential remarkably similar to those of β-
d
-3′-azido-3′-deoxythymidine (β-
d
-AZT), (−)-oxetanocin A, and (−)-carbovir. Such correlations involving EPS data and anti-HIV activity were also found with many other active nucleosides. Conversely, inactive compounds had EPS different from those of compounds in the same series that were active. For example, apio-ddNs, which are inactive against HIV, exhibit clear differences in electrostatic potential and 3-D electron density shape from isoddNs that are active against HIV. Additionally, the inactivity of (
S,S
)-isoddC and (
S,S
)-isoddT can be correlated convincingly with a combination of their EPS data and their conformational energy maps. The electrostatic potential distributions of active nucleoside triphosphates show remarkable correlations. For example, (
S,S
)-isoddATP, AZT triphosphate (AZTTP), and oxetanocin A TP have similar 3-D electron density surface patterns and similar high and low regions of electrostatic potential, which may suggest that these compounds proceed through related mechanisms in their interactions with, and inhibition of, HIV reverse transcriptase (RT). Docking of AZTTP, (
S,S
)-isoddATP, and other active triphosphates into the active site of HIV RT and calculation of the EPS of both the nucleotide and the active site show that there is excellent matching between inhibitor and enzyme binding site EPS data. The structure-activity profile discovered has contributed to the development of a first predictive quantitative structure-activity relationship analysis in the area.
Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro
Authors: Heaton Victoria J. , Ambler Jane E. , Fisher L. Mark
Published: July 2002

We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of
Streptococcus pneumoniae
to gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against
S. pneumoniae
7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either
parC
or
gyrA
quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-step
gyrA
mutants of
S. pneumoniae
7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in
parC
resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either
S. pneumoniae
gyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in
S. pneumoniae
, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.
Helicobacter pylori
Uptake and Efflux: Basis for Intrinsic Susceptibility to Antibiotics In Vitro
Authors: Bina J. E. , Alm R. A. , Uria-Nickelsen M. , Thomas S. R. , Trust T. J. , Hancock R. E. W.
Published: July 2002

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567–1572, 1995) that
Helicobacter pylori
has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels.
H. pylori
is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that
H. pylori
is able to take up higher basal levels of the hydrophobic fluorescent probe 1-
N
-phenylnaphthylamine (NPN) than
Pseudomonas aeruginosa
or
Escherichia coli
, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in
H. pylori
(6 to 8 μg/ml) than in
P. aeruginosa
or
E. coli
(0.3 to 0.5 μg/ml), suggesting that
H. pylori
has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic β-lactamase (which
H. pylori
lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in
H. pylori
11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that
H. pylori
contained only these three putative RND efflux systems, named here
hefABC
,
hefDEF
, and
hefGHI
, and that the
hefGHI
system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in
H. pylori
. Independent mutagenesis of the three putative RND efflux operons in the chromosome of
H. pylori
had no effect on the in vitro susceptibility of
H. pylori
to 19 antibiotics. These results, in contrast to what is observed in
E. coli
,
P. aeruginosa
, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of
H. pylori
to antibiotics.
Influence of Shaking on Antifungal Susceptibility Testing of
Cryptococcus neoformans
: a Comparison of the NCCLS Standard M27A Medium, Buffered Yeast Nitrogen Base, and RPMI–2% Glucose
Authors: Rodríguez-Tudela Juan L. , Martín-Díez Francisco , Cuenca-Estrella Manuel , Rodero Laura , Carpintero Yolanda , Gorgojo Begoña
Published: July 2002

Cryptococcus neoformans
is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640–2% glucose, and buffered yeast nitrogen base [BYNB]) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35°C for 48 h were determined. The results indicated that shaking and an inoculum size of 10
5
CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of
C. neoformans
and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI–2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the
C. neoformans
strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for
C. neoformans
.
Prevalence of
gyrA
,
gyrB
,
parC
, and
parE
Mutations in Clinical Isolates of
Streptococcus pneumoniae
with Decreased Susceptibilities to Different Fluoroquinolones and Originating from Worldwide Surveillance Studies during the 1997-1998 Respiratory Season
Authors: Jones Mark E. , Sahm Daniel F. , Martin Nele , Scheuring Sibylle , Heisig Peter , Thornsberry Clyde , Köhrer Karl , Schmitz Franz-Josef
Published: July 2002

From 8,419 worldwide clinical isolates of
Streptococcus pneumoniae
obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, only
parC
(Ser-79→Tyr) and
gyrA
(Ser-81→Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxacin/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.
Inactivation of the
ampD
Gene in
Pseudomonas aeruginosa
Leads to Moderate-Basal-Level and Hyperinducible AmpC β-Lactamase Expression
Authors: Langaee Taimour Yousef , Gagnon Luc , Huletsky Ann
Published: July 2002

It has been shown in enterobacteria that mutations in
ampD
provoke hyperproduction of chromosomal β-lactamase, which confers to these organisms high levels of resistance to β-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC β-lactamase expression of selected clinical isolates and laboratory mutants of
Pseudomonas aeruginosa
. The sequences of the
ampD
genes and promoter regions from these strains were determined and compared to that of wild-type
ampD
from
P. aeruginosa
PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the
ampD
genes of the
P. aeruginosa
mutants transcomplemented
Escherichia coli ampD
mutants to the same levels of β-lactam resistance and β-lactamase expression as wild-type
ampD
. Furthermore, complementation of the
P. aeruginosa
mutants with wild-type
ampD
did not restore the inducibility of β-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC β-lactamase expression in the
P. aeruginosa
mutants. The effects of AmpD inactivation in
P. aeruginosa
PAO1 were further investigated by gene replacement. This resulted in moderate-basal-level and hyperinducible expression of β-lactamase accompanied by high levels of β-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the
P. aeruginosa
mutants studied in this work.
The Biopesticide
Paenibacillus popilliae
Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene Cluster
Authors: Patel Robin , Piper Kerryl , Cockerill Franklin R. , Steckelberg James M. , Yousten Allan A.
Published: July 2002

We have previously identified, in
Paenibacillus popilliae
, a 708-bp sequence which has homology to the sequence of the enterococcal
vanA
gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in
P. popilliae
. The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in
P. popilliae
may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of
P. popilliae
biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens.
Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in
Chryseobacterium meningosepticum
Authors: Bellais Samuel , Aubert Daniel , Naas Thierry , Nordmann Patrice
Published: July 2002

Although the carbapenem-hydrolyzing β-lactamase (CHβL) BlaB-1 is known to be in
Chryseobacterium meningosepticum
NCTC 10585, a second CHβL gene,
bla
GOB-1
, was cloned from another
C. meningosepticum
clinical isolate (PINT). The G+C content of
bla
GOB-1
(36%) indicated the likely chromosomal origin of this gene. Its expression in
Escherichia coli
DH10B yields a mature CHβL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In
E. coli
, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHβLs, sharing 18% with the closest, L-1 from
Stenotrophomonas maltophilia
, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHβLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like β-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated
C. meningosepticum
isolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like β-lactamases was not found in two different isolates.
C. meningosepticum
is a bacterial species with two types of unrelated chromosome-borne class B CHβLs that can be expressed in
E. coli
and, thus, may represent a clinical threat if spread in gram-negative aerobes.
Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase
Authors: Iyobe Shizuko , Kusadokoro Haruko , Ozaki Junko , Matsumura Naoki , Minami Shinzaburo , Haruta Shin , Sawai Tetsuo , O'Hara Koji
Published: July 2002

In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene
bla
IMP
in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene
bla
IMP-3
was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of
bla
IMP-3
was identical to that of
bla
IMP-1
, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid
bla
genes were constructed from
bla
IMP-3
and
bla
IMP-1
by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the
bla
IMP-3
gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene
bla
IMP-3
is an ancestor of
bla
IMP-1
.
Quinolone Resistance Mutations in
Streptococcus pneumoniae
GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins
Authors: Pan Xiao-Su , Yague Genoveva , Fisher L. Mark
Published: July 2002

Mutations in DNA gyrase and/or topoisomerase IV genes are frequently encountered in quinolone-resistant mutants of
Streptococcus pneumoniae
. To investigate the mechanism of their effects at the molecular and cellular levels, we have used an
Escherichia coli
system to overexpress
S
.
pneumoniae
gyrase
gyrA
and topoisomerase IV
parC
genes encoding respective Ser81Phe and Ser79Phe mutations, two changes widely associated with quinolone resistance. Nickel chelate chromatography yielded highly purified mutant His-tagged proteins that, in the presence of the corresponding GyrB and ParE subunits, reconstituted gyrase and topoisomerase IV complexes with wild-type specific activities. In enzyme inhibition or DNA cleavage assays, these mutant enzyme complexes were at least 8- to 16-fold less responsive to both sparfloxacin and ciprofloxacin. The ciprofloxacin-resistant (Cip
r
) phenotype was silent in a sparfloxacin-resistant (Spx
r
)
S
.
pneumoniae gyrA
(Ser81Phe) strain expressing a demonstrably wild-type topoisomerase IV, whereas Spx
r
was silent in a Cip
r
parC
(Ser79Phe) strain. These epistatic effects provide strong support for a model in which quinolones kill
S
.
pneumoniae
by acting not as enzyme inhibitors but as cellular poisons, with sparfloxacin killing preferentially through gyrase and ciprofloxacin through topoisomerase IV. By immunoblotting using subunit-specific antisera, intracellular GyrA/GyrB levels were a modest threefold higher than those of ParC/ParE, most likely insufficient to allow selective drug action by counterbalancing the 20- to 40-fold preference for cleavable-complex formation through topoisomerase IV observed in vitro. To reconcile these results, we suggest that drug-dependent differences in the efficiency by which ternary complexes are formed, processed, or repaired in
S
.
pneumoniae
may be key factors determining the killing pathway.
Streptococcus pneumoniae
Response to Repeated Moxifloxacin or Levofloxacin Exposure in a Rabbit Tissue Cage Model
Authors: Xuan Dawei , Zhong Mingkang , Mattoes Holly , Bui Khanh Q. , McNabb Jocarol , Nicolau David P. , Quintiliani Richard , Nightingale Charles H.
Published: July 2002

The role of moxifloxacin and levofloxacin pharmacokinetics (PK) in antimicrobial efficacy and in the selection of fluoroquinolone-resistant
Streptococcus pneumoniae
strains was investigated using the rabbit tissue cage abscess model. A rabbit tissue cage was created by insertion of sterile Wiffle balls in the dorsal cervical area. Animals orally received a range of moxifloxacin or levofloxacin doses that simulate human PK for 7 days 48 h after the Wiffle balls were inoculated with fluoroquinolone-sensitive
S. pneumoniae
(10
7
CFU). Abscess fluid was collected on a daily basis over 14 days to measure bacterial density and MICs. Moxifloxacin regimens produced a range of area under the concentration-time curve (AUC)/MIC ratios ranging from 9.2 to 444 and peak/MIC ratios ranging from 1.3 to 102. Levofloxacin doses produced AUC/MIC ratios of 5.1 to 85.5 and peak/MIC ratio of 0.9 to 14.8. Moxifloxacin at 6.5, 26, and 42 mg/kg reduced the bacterial log CFU per milliliter in abscess fluid (percentage of that in a sterile animal) by 4.2 ± 2.2 (20%), 5.8 ± 0.4 (100%), and 5.4 ± 0.4 (100%), respectively, over the dosing period. Levofloxacin at 5.5, 22, and 32 mg/kg reduced the log CFU per milliliter in abscess fluid (percentage of that in a sterile animal) by 2.8 ± 0.7 (20%), 5.1 ± 1.3 (80%), and 4.6 ± 1.3 (60%), respectively. Moxifloxacin has a greater bactericidal rate as determined by regression of log CFU versus time data. The AUC/MIC and peak/MIC ratios correlated with the efficacy of both drugs (
P
< 0.05). Resistance to either drug did not develop with any of the doses as assessed by a change in the MIC. In conclusion, data derived from this study show that moxifloxacin and levofloxacin exhibit rapid bactericidal activity against
S. pneumoniae
in vivo, and moxifloxacin exhibits enhanced bactericidal activity compared to levofloxacin, with AUC/MIC and peak/MIC ratios correlated with antimicrobial efficacy for both drugs. The development of fluoroquinolone-resistant
S. pneumoniae
was not observed with either drug in this model.
Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain ofKlebsiella pneumoniae
Authors: Yigit Hesna , Queenan Anne Marie , Anderson Gregory J. , Domenech-Sanchez Antonio , Biddle James W. , Steward Christine D. , Alberti Sebastian , Bush Karen , Tenover Fred C.
Published: July 2002

AKlebsiella pneumoniaeisolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence ofblaSHVandblaTEMgenes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia colishowed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniaecarbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,blaKPC-1, was cloned inE. coliand shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescensS6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parentK. pneumoniaestrain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance inK. pneumoniaestrain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.
Resistance of
Streptococcus pneumoniae
to Deformylase Inhibitors Is Due to Mutations in
defB
Authors: Margolis Peter , Hackbarth Corinne , Lopez Sara , Maniar Mita , Wang Wen , Yuan Zhengyu , White Richard , Trias Joaquim
Published: July 2002

Resistance to peptide deformylase inhibitors in
Escherichia coli
or
Staphylococcus aureus
is due to inactivation of transformylase activity. Knockout experiments in
Streptococcus pneumoniae
R6x indicate that the transformylase (
fmt
) and deformylase (
defB
) genes are essential and that a
def
paralog (
defA
) is not. Actinonin-resistant mutants of
S. pneumoniae
ATCC 49619 harbor mutations in
defB
but not in
fmt
. Reintroduction of the mutated
defB
gene into wild-type
S. pneumoniae
R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
Sulfonamide Resistance in
Haemophilus influenzae
Mediated by Acquisition of
sul2
or a Short Insertion in Chromosomal
folP
Authors: Enne Virve I. , King Anna , Livermore David M. , Hall Lucinda M. C.
Published: July 2002

Determinants of sulfonamide resistance were investigated in clinical isolates of
Haemophilus influenzae
from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in
H. influenzae
has not previously been reported. Eight isolates requiring at least 1,024 μg of sulfamethoxazole per ml for inhibition carried the
sul2
gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase,
folP
, was found to carry an insertion of 15 bp together with other missense mutations relative to
folP
of
H. influenzae
strain Rd RM118 (MIC, 8 μg/ml); the
folP
sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 μg/ml (although these exhibited considerable divergence in
folP
sequence) and in highly resistant isolates carrying
sul2
. Transformation with a 599-bp fragment of
folP
containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by
sul2
and modified
folP
, were detected in isolates from both the United Kingdom and Kenya.
Use of Combined Microautoradiography and Fluorescence In Situ Hybridization To Determine Carbon Metabolism in Mixed Natural Communities of Uncultured Bacteria from the Genus
Achromatium
Authors: Gray N. D. , Howarth R. , Pickup R. W. , Jones J. Gwyn , Head I. M.
Published: July 2002

Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus
Achromatium
. All of the
Achromatium
species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [
14
C]bicarbonate and [
14
C]acetate. This extends previous findings that
Achromatium
spp. present at another location could only utilize organic carbon sources.
Achromatium
spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g.,
Beggiatoa
spp.).
Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of
Clonostachys rosea
and Development of a Strain-Specific PCR Detection Assay
Authors: Bulat Sergey A. , Lübeck Mette , Alekhina Irina A. , Jensen Dan Funck , Knudsen Inge M. B. , Lübeck Peter Stephensen
Published: July 2002

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of
Clonostachys rosea
(syn.
Gliocladium roseum
) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.
xylP
Promoter-Based Expression System and Its Use for Antisense Downregulation of the
Penicillium chrysogenum
Nitrogen Regulator NRE
Authors: Zadra Ivo , Abt Beate , Parson Walther , Haas Hubertus
Published: July 2002

A highly inducible fungal promoter derived from the
Penicillium chrysogenum
endoxylanase (
xylP
) gene is described. Northern analysis and the use of a β-glucuronidase (
uidA
) reporter gene strategy showed that
xylP
expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the
niaD
locus in
P. chrysogenum
and at the
argB
locus in
Aspergillus nidulans
demonstrated that the
xylP
promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the
Aspergillus
species. The
xylP
promoter was found to be 65-fold more efficient than the isopenicillin-
N
-synthetase (
pcbC
) promoter in
Penicillium
and 23-fold more efficient than the nitrate reductase (
niaD
) promoter in
Aspergillus
under induced conditions. Furthermore, the
xylP
promoter was used for controllable antisense RNA synthesis of the
nre
-encoded putative major nitrogen regulator of
P. chrysogenum
. This approach led to inducible downregulation of the steady-state mRNA level of
nre
and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of
nreB
, which encodes a negative-acting nitrogen regulatory GATA factor of
Penicillium
, was found to be subject to regulation by NRE. Our data are the first direct evidence that
nre
indeed encodes an activator in the nitrogen regulatory circuit in
Penicillium
and indicate that cross regulation of the controlling factors occurs.
Genetic Linkage Map of the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Pérez Gúmer , Ritter Enrique , Pisabarro Antonio G. , Ramı́rez Lucı́a
Published: July 2002

We have constructed a genetic linkage map of the edible basidiomycete
Pleurotus ostreatus
(var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (
est1
); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of
P. ostreatus
, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of
P. ostreatus
.