bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐term motor cortex reorganization after facial nerve severing in newborn rats
Authors: Franchi Gianfranco , Veronesi Carlo
Published: December 2004

Using the model of facial nerve injury, we have compared the effect of injury in newborn and adult rats on the adult rat motor cortex (M1). To this end, the facial nerve was severed in 10 newborn rats 2 days after birth (Newborn group) and in 10 adult rats (Adult group). In both the Control (contralateral to untouched nerve) and the Experimental (contralateral to severed nerve) hemisphere of each rat, the M1 output organization was assessed by intracortical microstimulation. Our findings demonstrated that: (i) there is no statistical difference in the percentage of movement sites and in current thresholds required to evoke movement in Control hemispheres between the Adult and Newborn groups of rats; (ii) in Adult Experimental hemispheres, neck sites expand in the medial part of the vibrissae representation more extensively than shown in Newborn Experimental hemispheres; (iii) in Newborn Experimental hemispheres eye sites expand in the medial part of the vibrissae representation more extensively than in Adult Experimental hemispheres (these sites overlap the cortical region where electrical stimulation evokes neck movement in Adult Experimental hemispheres) and (iv) in both Newborn and Adult Experimental hemispheres, forelimb sites expand similarly thereby overlapping the same cortical region, corresponding to the lateral part of the vibrissae representation. We conclude that, when the facial nerve injury is performed in the newborn rat, the pattern of movement representation differs from that obtained with the same lesion in the mature brain only in the frontal cortex corresponding to the medial part of the normal vibrissae representation.
Death of dopaminergic neurones in the rat substantia nigra can be induced by damage to globus pallidus
Authors: Wright A. K. , Atherton J. F. , Norrie L. , Arbuthnott G. W.
Published: December 2004

Parkinson's disease is a debilitating disorder that results from the death of dopaminergic neurones in the substantia nigra. Subthalamic nucleus neurones use glutamate as their neurotransmitter and send excitatory projections to the substantia nigra. Changes in both the mean firing rate and firing pattern of neurones of the subthalamic nucleus have been found in patients with this disease. This has led to the suggestion that hyperactivity of the subthalamic nucleus may be involved in the pathology of the dopaminergic neurones. Subthalamic nucleus lesions or treatment with glutamatergic antagonists can be neuroprotective in animal models of Parkinson's disease but until now there has been no direct evidence that hyperactivity of subthalamic nucleus neurones can lead to downstream cell death. Here we show that lesions of the rat globus pallidus (a treatment that has been shown to increase subthalamic nucleus neuronal activity) result in a significant reduction of the number of dopaminergic neurones in the substantia nigra.
Topography of cortico‐striatal connections in man: anatomical evidence for parallel organization
Authors: Wiesendanger E. , Clarke S. , Kraftsik R. , Tardif E.
Published: December 2004

Tracing studies in non‐human primates support the existence of several parallel neuronal circuits involving cerebral cortex, basal ganglia and thalamus. Distinct functional loops were proposed to underlie multiple aspects of normal and pathological behaviour in man. We present here the first anatomical evidence for separate corticostriatal systems in humans. Neural connections of the sensorimotor and prefrontal cortex to the striatum were studied in one human brain using the Nauta method for anterogradely degenerating axons. Axons originating from a lesion in the left sensorimotor cortex, including the face area, were found to terminate in the superolateral part of the ipsilateral putamen, forming a narrow band in its posterior part. Inside the band, the distribution of degenerating axons was inhomogeneous; high‐density clusters of approximately 2.5 mm in diameter were separated by regions with less dense cortical projections. Axons originating from a small lesion in the fundus of the right superior frontal sulcus were found in the upper part of the ipsilateral caudate nucleus. The existence of discrete and anatomically segregated terminal patches originating from distinct cortical regions suggests parallel organization of cortico‐striatal connections in man.
Caspase‐1 and poly (ADP‐ribose) polymerase inhibitors may protect against peroxynitrite‐induced neurotoxicity independent of their enzyme inhibitor activity
Authors: Zhang Yumin , Rosenberg Paul A.
Published: December 2004

We investigated the mechanism of 3‐morpholinosyndnomine (SIN‐1) neurotoxicity in nearly pure neuronal cultures. In a simple saline solution, SIN‐1 neurotoxicity was found to be mediated by peroxynitrite and independent of glutamate receptor activation [Y. Zhang & P.A. Rosenberg (2002) Eur. J. Neurosci, 16, 1015–1024]. To further study the mechanism of peroxynitrite toxicity to neurons we investigated the role of caspases and poly (ADP‐ribose) polymerase (PARP) in this model system. Ac‐Tyr‐Val‐Ala‐Asp‐chloromethyl ketone (Ac‐YVAD‐cmk), a specific caspase‐1 inhibitor, completely blocked neurotoxicity as well as ATP depletion induced by SIN‐1. However, a caspase‐3 inhibitor and a pan‐caspase inhibitor were both without effect. These results suggested that the protection of Ac‐YVAD‐cmk might not be due to its inhibition of caspase‐1. Indeed, Western blot analysis and assay of caspase activity indicated that caspase activation was not involved in SIN‐1 toxicity. Ac‐YVAD‐cmk also completely blocked in vitro protein nitration induced by SIN‐1 or peroxynitrite, suggesting that Ac‐YVAD‐cmk may interact with peroxynitrite directly. Similarly, although activation of PARP is thought to be a major cause of peroxynitrite‐induced ATP depletion, and two PARP inhibitors, 1,5‐dihydroxyisoquinoline (DHQ) and 3‐aminobenzamide (3‐AB), completely prevented ATP depletion and neurotoxicity induced by SIN‐1, SIN‐1 did not increase poly (ADP‐ribosyl)ation and PARP activity. Furthermore, DHQ and 3‐AB completely prevented in vitro protein nitration induced by peroxynitrite, indicating that DHQ and 3‐AB directly interact with peroxynitrite. Taken together, these results suggest that in the model system used here peroxynitrite neurotoxicity is independent of caspase and PARP activation, and therefore implicate a novel mechanism.
Posttranscriptional regulation of the immediate‐early gene EGR1 by light in the mouse retina
Authors: Simon Perikles , Schott Klaus , Williams Robert W. , Schaeffel Frank
Published: December 2004

Synaptic plasticity is modulated by differential regulation of transcription factors such as EGR1 which binds to DNA via a zinc finger binding domain. Inactivation of EGR1 has implicated this gene as a key regulator of memory formation and learning. However, it remains puzzling how synaptic input can lead to an up‐regulation of the EGR‐1 protein within only a few minutes. Here, we show by immunohistochemical staining that the EGR‐1 protein is localized in synapses throughout the mouse retina. We demonstrate for the first time that two variants of Egr‐1 mRNA are produced in the retina by alternative polyadenylation, with the longer version having an additional 293 base pairs at the end of the 3′UTR. Remarkably, the use of the alternative polyadenylation site is controlled by light. The additional 3′UTR sequence of the longer variant displays an even higher level of phylogenetic conservation than the coding region of this highly conserved gene. Additionally, it harbours a cytoplasmic polyadenylation element which is known to respond to NMDA receptor activation. The longer version of the Egr‐1 mRNA could therefore rapidly respond to excitatory stimuli such as light or glutamate release whereas the short variant, which is predominantly expressed and contains the full coding sequence, lacks the regulatory elements for cytoplasmic polyadenylation in its 3′UTR.
Relevance of ammonium oxidation within biological soil crust communities
Authors: Johnson Shannon L. , Budinoff Charles R. , Belnap Jayne , Garcia‐Pichel Ferran
Published: December 2004

Thin, vertically structured topsoil communities that become ecologically important in arid regions (biological soil crusts or BSCs) are responsible for much of the nitrogen inputs into pristine arid lands. We studied N2 fixation and ammonium oxidation (AO) at subcentimetre resolution within BSCs from the Colorado Plateau. Pools of dissolved porewater nitrate/nitrite, ammonium and organic nitrogen in wetted BSCs were high in comparison with those typical of aridosoils. They remained stable during incubations, indicating that input and output processes were of similar magnitude. Areal N2 fixation rates (6.5–48 µmol C2H2 m−2 h−1) were high, the vertical distribution of N2 fixation peaking close to the surface if populations of heterocystous cyanobacteria were present, but in the subsurface if they were absent. Areal AO rates (19–46 µmol N m−2 h−1) were commensurate with N2 fixation inputs. When considering oxygen availability, AO activity invariably peaked 2–3 mm deep and was limited by oxygen (not ammonium) supply. Most probable number (MPN)‐enumerated ammonia‐oxidizing bacteria (6.7–7.9 × 103 cells g−1 on average) clearly peaked at 2–3 mm depth. Thus, AO (hence nitrification) is a spatially restricted but important process in the nitrogen cycling of BSC, turning much of the biologically fixed nitrogen into oxidized forms, the fate of which remains to be determined.
The Use of USPTO “Continuation” Applications in the Patenting of Software: Implications for Free and Open Source*
Authors: GRAHAM STUART J. H. , MOWERY DAVID C.
Published: December 2004

This paper examines the role played by “continuations” (procedural revisions of patent applications) within software patenting and the implications that the use by patentees of continuations has for free and open‐source software design. Our research analyzes data on continuations in software patenting 1987–99, providing information on the effects of changes made to the U.S. patent laws in 1995 intended to curb “submarine patenting.” Our analysis of all U.S. patents issued 1987–99 shows that the use of continuations grew steadily in overall U.S. patenting through 1995, with particularly rapid growth in continuations in software patenting. Sharp reversals in these growth rates after 1995 suggest that changes in the U.S. patent law were effective. We analyze the role of continuation patents in creating opportunities for patentees to engage in strategic “hold‐up” of software adopters and follow‐on software innovators, and extend the analysis to open‐source software.
The Human Genome Diversity Project: The Politics of Patents at the Intersection of Race, Religion, and Research Ethics
Authors: AMANI BITA , COOMBE ROSEMARY J.
Published: December 2004

The patenting of human genetic materials provokes wide‐ranging misgivings about the appropriate place and scope of intellectual property protections. The issues implicated range from anti‐competitive practices in the market, the imposition of limits on biomedical research, increasing costs for health care, research ethics, potentials for racial discrimination, and various violations of human rights. Exploring controversies around the Human Genome Diversity Project, patents on genetic sequences, and patents on higher life forms such as the so‐called “Harvard mouse,” the authors find that North American patent policy has developed in the absence of necessary political debate. They link this de‐politicization to the hegemony of neo‐liberal principles most fully demonstrated by the incorporation of intellectual property under international trade negotiations. They point, however, to the recent emergence and increasing audibility of new social movements that seek to reposition issues of intellectual property in larger debates about human rights, distributional equalities, and social justice.
An intelligent logistics support system for enhancing the airfreight forwarding business
Authors: Lau H.C.W. , Choy K.L. , Lau Peter K.H. , Tsui W.T. , Choy L.C.
Published: December 2004

Abstract: Recent research related to the aircraft container loading and scheduling problem for airfreight forwarding business has seen significant advances in terms of load plan optimization, taking into account the cost and volume of packed boxes. In today's competitive industrial environment, it is essential that freight forwarders are able to collaborate with carriers (airline companies) to achieve the best possible selection of logistics workflow. However, study of contemporary research publications indicates that there is a dearth of articles related to the design and implementation of an intelligent logistics system to support decision‐making on carrier selection, aircraft container loading plans as well as carrier benchmarking.

This paper presents an intelligent logistics support system (ILSS) which is able to provide expert advice related to the airfreight forwarding business, enhancing the logistics operations in relevant activities within the value chain of tasks. ILSS comprises a heuristics‐based intelligent expert system which supports carrier searching and cargo trading planning as well as load plan generation. The proposed approach is meant to enhance various operations in the airfreight forwarding business, adopting computational intelligence technologies such as rule‐based reasoning to provide domain advice and heuristics to support the generation of load plans. After potential outcomes are generated by the heuristics‐based intelligent expert system, a neural network engine is applied to support prediction of unexpected events. To validate the viability of this approach, a production system using the ILSS has been developed and subsequently applied in an emulated airfreight forwarding environment. The application results indicate that the operation time from searching for potential carriers to the execution of the order is greatly reduced. In this paper, details related to the structure, design and implementation of the ILSS are also covered with the inclusion of the actual program codes for building the prototype.
Herpes B‐virus seroreactivity in a colony of Macaca mulatta: data from the Sabana Seca Field Station, a new Specific Pathogen‐Free Program
Authors: Sariol Carlos A. , Arana Teresa , Maldonado Elizabeth , Gerald Melissa , Gonzalez‐Martinez Janis , Rodriguez Maria , Kraiselburd Edmundo N.
Published: December 2004

Abstract:  The demand for B‐virus‐free animals for biomedical research is increasing, while at the same time the availability of such animals is decreasing. The establishment of Specific Pathogen‐Free (SPF) breeding macaque colonies is a priority of the National Institutes of Health. Nevertheless, it is well known that seroreactivity to B‐virus can be difficult to interpret, particularly as it can vary over time in a single animal. The aim of the present study was to implement a reliable algorithm to examine B‐virus reactivity among the rhesus monkey population of the Caribbean Primate Research Center. The sensitivity and specificity of our assay were determined using reports from two different laboratories as references. Whereas 95.4% of animals showed consistent serological status and 4.6% of animals recruited to this SPF program showed serovariability to B‐virus over the initial 2 years of examination. Implications for all SPF programs are discussed in this article.
Removal of Group B Streptococci Colonizing the Vagina and Oropharynx of Mice with a Bacteriophage Lytic Enzyme
Authors: Cheng Qi , Nelson Daniel , Zhu Shiwei , Fischetti Vincent A.
Published: December 2004

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. The current treatment strategy is limited to intrapartum antibiotic prophylaxis in pregnant women to prevent early-onset neonatal diseases, but considering the potential for antibiotic resistance, the risk of losing control over the disease is high. To approach this problem, we have developed a bacteriophage (phage) lytic enzyme to remove colonizing GBS. Bacteriophage muralytic enzymes, termed lysins, are highly evolved molecules designed to degrade the cell wall of host bacteria to release phage particles from the bacterial cytoplasm. Several different lysins have been developed to specifically kill bacterial pathogens both on mucosal surfaces and in blood and represent a novel approach to control infection. A lysin cloned from a phage infecting GBS was found to contain two putative catalytic domains and one putative binding domain, which is similar to the domain organization of some staphylococcal phage lysins. The lysin (named PlyGBS) was recombinantly expressed in
Escherichia coli
, and purified PlyGBS efficiently killed all tested GBS serotypes in vitro. In a mouse model, a single dose of PlyGBS significantly reduced bacterial colonization in both the vagina and oropharynx. As an alternative strategy for intrapartum antibiotic prophylaxis, this approach may be used to reduce vaginal GBS colonization in pregnant women before delivery or to decontaminate newborns, thus reducing the incidence of GBS-associated neonatal meningitis and sepsis.
Emergence and Spread of
Neisseria gonorrhoeae
Clinical Isolates Harboring Mosaic-Like Structure of Penicillin-Binding Protein 2 in Central Japan
Authors: Ito Masayasu , Deguchi Takashi , Mizutani Koh-Suke , Yasuda Mitsuru , Yokoi Shigeaki , Ito Shin-Ichi , Takahashi Yoshito , Ishihara Satoshi , Kawamura Yoshiaki , Ezaki Takayuki
Published: December 2004

Of 150 clinical isolates of
Neisseria gonorrhoeae
recovered in 2001, we examined 55 clinical isolates of
N. gonorrhoeae
for which cefixime MICs were ≥0.125 μg/ml and randomly selected 15 isolates for which cefixime MICs were ≤0.06 μg/ml for analysis of alterations in the penicillin-binding protein 2 (PBP 2) gene. We found insertion of an extra codon (Asp-345a) in the transpeptidase domain of PBP 2, and this insertion occurred alone or in conjunction with other amino acid substitutions. We also found a mosaic PBP 2 that was composed of fragments of the PBP 2 proteins from
Neisseria cinera
and
Neisseria perflava
. This mosaic PBP 2 was significantly associated with decreased susceptibilities to penicillin and cephalosporins, especially oral cephalosporins. For most of the isolates with a mosaic PBP 2, the cefixime MICs were ≥0.5 μg/ml and the cefdinir MICs were ≥1 μg/ml. Analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis revealed that most isolates with the mosaic PBP 2 were genetically similar. The recombination events that generated the mosaic PBP 2 would likely have contributed to the decreased sensitivities to cephalosporins. Isolates with the mosaic PBP 2 appear to threaten the efficacy of the currently recommended regimen with cefixime. The emergence of such strains may be the result of the in vivo generation of clones in which interspecies recombination occurred between the
penA
genes of
N. gonorrhoeae
and commensal
Neisseria
species.
Efficacy and Safety of Pharmacokinetically Enhanced Amoxicillin-Clavulanate at 2,000/125 Milligrams Twice Daily for 5 Days versus Amoxicillin-Clavulanate at 875/125 Milligrams Twice Daily for 7 Days in the Treatment of Acute Exacerbations of Chronic Bronchitis
Authors: Sethi Sanjay , Breton John , Wynne Brian
Published: December 2004

This randomized, controlled trial was designed to show that a short, 5-day course of pharmacokinetically enhanced amoxicillin-clavulanate at 2,000/125 mg (Augmentin XR) is as effective clinically as a longer, 7-day course of conventional amoxicillin-clavulanate at 875/125 mg (both given twice daily) in the treatment of acute exacerbations of chronic bronchitis (AECB). Amoxicillin-clavulanate at 2,000/125 mg was designed to extend the therapeutic levels of amoxicillin in serum over the 12-h dosing interval, compared with conventional formulations, to eradicate bacterial strains for which amoxicillin MICs were ≤4 μg/ml while retaining efficacy against β-lactamase-producing pathogens. A total of 893 patients were randomized and received study medication (amoxicillin-clavulanate at 2,000/125 mg for 443 patients and 875/125 mg for 450 patients). Overall, 141 patients receiving amoxicillin-clavulanate at 2,000/125 mg and 135 receiving the comparator formulation had at least one pathogen identified at screening. Amoxicillin-clavulanate at 2,000/125 mg was as effective clinically in the per-protocol (PP) population at the test of cure (days 14 to 21, primary efficacy endpoint) as amoxicillin-clavulanate at 875/125 mg (clinical success rates of 93.0 and 91.2%, respectively; treatment difference, 1.8; 95% confidence interval [CI], −2.2, 5.7). Bacteriological success in the bacteriology PP population was high for both formulations (amoxicillin-clavulanate at 2,000/125 mg, 76.7%; amoxicillin-clavulanate at 875/125 mg, 73.0%; treatment difference, 3.8; 95% CI, −7.5, 15.0). Both therapies were well tolerated, with a similar incidence of adverse events. Fewer than 5% of patients in each group withdrew from the study due to adverse events. The shorter, 5-day course of amoxicillin-clavulanate at 2,000/125 mg was shown to be as effective clinically as a longer, 7-day course of amoxicillin-clavulanate at 875/125 mg, with high bacteriological efficacy and no difference in tolerability.
Induction of β-Defensin Resistance in the Oral AnaerobePorphyromonas gingivalis
Authors: Shelburne Charles E. , Coulter Wilson A. , Olguin De'Avlin , Lantz Marilyn S. , Lopatin Dennis E.
Published: December 2004

Induction of resistance of oral anaerobes to the effects of human β-defensin 1 (hβD-1) to hβD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures ofPorphyromonas gingivaliswere (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42°C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 μl) were distributed among the wells of sterile 384-well plates containing hβD-1 to -4 (100 μg/ml). Plates were incubated at 37°C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins forP. gingivaliswere 3 to 12 μg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 μg of defensin per ml inP. gingivalis. Resistance induced by sublethal levels of hβD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hβD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogenP. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.
Pharmacokinetics of Tigecycline after Single and Multiple Doses in Healthy Subjects
Authors: Muralidharan Gopal , Micalizzi Marlynne , Speth John , Raible Donald , Troy Steven
Published: December 2004

Tigecycline, a novel glycylcycline antibiotic, exhibits strong activity against gram-positive, gram-negative, aerobic, anaerobic, and atypical bacterial species, including many resistant pathogens, i.e., vancomycin-resistant enterococci, methicillin-resistant
Staphylococcus aureus
and penicillin-resistant
Streptococcus pneumoniae
. The safety and tolerability of tigecycline administered as single or multiple doses or at various infusion rates were explored in three phase 1, randomized, double-blind, placebo-controlled studies in healthy subjects. Full pharmacokinetic profiles of tigecycline were determined in two of these studies. Subjects in the single-dose study received 12.5 to 300 mg of tigecycline, which differed with respect to the duration of infusion, subjects' feeding status, and ondansetron pretreatment. Subjects in the ascending multiple-dose study received 25 to 100-mg doses of tigecycline as a 1-h infusion every 12 h. The variable volume and infusion rate study consisted of administration of 100-mg loading dose of tigecycline, followed by 50 mg every 12 h for 5 days. Serum samples were analyzed for tigecycline by validated high-pressure liquid chromatography or liquid chromatography/tandem mass spectrometry methods. Systemic clearance ranged from 0.2 to 0.3 liters/h/kg, and the tigecycline half-life ranged from 37 to 67 h. Tigecycline had a large volume of distribution (7 to 10 liters/kg), indicating extensive distribution into the tissues. Food increased the maximum tolerated single-dose from 100 to 200 mg, but the duration of infusion did not affect tolerability. Side effects, mainly nausea and vomiting, which are common to the tetracycline class of antimicrobial agents, were seen in these studies. Tigecycline exhibits linear pharmacokinetics and is safe and well tolerated in the dose ranges examined.
Overexpression of the Multidrug Efflux Operon
acrEF
by Insertional Activation with IS
1
or IS
10
Elements in
Salmonella enterica
Serovar Typhimurium DT204
acrB
Mutants Selected with Fluoroquinolones
Authors: Olliver Anne , Vallé Michel , Chaslus-Dancla Elisabeth , Cloeckaert Axel
Published: December 2004

High-level fluoroquinolone (FQ) resistance in
Salmonella enterica
serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant
acrB
-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204
acrB
mutant strains, an insertion sequence (IS
1
or IS
10
) was found integrated upstream of the
acrEF
operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS
1
caused partial deletion of
acrS
, the putative local repressor gene of the
acrEF
operon. Sequence analysis showed that both IS
1
and IS
10
elements contain putative promoter sequences that might alter the expression of adjacent
acrEF
genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of
acrF
in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since
acrF
deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of
acrS
did not cause any
acrF
overexpression or any decrease in drug susceptibility, suggesting that
acrEF
overexpression is mediated solely by the IS
1
and IS
10
promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS
1
and IS
10
elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS
1
and/or IS
10
elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS
1
- or IS
10
- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.
Molecular Characterization of Cefoxitin-Resistant
Escherichia coli
from Canadian Hospitals
Authors: Mulvey Michael R. , Bryce Elizabeth , Boyd David A. , Ofner-Agostini Marianna , Land Allison M. , Simor Andrew E. , Paton Shirley
Published: December 2004

A study designed to gain baseline information on strains of
Escherichia coli
displaying resistance to cefoxitin in Canada is described. A total of 29,323
E. coli
isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative. A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. Two hundred thirty-two isolates were identified as resistant to cefoxitin. All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable. PCR and sequence analysis of the
ampC
promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters. Three promoter regions were interrupted by insertion elements, two contained IS
10
elements, and one contained an IS
911
variant. PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters. The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting.
IS
Ecp1B
-Mediated Transposition of
bla
CTX-M
in
Escherichia coli
Authors: Poirel Laurent , Lartigue Marie-Frédérique , Decousser Jean-Winoc , Nordmann Patrice
Published: December 2004

Several expanded-spectrum β-lactamase
bla
CTX-M
genes are associated with IS
Ecp1
-like elements in
Enterobacteriaceae
. We found that IS
Ecp1B
was able to mobilize the adjacent
bla
CTX-M-19
gene by a transpositional mechanism in
Escherichia coli
by recognizing a variety of DNA sequences as right inverted repeats.
The
trans
-Sialidase from
Trypanosoma cruzi
Induces Thrombocytopenia during Acute Chagas' Disease by Reducing the Platelet Sialic Acid Contents
Authors: Tribulatti María Virginia , Mucci Juan , Van Rooijen Nico , Leguizamón María Susana , Campetella Oscar
Published: December 2004

Strong thrombocytopenia is observed during acute infection with
Trypanosoma cruzi
, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds
trans
-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of
trans
-sialidase into naïve mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43
−/−
mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with
trans
-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naïve animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of
trans
-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more
trans
-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a
trans
-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the
trans
-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.
Fungal Metabolite Gliotoxin Targets Flavocytochrome
b
558
in the Activation of the Human Neutrophil NADPH Oxidase
Authors: Nishida Satoshi , Yoshida Lucia S. , Shimoyama Takashi , Nunoi Hiroyuki , Kobayashi Toshihiro , Tsunawaki Shohko
Published: December 2004

Fungal gliotoxin (GT) is a potent inhibitor of the O
2
−
-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47

phox

, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C βII with p47

phox

on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O
2
−
-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome
b
558
is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome
b
558
and cytosolic components (native cytosol or recombinant p67

phox

, p47

phox

, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 μM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67

phox

, p47

phox

, and Rac2 with flavocytochrome
b
558
is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome
b
558
's ability to reconstitute O
2
−
generation, supporting the conclusion at the cellular level. The flavocytochrome
b
558
heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome
b
558
, which is accessible only before oxidase activation.
Association of a Major Protein Antigen of
Mycoplasma arthritidis
with Virulence
Authors: Tu A.-H. T. , Clapper B. , Schoeb T. R. , Elgavish A. , Zhang J. , Liu L. , Yu H. , Dybvig K.
Published: December 2004

Mycoplasma arthritidis
causes acute polyarthritis in rats and chronic proliferative arthritis in mice.
M. arthritidis
-induced arthritis serves as a model for arthritis caused by infectious agents and as a model for examining the role of the superantigen MAM (
M. arthritidis
T-cell mitogen) in the development of autoimmunity.
M. arthritidis
strain 158-1 is a spontaneous mutant of strain 158 that has a drastic reduction in virulence. We show that the mutant is missing a major antigen of 47 kDa (P47) and has acquired a protein of 67 kDa (P67). P47 and P67 partitioned into the detergent phase by extraction with Triton X-114. Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels show that P67 is produced in abundance. Analysis of gel-purified P67 by mass spectrometry led to its identification as a lipoprotein (the open reading frame [ORF] 619 gene product) predicted from the genome sequence of
M. arthritidis
. PCR analysis of genomic DNA from 158 and 158-1 indicates that P47 and P67 are encoded by the same ORF 619 gene and differ only in the number of repeats in a tandem repeat region. By two-dimensional polyacrylamide gel analysis, no protein differences were detectable between 158 and 158-1 other than P47 and P67. Collectively, the data suggest that the tandem repeat region of P47 and P67 influences disease outcome.
Invasiveness of Serotypes and Clones of
Streptococcus pneumoniae
among Children in Finland
Authors: Hanage William P. , Kaijalainen Tarja H. , Syrjänen Ritva K. , Auranen Kari , Leinonen Maija , Mäkelä P. Helena , Spratt Brian G.
Published: December 2004

Streptococcus pneumoniae
(the pneumococcus) causes diseases from otitis media to life-threatening invasive infection. The species is extremely antigenically and clonally diverse. We wished to determine odds ratios (ORs) for serotypes and clones of
S. pneumoniae
that cause invasive disease in Finland. A total of 224 isolates of
S. pneumoniae
from cases of invasive disease in children <2 years of age in Finland between 1995 and 1999 were serotyped, and sequence types (STs) were determined by multilocus sequence typing. These STs were compared with a previously published carriage data set. STs from invasive disease were significantly less diverse than those from carriage (invasive disease, 0.038 ± 0.01; carriage, 0.019 ± 0.005). The ORs of serotypes 14, 18C, 19A, and 6B were significantly greater than 1, indicating association with invasive disease. The ORs of 6A and 11A were significantly less than 1. The difference between 6A and 6B is significant, which suggests that relatively subtle changes in the capsule may have a dramatic effect upon disease potential. We found that ST 156, the Spain
9V
-3 clone which mainly expressed serotype 14 in Finland, is strongly associated with invasive disease (OR, 10.1; 95% confidence interval, 1.3 to 79.5). Significant associations with invasive disease were also detected for STs 482, 191, 124, and 138, and associations with carriage were detected for STs 485 and 62. These results demonstrate the invasive phenotype of the serotype 14 variant of the Spain
9V
-3 clone and differences between members of the same serogroup in invasive disease potential.
Cytolethal Distending Toxin from Shiga Toxin-Producing
Escherichia coli
O157 Causes Irreversible G
2
/M Arrest, Inhibition of Proliferation, and Death of Human Endothelial Cells
Authors: Bielaszewska Martina , Sinha Bhanu , Kuczius Thorsten , Karch Helge
Published: December 2004

Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing
Escherichia coli
(STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H
−
(strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G
2
/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G
2
/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G
2
/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G
2
/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G
2
/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases.
A Mutation in the
sap
Operon Attenuates Survival of Nontypeable
Haemophilus influenzae
in a Chinchilla Model of Otitis Media
Authors: Mason Kevin M. , Munson Robert S. , Bakaletz Lauren O.
Published: December 2004

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The
sap
(sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of
Salmonella
. We have recently shown that
sapA
gene expression is upregulated in the middle ear in a chinchilla model of nontypeable
Haemophilus influenzae
(NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the
sapA
promoter and demonstrated early yet transient expression of the
sap
operon within sites of the chinchilla upper airway upon infection. We hypothesized that the
sap
operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the
sapA
gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The
sapA
mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla β-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The
sapA
mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the
sap
operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.
Truncated Internalin A and Asymptomatic
Listeria monocytogenes
Carriage: In Vivo Investigation by Allelic Exchange
Authors: Olier Maïwenn , Garmyn Dominique , Rousseaux Sandrine , Lemaître Jean-Paul , Piveteau Pascal , Guzzo Jean
Published: December 2004

Allelic exchange of the region coding for the C terminus of InlA between one epidemic (with an 80-kDa InlA) and one asymptomatic (with a 47-kDa InlA) carriage
Listeria monocytogenes
strain confirmed the need for this region for internalin entry in vitro. Interestingly, restoration of internalin A functionality did not result in full virulence in chicken embryo assays.
LENS KNOTS, PERIODIC LINKS AND VASSILIEV INVARIANTS
Authors: JEONG MYEONG-JU , PARK CHAN-YOUNG
Published: December 2004

In this paper, we study lens knots and periodic knots by using integral Vassiliev invariants. Knot polynomials such as the Jones, HOMFLY, Kauffman polynomials give infinitely many integral Vassiliev invariants and we get some necessary conditions for a link to be a lens knot or a periodic link by using these polynomial invariants.
Blockade of brain mineralocorticoid receptors or Na+ channels prevents sympathetic hyperactivity and improves cardiac function in rats post-MI
Authors: Huang Bing S. , Leenen Frans H. H.
Published: December 2004

In rats post-myocardial infarction (MI), sympathetic hyperactivity can be prevented by blockade of brain mineralocorticoid receptors (MR). Stimulatory responses to central infusion of aldosterone can be blocked by benzamil and therefore appear to be mediated via Na+ channels, presumably epithelial Na+ channels (ENaC), in the brain. To evaluate this concept of endogenous mineralocorticoids in Wistar rats post-MI, we examined effects of blockade of MR and Na+ channels in the brain. At 3 days after coronary artery ligation, intracerebroventricular infusions were started with spironolactone (400 ng·kg−1·h−1) or its vehicle, or with benzamil (4 μg·kg−1·h−1) or its vehicle, using osmotic minipumps. Rats with sham ligation served as control. After 4 wk, in conscious rats, mean arterial pressure, heart rate, and renal sympathetic nerve activity were recorded at rest and in response to air-jet stress, intracerebroventricular injection of the α2-adrenoceptor agonist guanabenz, and intravenous infusion of phenylephrine and nitroprusside for baroreflex function. MI size was similar among the four groups of rats (∼31%). In rats treated post-MI with vehicles, cardiac function was decreased, sympathetic reactivity was enhanced, and baroreflex function was impaired. Blockade of brain Na+ channels or brain MR similarly prevented sympathetic hyperactivity and impairment of baroreflex function and improved cardiac function. These findings suggest that in rats post-MI, increased binding of endogenous agonists to MR increases ENaC activity in the brain and thereby leads to sympathetic hyperactivity and progressive left ventricular dysfunction.
Both Kappa and Mu Opioid Agonists Inhibit Glutamatergic Input to Ventral Tegmental Area Neurons
Authors: Margolis Elyssa B. , Hjelmstad Gregory O. , Bonci Antonello , Fields Howard L.
Published: December 2004

The ventral tegmental area (VTA) plays a critical role in motivation and reinforcement. Kappa and μ opioid receptor (KOP-R and MOP-R) agonists microinjected into the VTA produce powerful and largely opposing motivational actions. Glutamate transmission within the VTA contributes to these motivational effects. Therefore information about opioid control of glutamate release onto VTA neurons is important. To address this issue, we performed whole cell patch-clamp recordings in VTA slices and measured excitatory postsynaptic currents (EPSCs). There are several classes of neuron in the VTA: principal, secondary, and tertiary. The KOP-R agonist ( trans)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593; 1 μM) produced a small reduction in EPSC amplitude in principal neurons (14%) and a significantly larger inhibition in secondary (47%) and tertiary (33%) neurons. The MOP-R agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO; 3 μM) inhibited glutamate release in principal (42%), secondary (45%), and tertiary neurons (35%). Unlike principal and tertiary neurons, in secondary neurons, the magnitude of the U69593 EPSC inhibition was positively correlated with that produced by DAMGO. Finally, DAMGO did not occlude the U69593 effect in principal neurons, suggesting that some glutamatergic terminals are independently controlled by KOP and MOP receptor activation. These findings show that MOP-R and KOP-R agonists regulate excitatory input onto each VTA cell type.
Pharmacology of Acetylcholine-Mediated Cell Signaling in the Lateral Line Organ Following Efferent Stimulation
Authors: Dawkins Rosie , Keller Sarah L. , Sewell William F.
Published: December 2004

Cholinergic efferent fibers modify hair cell responses to mechanical stimulation. It is hypothesized that calcium entering the hair cell through a nicotinic receptor activates a small-conductance (SK), calcium-activated potassium channel to hyperpolarize the hair cell. The calcium signal may be amplified by calcium-induced calcium release from the synaptic cisternae. Pharmacological tests of these ideas in the intact cochlea have been technically difficult because of the complex and fragile structure of the mammalian inner ear. We turned to the Xenopus laevis lateral line organ, whose simplicity and accessibility make it a model for understanding hair cell organ function in a relatively intact system. Drugs were applied to the inner surface of the skin while monitoring the effects of efferent stimulation on afferent fiber discharge rate. Efferent effects were blocked by antagonists of SK channels including apamin (EC50= 0.5 μM) and dequalinium (EC50= 12 μM). The effect of apamin was not enhanced by co-administration of phenylmethylsulfonyl fluoride, a proteolysis inhibitor. Efferent effects were attenuated by ryanodine, an agent that can interfere with calcium-induced calcium release, although relatively high (mM) concentrations of ryanodine were required. Fluorescent cationic styryl dyes, 4-di-2-asp and fm 1–43, blocked efferent effects, although it was not possible to observe specific entry of the dye into the base of hair cells. These pharmacological findings in the Xenopus lateral line organ support the hypothesis that effects of efferent stimulation are mediated by calcium entry through the nicotinic receptor via activation of SK channels and suggest the generality of this mechanism in meditating cholinergic efferent effects.