bluuebunny/crossref_metadata_2025 huggingface dataset:
Orexins induce increased Excitability and Synchronisation of Rat Sympathetic Preganglionic Neurones
Authors: van den Top Marco , Nolan Matthew F. , Lee Kevin , Richardson Peter J. , Buijs Ruud M. , Davies Ceri H. , Spanswick David
Published: April 2003

The neuropeptides orexin A and B are synthesised by perifornical and lateral hypothalamic (LH) neurones and exert a profound influence on autonomic sympathetic processes. LH neurones project to spinal areas containing sympathetic preganglionic neurones (SPNs) and therefore may directly modulate sympathetic output. In the present study we examined the possibility that orexinergic inputs from the LH influence SPN activity. Orexin‐positive neurones in the LH were labelled with pseudorabies virus injected into the liver of parasympathetically denervated animals and orexin fibres were found adjacent to the soma and dendrites of SPNs. Orexin A or B (10–1000 nm) directly and reversibly depolarised SPNs in spinal cord slices. The response to orexin A was significantly reduced in the presence of the orexin receptor 1 (OX1R) antagonist SB334867A at concentrations of 1–10 μm. Single cell reverse transcriptase‐polymerase chain reaction revealed expression of mRNA for both OX1R and OX2R in the majority of orexin‐sensitive SPNs. The orexin‐induced depolarisation involved activation of pertussis toxin‐sensitive G‐proteins and closure of a K+ conductance via a protein kinase A (PKA)‐dependent pathway that did not require an increase in intracellular Ca2+. Orexins also induced biphasic subthreshold membrane potential oscillations and synchronised activity between pairs of electrically coupled SPNs. Coupling coefficients and estimated junctional conductances between SPNs were not altered indicating synchronisation is due to activation of previously silent coupled neurones rather than modulation of gap junctions. These findings are consistent with a direct excitation and synchronisation of SPNs by orexinergic neurones that in vivo could increase the frequency and coherence of sympathetic nerve discharges and mediate LH effects on sympathetic components of energy homeostasis and cardiovascular control.
Disruptive effects of Anion Secretion Inhibitors on Airway Mucus Morphology in Isolated Perfused Pig Lung
Authors: Trout Laura , Townsley Mary I. , Bowden Amy L. , Ballard Stephen T.
Published: April 2003

Since anion secretion inhibitors reproduce important aspects of cystic fibrosis (CF) lung disease, the effects of these antagonists on airway mucus morphology were assessed in isolated perfused pig lungs. Maximal inhibitory concentrations of bumetanide and dimethylamiloride, which respectively block Cl− and HCO3− secretion in porcine airways, induced the formation of dense ‘plastered’ mucus on the airway surface, depletion of periciliary fluid and collapse of cilia. This effect was more pronounced when lungs were also exposed to bethanechol to stimulate submucosal gland secretion, when plastered mucus covered > 98% of the airway surface. Bethanechol also reduced gland duct mucin content in the absence, but not presence, of the anion secretion inhibitors. Anion secretion inhibitors did not induce measurable increases in goblet cell degranulation. We conclude that inhibition of anion and liquid secretion in porcine lungs disrupts the normal morphology of airway surface mucus, providing further evidence that impaired anion secretion alone could account for critical aspects of CF lung disease.
Synaptically Activated Ca2+ Waves in Layer 2/3 and Layer 5 Rat Neocortical Pyramidal Neurons
Authors: Larkum Matthew E. , Watanabe Shigeo , Nakamura Takeshi , Lasser‐Ross Nechama , Ross William N.
Published: April 2003

Calcium waves in layer 2/3 and layer 5 neocortical somatosensory pyramidal neurons were examined in slices from 2‐ to 8‐week‐old rats. Repetitive synaptic stimulation evoked a delayed, all‐or‐none [Ca2+]i increase primarily on the main dendritic shaft. This component was blocked by 1 mm (R,S)‐α‐methyl‐4‐carboxyphenylglycine (MCPG), 10 μm ryanodine, 1 mg ml−1 internal heparin, and was not blocked by 400 μm internal Ruthenium Red, indicating that it was due to Ca2+ release from internal stores by inositol 1,4,5‐trisphosphate (IP3) mobilized via activation of metabotropic glutamate receptors. Calcium waves were initiated on the apical shaft at sites between the soma to around the main branch point, mostly at insertion points of oblique dendrites, and spread in both directions along the shaft. In the proximal dendrites the peak amplitude of the resulting [Ca2+]i change was much larger than that evoked by a train of Na+ spikes. In distal dendrites the peak amplitude was comparable to the [Ca2+]i change due to a Ca2+ spike. IP3‐mediated Ca2+ release also was observed in the presence of the metabotropic agonists t‐ACPD and carbachol when backpropagating spikes were generated. Ca2+ entry through NMDA receptors was observed primarily on the oblique dendrites. The main differences between waves in neocortical neurons and in previously described hippocampal pyramidal neurons were, (a) Ca2+ waves in L5 neurons could be evoked further out along the main shaft, (b) Ca2+ waves extended slightly further out into the oblique dendrites and (c) higher concentrations of bath‐applied t‐ACPD and carbachol were required to generate Ca2+ release events by backpropagating action potentials.
Oxygen Partial Pressure in Outer Layers of Skin of Human Finger Nail Folds
Authors: Wang W. , Winlove C. P. , Michel C. C.
Published: April 2003

To gain insight into oxygen transport by the cutaneous microcirculation, we have developed oxygen‐sensitive microelectrodes (tip diameter ∼5 μm) to measure the distribution of PO2 in dermal papillae of the finger nail folds of healthy human subjects. Oxygen entry into the tissue was minimised by covering the skin with a layer of paraffin oil. The finger was held under a dissecting microscope and microelectrodes were guided into position. PO2 varied from 5–25 % of its atmospheric value, Pair (∼160 mmHg), depending on the location within the papilla. Along the axis of a papillary loop, PO2 decreased from 40.0 ± 4.8 mmHg (mean ±s.e.m., n= 6) at the base to 30.4 ± 5.2 mmHg (n= 6) at the tip. The lowest values of PO2, in the range of 5 % of Pair, were measured in the epidermis where the metabolism of cells was highest and the steepest PO2 gradients were recorded in the vicinity of the epidermal–dermal boundary. When the local circulation was abruptly reduced or stopped, PO2 fell exponentially with time, with a time constant of 8.4 ± 1.5 s (n= 7). When flow was reinstated, PO2 rose exponentially to a new value with a time constant of 4.8 ± 0.8 s (n= 6). The steady state PO2 following reperfusion was ∼23 % higher than the pre‐occlusion value (P < 0.05, ANOVA and two‐tailed Student's t test) indicating localised reactive hyperaemia.
Central Nervous System Prostaglandin Endoperoxide Synthase‐1 and −2 Responses to Oestradiol and Cerebral Hypoperfusion in Late‐Gestation Fetal Sheep
Authors: Wood Charles E. , Giroux Damian
Published: April 2003

Previous work in this laboratory has demonstrated that cerebral hypoperfusion increases the expression of prostaglandin endoperoxide synthase‐2 (PGHS‐2) in ovine fetal brain regions. Endogenously produced prostaglandins, in turn, partially mediate the fetal hypothalamus‐ pituitary‐adrenal (HPA) axis response to arterial hypotension. In separate experiments, we have found that oestradiol stimulates fetal HPA axis activity. The present experiments were designed to test the hypothesis that oestradiol increases the expression of PGHS isoforms, and that oestradiol augments the PGHS response to cerebral hypoperfusion. Sixteen fetal sheep of known gestational ages (124–128 days' gestation at the time of study) were subjected to chronic catheterization and implantation of extravascular occluder around the brachiocephalic artery. Eight fetuses were subjected to subcutaneous implantation of a pellet containing 17β‐oestradiol (release rate 5 mg (21 days)−1). Brachiocephalic occlusion (BCO) stimulated adrenocorticotropin (ACTH), cortisol and arginine vasopressin (AVP) secretion, responses that were augmented by oestradiol. One hour after the beginning of a 10 min period of BCO, PGHS‐1 mRNA was increased in fetal brainstem and hypothalamus, and PGHS‐2 mRNA was increased in fetal brainstem. Oestradiol, by itself, increased the abundance of PGHS‐2 mRNA in brainstem and cerebellum, and augmented the PGHS‐2 mRNA response to BCO in brainstem. In contrast, oestradiol had no significant effect on the abundance of PGHS‐1 mRNA in any brain region. PGHS‐1 and PGHS‐2 protein levels did not reflect the changes in the respective mRNAs. The abundance of both proteins was increased in cerebral cortex in response to BCO, and the abundance of PGHS‐2 protein was increased by both oestradiol and BCO in the hippocampus. The results of this study confirm and extend the results of our previous studies, demonstrating an effect of cerebral hypoperfusion on the expression of both isoforms of PGHS. We conclude that oestradiol increases the expression of PGHS‐2 in specific fetal brain regions, and that there is an interaction between oestradiol and BCO in the control of PGHS‐2 expression in the fetal brainstem. We expect that at later time‐points, the changes in mRNA would be followed by similar changes in enzyme abundance at the protein level. We speculate that at least a part of the effect of oestradiol on fetal HPA axis function is mediated by an interaction between oestradiol and prostaglandin biosynthesis in the fetal brain.
KCNQ1 Channels Sense Small Changes in Cell Volume
Authors: Grunnet Morten , Jespersen Thomas , MacAulay Nanna , Jørgensen Nanna K. , Schmitt Nicole , Pongs Olaf , Olesen Søren‐Peter , Klaerke Dan A.
Published: April 2003

Many important physiological processes involve changes in cell volume, e.g. the transport of salt and water in epithelial cells and the contraction of cardiomyocytes. In this study, we show that voltage‐gated KCNQ1 channels, which are strongly expressed in epithelial cells or cardiomyocytes, and KCNQ4 channels, expressed in hair cells and the auditory tract, are tightly regulated by small cell volume changes when co‐expressed with aquaporin 1 water‐channels (AQP1) in Xenopus oocytes. The KCNQ1 and KCNQ4 current amplitudes precisely reflect the volume of the oocytes. By contrast, the related KCNQ2 and KCNQ3 channels, which are prominently expressed in neurons, are insensitive to cell volume changes. The sensitivity of the KCNQ1 and KCNQ4 channels to cell volume changes is independent of the presence of the auxiliary KCNE1–3 subunits, although modulated by KCNE1 in the case of KCNQ1. Incubation of the oocytes in cytochalasin D and experiments with truncated KCNQ1 channels suggest that KCNQ1 channels sense cell volume changes through interactions between the cytoskeleton and the N‐terminus of the channel protein. From our results we propose that KCNQ1 and KCNQ4 channels play an important role in cell volume control, e.g. during transepithelial transport of salt and water.
Recurrent Inhibition of Wrist Extensor Motoneurones: A Single Unit Study on a Deafferented Patient
Authors: Mattei Benjamin , Schmied Annie , Vedel Jean‐Pierre
Published: April 2003

In order to document the effects of recurrent inhibition on the firing times of human α‐motoneurones during natural motor behaviour, a case study was performed on a deafferented patient. The fact that this subject had completely lost the large‐diameter sensory afferents provided us with a unique opportunity of selectively stimulating the motor axons in the nerves. The tonic activity of single motor units (n= 21) was recorded in the extensor carpi radialis muscles while applying randomly timed antidromic electrical stimuli to the radial nerve. The peristimulus time histogram analysis showed the presence of biphasic inhibitory effects, including an early, short‐lasting component followed by a longer‐lasting component occurring 20–40 ms later. The interspike interval (ISI) during which the stimulation occurred was generally lengthened as compared to the previous ISIs. The stimulation was most effective when delivered early (20–30 ms) after a spike. It was also effective, although less so, when delivered at the end of the ISI (70–100 ms after a spike). The lengthening effect sometimes extended over one or two of the subsequent ISIs. The lengthening effect of the motor axon stimulation was followed by an excitatory‐like effect, which took the form of a shortening that affected up to five ISIs after the stimulation. The biphasic inhibitory effects and the subsequent facilitatory effects are discussed in terms of the dual nature of the synaptic processes involved in the recurrent inhibitory network, the postactivation facilitation/depression processes and the mutual inhibition occurring between Renshaw cells.
Upregulation of Swelling‐Activated Cl− Channel Sensitivity to Cell Volume by Activation of EGF Receptors in Murine Mammary Cells
Authors: Abdullaev Iskandar F. , Sabirov Ravshan Z. , Okada Yasunobu
Published: April 2003

Whole‐cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl− currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell‐attached single‐channel recordings showed that BPV‐induced augmentation of the peak amplitude of the whole‐cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV‐transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole‐cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV‐transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet‐derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume‐sensitive outwardly rectifying (VSOR) Cl− currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl− currents in control and BPV‐transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl− current in control cells. However, they failed to induce further augmentation of the current in BPV‐transfected cells. VSOR Cl− currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV‐transfected cells. The IC50 value in BPV‐transfected cells (12 μm) was lower than that in control cells (31 μm). However, the VSOR Cl− currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was markedly diminished by tyrphostin B46 but not significantly affected by tyrphostin AG1296. We thus conclude that the EGF receptor tyrosine kinase upregulates the activity of the VSOR Cl− channel, mainly by enhancing the volume sensitivity.
Functional Properties of Spontaneous EPSCs and non‐NMDA Receptors in Rod Amacrine (AII) Cells in the Rat Retina
Authors: Veruki Margaret Lin , Mørkve Svein Harald , Hartveit Espen
Published: April 2003

The functional properties of spontaneous, glutamatergic EPSCs and non‐NMDA receptors in AII amacrine cells were studied in whole cells and patches from slices of the rat retina using single and dual electrode voltage clamp recording. Pharmacological analysis verified that the EPSCs (Erev∼0 mV) were mediated exclusively by AMPA‐type receptors. EPSCs displayed a wide range of waveforms, ranging from simple monophasic events to more complex multiphasic events. Amplitude distributions of EPSCs were moderately skewed towards larger amplitudes (modal peak 23 pA). Interevent interval histograms were best fitted with a double exponential function. Monophasic, monotonically rising EPSCs displayed very fast kinetics with an average 10–90 % rise time of ∼340 μs and a decay phase well fitted by a single exponential (τdecay∼760 μs). The specific AMPA receptor modulator cyclothiazide markedly slowed the decay phase of spontaneous EPSCs (τdecay∼3 ms). An increase in temperature decreased both 10–90 % rise time and τdecay with Q10 values of 1.3 and 1.5, respectively. The decay kinetics were slower at positive membrane potentials compared to negative membrane potentials (205 mV/e‐fold change in τdecay). Step depolarization of individual presynaptic rod bipolar cells or OFF‐cone bipolar cells evoked transient, CNQX‐sensitive responses in AII amacrine cells with average peak amplitudes of ∼330 pA. Ultrafast application of brief (∼1 ms) or long (∼500 ms) pulses of glutamate to outside‐out patches evoked strongly desensitizing responses with very fast deactivation and desensitization kinetics, well fitted by single (τdecay∼1.1 ms) and double exponential (τ1∼3.5 ms; τ2∼21 ms) functions, respectively. Double‐pulse experiments indicated fast recovery from desensitization (τ∼12.4 ms). Our results indicate that spontaneous, AMPA receptor‐mediated EPSCs in AII amacrine cells have very fast, voltage‐dependent kinetics that can be well accounted for by the kinetic properties of the AMPA receptors themselves.
The Effect of Dehydroepiandrosterone on Coronary Blood Flow in Prepubertal Anaesthetized Pigs
Authors: Molinari C. , Battaglia A. , Grossini E. , Mary D. A. S. G. , Vassanelli C. , Vacca G.
Published: April 2003

Extensive research suspecting an association between plasma levels of dehydroepiandrosterone and the risk of coronary heart disease has not been conclusive. The present study was designed to investigate the effect of dehydroepiandrosterone on the coronary circulation and to determine the mechanisms involved. In prepubertal pigs of both sexes anaesthetized with sodium pentobarbitone, changes in left circumflex or anterior descending coronary flow caused by intravenous infusion of dehydroepiandrosterone were assessed using an electromagnetic flowmeter. Changes in heart rate and arterial pressure were prevented by atrial pacing and by connecting the arterial system to a pressurized reservoir containing Ringer solution. In 20 pigs, infusion of 1 mg h−1 of dehydroepiandrosterone caused a decrease in coronary flow without affecting left ventricular dP/dtmax (rate of change of left ventricular systolic pressure) and filling pressures of the heart. In a further eight pigs, a dose–response curve was obtained by graded increases in the infused dose of hormone between 0.03 and 4 mg h−1. The mechanisms of the above response were studied in the 20 pigs by repeating the experiment after haemodynamic variables had returned to the control values observed before infusion. Blockade of muscarinic cholinoceptors with intravenous atropine (five pigs) and of α‐adrenoceptors with intravenous phentolamine (five pigs) did not affect the dehydroepiandrosterone‐induced coronary vasoconstriction. This response was abolished by blockade of β‐adrenoceptors with intravenous propranolol (five pigs) and of coronary nitric oxide synthase with intracoronary injection of Nω‐nitro‐l‐arginine methyl ester (five pigs) even after reversing the increase in arterial pressure and coronary vascular resistance caused by the two blocking agents with intravenous infusion of papaverine. The present study showed that intravenous infusion of dehydroepiandrosterone primarily caused coronary vasoconstriction. The mechanisms of this response were shown to involve the inhibition of a vasodilatory β‐adrenergic receptor‐mediated effect related to the release of nitric oxide.
Contribution of Adenosine to the Depression of Sympathetically Evoked Vasoconstriction induced by Systemic Hypoxia in the Rat
Authors: Coney Andrew M. , Marshall Janice M.
Published: April 2003

Previous studies have shown that systemic hypoxia evokes vasodilatation in skeletal muscle that is mediated mainly by adenosine acting on A1 receptors, and that the vasoconstrictor effects of sympathetic nerve activity are depressed during hypoxia. The aim of the present study was to investigate the role of adenosine in this depression. In anaesthetised rats, increases in femoral vascular resistance (FVR) evoked by stimulation of the lumbar sympathetic chain with bursts of impulses at 40 or 20 Hz were greater than those evoked by continuous stimulation at 2 Hz with the same number of impulses (120) over 1 min. All of these responses were substantially reduced by infusion of adenosine or by graded systemic hypoxia (breathing 12, 10 or 8 % O2), increases in FVR evoked by continuous stimulation at 2 Hz being most vulnerable. Blockade of A1 receptors ameliorated the depression caused by adenosine infusion of the increase in FVR evoked by 2 Hz only and did not ameliorate the depression caused by 8 % O2 of increases in FVR evoked by any pattern of sympathetic stimulation. A2A receptor blockade accentuated hypoxia‐induced depression of the increase in FVR evoked by burst stimulation at 40 Hz, but had no other effect. Neither A1 nor A2A receptor blockade affected the depression caused by hypoxia (8 % O2) of the FVR increase evoked by noradrenaline infusion. These results indicate that endogenously released adenosine is not responsible for the depression of sympathetically evoked muscle vasoconstriction caused by systemic hypoxia; adenosine may exert a presynaptic facilitatory influence on the vasoconstrictor responses evoked by bursts at high frequency.
Glucose Ingestion Attenuates Interleukin‐6 Release from Contracting Skeletal Muscle in Humans
Authors: Febbraio Mark A. , Steensberg Adam , Keller Charlotte , Starkie Rebecca L. , Nielsen Henning B. , Krustrup Peter , Ott Peter , Secher Niels H. , Pedersen Bente K.
Published: April 2003

To examine whether glucose ingestion during exercise affects the release of interleukin‐6 (IL‐6) from the contracting limb, seven men performed 120 min of semi‐recumbent cycling on two occasions while ingesting either 250 ml of a 6.4 % carbohydrate (GLU trial) or sweet placebo (CON trial) beverage at the onset of, and at 15 min intervals throughout, exercise. Muscle biopsies obtained before and immediately after exercise were analysed for glycogen and IL‐6 mRNA expression. Blood samples were simultaneously obtained from a brachial artery and a femoral vein prior to and during exercise and leg blood flow was measured by thermodilution in the femoral vein. Net leg IL‐6 release, and net leg glucose and free fatty acid (FFA) uptake, were calculated from these measurements. The arterial IL‐6 concentration was lower (P < 0.05) after 120 min of exercise in GLU, but neither intramuscular glycogen nor IL‐6 mRNA were different when comparing GLU with CON. However, net leg IL‐6 release was attenuated (P < 0.05) in GLU compared with CON. This corresponded with an enhanced (P < 0.05) glucose uptake and a reduced (P < 0.05) FFA uptake in GLU. These results demonstrate that glucose ingestion during exercise attenuates leg IL‐6 release but does not decrease intramuscular expression of IL‐6 mRNA.
The Answer Is Forty-Two — Many Mechanics Problems, Only One Answer
Authors: Romer Robert H.
Published: April 2003

The answer is 42, actually 42 minutes, but what was the question? There are, in fact, a number of physics questions, not at first glance closely related to one another, to which 42 minutes (or perhaps 84 minutes) is the answer. This paper was prompted by one such question, a Figuring Physics item in a recent issue of this journal,1 which called for a description of the motion of a block released on a flat frictionless plate tangent to the Earth's surface. The correct answer given in Figuring Physics was that “the block will oscillate to and fro,”2 but I was disappointed to see that the time of oscillation was not called for. So let us refine the question: “For small-amplitude oscillations, how long does it take for the block to travel from one extreme to the other, i.e., what is the half-period of the motion?” This is the question (actually just one of the questions) to which the answer is 42 minutes, as we can readily calculate. Those who are familiar with The Hitchhiker's Guide to the Galaxy [Douglas Adams (Harmony Books, New York, 1979)] will understand why I chose to ask initially for the half period (42 min) rather than asking the more obvious question, “What is the period?”3
Role of 16S rRNA Helix 44 in Ribosomal Resistance to Hygromycin B
Authors: Pfister P. , Risch M. , Brodersen D. E. , Böttger E. C.
Published: April 2003

Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium
Mycobacterium smegmatis
for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin B in eubacteria. Resistance mutations were found to localize exclusively in 16S rRNA. The mutations observed, i.e., 16S rRNA U1406C, C1496U, and U1498C (
E. coli
numbering), are in close proximity to the hygromycin B binding site located in conserved helix 44 of 16S rRNA. The 16S rRNA positions involved in hygromycin B resistance are highly conserved in all three domains of life, explaining the lack of specificity and general toxicity of hygromycin B.
Prevention of Human Rhinovirus Infection by Multivalent Fab Molecules Directed against ICAM-1
Authors: Charles Catherine H. , Luo Guang X. , Kohlstaedt Lori A. , Morantte Ianessa G. , Gorfain Elliott , Cao Linguang , Williams Jimmy H. , Fang Fang
Published: April 2003

We have developed a technology for improving avidity by making bivalent, trivalent, or tetravalent recombinant polypeptides. We designed tripartite proteins consisting of the Fab fragment of an antibody fused with a hinge derived from human immunoglobulin D that was further linked to polymerization domains derived from human coiled-coil proteins. We report here on the application of this method with a Fab domain directed against the major human rhinovirus receptor, intercellular adhesion molecule 1 (ICAM-1). Multivalent anti-ICAM-1 molecules were produced in bacteria and purified as soluble preassembled homogeneous proteins at high yield. These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer. The diminished dissociation rate of these multivalent antibodies and their improved efficacy in preventing rhinovirus infection provide a foundation for producing prophylactic and therapeutic molecules against human rhinovirus, the causative agent of the majority of common colds.
Adverse Effect of Rifampin on Quinine Efficacy in Uncomplicated Falciparum Malaria
Authors: Pukrittayakamee S. , Prakongpan S. , Wanwimolruk S. , Clemens R. , Looareesuwan S. , White N. J.
Published: April 2003

The effects of adding rifampin to quinine were assessed in adults with uncomplicated falciparum malaria. Patients were randomized to receive oral quinine either alone (
n
= 30) or in combination with rifampin (
n
= 29). Although parasite clearance times were shorter in the quinine-rifampin-treated patients (mean ± standard deviation, 70 ± 21 versus 82 ± 18 h;
P
= 0.023), recrudescence rates were five times higher (
n
= 15 of 23; 65%) than those obtained with quinine alone (
n
= 3 of 25; 12%),
P
< 0.001. Patients receiving rifampin had significantly greater conversion of quinine to 3-hydroxyquinine and consequently considerably lower concentrations of quinine in their plasma after the second day of treatment (median area under the plasma drug concentration-time curve from day zero to day 7 = 11.7 versus 47.5 μg/ml · day,
P
< 0.001). Rifampin significantly increases the metabolic clearance of quinine and thereby reduces cure rates. Rifampin should not be combined with quinine for the treatment of malaria, and the doses of quinine should probably be increased in patients who are already receiving rifampin treatment.
Novel Point Mutations in the Dihydrofolate Reductase Gene of
Plasmodium vivax
: Evidence for Sequential Selection by Drug Pressure
Authors: Imwong Mallika , Pukrittayakamee Sasithon , Rénia Laurent , Letourneur Franck , Charlieu Jean-Paul , Leartsakulpanich Ubolsree , Looareesuwan Sornchai , White Nicholas J. , Snounou Georges
Published: April 2003

Mutations in the dihydrofolate reductase (
dhfr
) genes of
Plasmodium falciparum
and
P. vivax
are associated with resistance to the antifolate antimalarial drugs.
P. vivax
dhfr
sequences were obtained from 55
P. vivax
isolates (isolates Belem and Sal 1, which are established lines originating from Latin America, and isolates from patient samples from Thailand [
n
= 44], India [
n
= 5], Iran [
n
= 2], and Madagascar [
n
= 2]) by direct sequencing of both strands of the purified PCR product and were compared to the
P. vivax dhfr
sequence from a
P. vivax
parasite isolated in Pakistan (isolate ARI/Pakistan), considered to represent the wild-type sequence. In total, 144
P. vivax dhfr
mutations were found at only 12 positions, of which 4 have not been described previously. An F→L mutation at residue 57 had been observed previously, but a novel codon (TTA) resulted in a mutation in seven of the nine mutated variant sequences. A new mutation at residue 117 resulted in S→T (S→N has been described previously). These two variants are the same as those observed in the
P. falciparum dhfr
gene at residue 108, where they are associated with different levels of antifolate resistance. Two novel mutations, I→L at residue 13 and T→M at residue 61, appear to be unique to
P. vivax.
The clinical, epidemiological, and sequence data suggest a sequential pathway for the acquisition of the
P. vivax dhfr
mutations. Mutations at residues 117 and 58 arise first when drug pressure is applied. Highly mutated genes carry the S→T rather than the S→N mutation at residue 117. Mutations at residues 57 and 61 then occur, followed by a fifth mutation at residue 13.
IMP-12, a New Plasmid-Encoded Metallo-β-Lactamase from a
Pseudomonas putida
Clinical Isolate
Authors: Docquier Jean-Denis , Riccio Maria Letizia , Mugnaioli Claudia , Luzzaro Francesco , Endimiani Andrea , Toniolo Antonio , Amicosante Gianfranco , Rossolini Gian Maria
Published: April 2003

A
Pseudomonas putida
strain showing broad-spectrum resistance to β-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-β-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new
bla
IMP
determinant, named
bla
IMP-12
. Plasmid pVA758 was not self-transferable by conjugation to either
Escherichia coli
or
Pseudomonas aeruginosa
but could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various β-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The
bla
IMP-12
determinant is carried on an integron-borne gene cassette whose
attC
recombination site is related to those present in cassettes containing
bla
IMP-1
,
bla
IMP-6
,
bla
IMP-7
,
bla
IMP-10
, and
bla
IMP-11
and unrelated to that present in cassettes containing
bla
IMP-2
and
bla
IMP-8
. IMP-12 was overproduced in
E. coli
by using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins (
k
cat
/

K
m

values, around 10
4
M
−1
· s
−1
) and a remarkably high

K
m

(around 900 μM) for imipenem.
The In Vivo Susceptibility of
Leishmania donovani
to Sodium Stibogluconate Is Drug Specific and Can Be Reversed by Inhibiting Glutathione Biosynthesis
Authors: Carter K. C. , Sundar S. , Spickett C. , Pereira O. C. , Mullen A. B.
Published: April 2003

Resistance to pentavalent antimonial (Sb
v
) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of
Leishmania donovani
strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb
v
/kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme γ-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment.
L. donovani
strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016
L. donovani
strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the
L. donovani
strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.
Chromosomal Integration of a Cephalosporinase Gene from
Acinetobacter baumannii
into
Oligella urethralis
as a Source of Acquired Resistance to β-Lactams
Authors: Mammeri Hedi , Poirel Laurent , Mangeney Nicole , Nordmann Patrice
Published: April 2003

Clinical
Oligella urethralis
isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into
Escherichia coli
and expression experiments gave recombinant clones expressing either an AmpC β-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing β-lactamase-type phenotype of resistance. The AmpC β-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of
Acinetobacter baumannii
. A 820-bp insertion sequence element, IS
Our1
, belonging to the IS
6
family of insertion sequence elements, was identified immediately upstream of
bla
ABA-1
, providing a −35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing β-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of
Ceu
I fragment I-restricted DNA fragments of
O. urethralis
COH-1 with
bla
ABA-1
-,
bla
CARB-8
-, and 16S rRNA-specific probes indicated the chromosomal integration of the β-lactamase genes. PCR and hybridization experiments failed to detect
bla
CARB-8
- and
bla
ABA-1
-like genes in three
O. urethralis
reference strains, indicating that the β-lactamase genes identified were the source of acquired resistance in
O. urethralis
COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring β-lactamase.
Functional Similarities and Differences between
Candida albicans
Cdr1p and Cdr2p Transporters
Authors: Gauthier Christian , Weber Sandra , Alarco Anne-Marie , Alqawi Omar , Daoud Roni , Georges Elias , Raymond Martine
Published: April 2003

The
Candida albicans CDR1
and
CDR2
genes code for highly homologous ATP-binding cassette (ABC) transporters which are overexpressed in azole-resistant clinical isolates and which confer resistance to multiple drugs by actively transporting their substrates out of the cells. These transporters are formed by two homologous halves, each with an intracellular domain containing an ATP-binding site followed by a membrane-associated domain. We have expressed Cdr1p and Cdr2p in
Saccharomyces cerevisiae
to investigate their functions. The two proteins were properly expressed and functional, as determined by Western blotting, drug susceptibility assays, and rhodamine efflux. Using total membrane proteins from these transformants, we showed that Cdr1p and Cdr2p bind to the photoreactive analogue of rhodamine 123, [
125
I]iodoaryl azido-rhodamine 123 (IAARh123). IAARh123 photoaffinity labeling of membranes prepared from cells expressing either the N half or the C half of Cdr2p, or both, demonstrated that both halves contribute to rhodamine binding and can bind to rhodamine independently. Interestingly, Cdr1p was found to confer hypersusceptibility to FK520, an immunosuppressant and antifungal agent, whereas Cdr2p conferred resistance to this compound, uncovering a major functional difference between the two transporters. Furthermore, when administered in combination with azoles, FK520 sensitized cells expressing
CDR1
but not those expressing
CDR2
. Finally, we showed that Cdr2p confers hypersusceptibility to hydrogen peroxide and resistance to diamide, while Cdr1p has no effect against these oxidative agents. Taken together, our results demonstrate that, despite a high level of structural conservation, Cdr1p and Cdr2p exhibit major functional differences, suggesting distinct biological functions.
Modification of Outer Membrane Protein Profile and Evidence Suggesting an Active Drug Pump in
Enterobacter aerogenes
Clinical Strains
Authors: Gayet Stéphane , Chollet Renaud , Molle Gérard , Pagès Jean-Marie , Chevalier Jacqueline
Published: April 2003

Two clinical strains of
Enterobacter aerogenes
that exhibited phenotypes of multiresistance to β-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide
m
-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in
Escherichia coli
cells.
Impact of Elements Containing Glycopeptide Resistance Genes on Expression of Virulence in
Enterococcus faecalis
Peritonitis: a Pilot Study with Rats
Authors: Plantefeve G. , Dupont H. , Hubert V. , Garry L. , Poüs C. , Carbon C. , Montravers P.
Published: April 2003

The relationship between virulence and chromosomal elements containing glycopeptide resistance genes was experimentally assessed for two transconjugant strains of
Enterococcus faecalis
(VanA and VanB phenotypes) and compared to that for a susceptible wild-type strain. Microbiologic and inflammatory effects were assessed in a polymicrobial rat model of peritonitis. Mean peritoneal enterococcus concentrations ± standard deviations at day 1 were 2.1 ± 1.9, 1.3 ± 1.1, and 1.7 ± 2.0 log
10
CFU/ml for susceptible, VanA, and VanB strains, respectively (
P
< 0.05). At day 3 also there were lower concentrations of glycopeptide-resistant enterococcal strains in peritoneal fluid (3.2 ± 3.4, 1.8 ± 1.8, and 2.1 ± 2.4 log
10
CFU/ml for susceptible, VanA, and VanB strains, respectively [
P
< 0.05]). Transconjugant glycopeptide-resistant strains were associated with increased peritoneal cell counts at the different evaluation times of the experiment (
P
< 0.001). Plasma α1-acid glycoprotein concentrations were lower in the presence of the susceptible strain (667 ± 189 mg/liter) than in the presence of the VanA or VanB strain (1,193 ± 419 or 1,210 ± 404 mg/liter, respectively [
P
< 0.05]), while concentrations of tumor necrosis factor alpha and interleukin-6 in peritoneal fluid remained similar for the strains. These results suggest a trend toward variation of virulence of transconjugant strains compared to the wild-type strain in this peritonitis model.
Fungicidal Synergism of Fluconazole and Cyclosporine in
Candida albicans
Is Not Dependent on Multidrug Efflux Transporters Encoded by the
CDR1
,
CDR2
,
CaMDR1
, and
FLU1
Genes
Authors: Marchetti Oscar , Moreillon Philippe , Entenza Josè M. , Vouillamoz Jacques , Glauser Michel P. , Bille Jacques , Sanglard Dominique
Published: April 2003

The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible
C. albicans
(O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the
CDR1
,
CDR2
,
CaMDR1
, and
FLU1
transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (
P
= 0.05 at 48 h with an inoculum of 10
3
CFU/ml) and a more rapid fungicidal effect of FLC-CY (
P
= 0.05 at 24 h with an inoculum of 10
3
CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (
P
= 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (
P
= 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a “dual-hit” mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by
CDR1
,
CDR2
,
CaMDR1
, and
FLU1
genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.
Presence of a
vanA
-Carrying Pheromone Response Plasmid (pBRG1) in a Clinical Isolate of
Enterococcus faecium
Authors: Magi Gloria , Capretti Roberta , Paoletti Claudia , Pietrella Marco , Ferrante Luigi , Biavasco Francesca , Varaldo Pietro Emanuele , Facinelli Bruna
Published: April 2003

Sex pheromone plasmids, frequently found in
Enterococcus faecalis
, have rarely been detected in
Enterococcus faecium
. pBRG1 is an approximately 50-kb
vanA
-carrying conjugative plasmid of an
E. faecium
clinical isolate (LS10) that is transferable to
E. faecalis
laboratory strains. In cell infection experiments,
E. faecium
LS10 exhibited remarkably high invasion efficiency and produced cytopathogenic effects in Caco-2 cell monolayers. Growth in the presence of sex pheromones produced by
E. faecalis
JH2-2 was found to cause self-aggregation of both
E. faecium
LS10 and
E. faecalis
JH-RFV(pBRG1) (a transconjugant obtained by transfer of pBRG1 to
E. faecalis
JH2-2) and to increase the cell adhesion and invasion efficiencies of both
E. faecium
LS10 and
E. faecalis
JH-RFV(pBRG1). Sex pheromone cCF10 caused clumping of
E. faecalis
OG1RF(pBRG1) (a transconjugant obtained by transfer of pBRG1 to
E. faecalis
OG1RF) at a concentration ∼100-fold higher than the one required for the control strain
E. faecalis
OG1RF(pCF10). PCR products of the expected sizes were obtained with primers internal to aggregation substance genes of
E. faecalis
pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to
ash701
of
E. faecium
pheromone plasmid pHKK701. These findings suggest that pBRG1 of
E. faecium
LS10 is a sex pheromone response plasmid.
Aminoglycoside Resistance Resulting from Tight Drug Binding to an Altered Aminoglycoside Acetyltransferase
Authors: Magnet Sophie , Smith Terry-Ann , Zheng Renjian , Nordmann Patrice , Blanchard John S.
Published: April 2003

The
aacA29b
gene, which confers an atypical aminoglycoside resistance pattern to
Escherichia coli
, was identified on a class 1 integron from a multidrug-resistant isolate of
Pseudomonas aeruginosa
. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6′-
N
-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside
N-
acetyltransferase AAC(6′)-I, except netilmicin. The level of resistance conferred by
aacA29b
correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6′)-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (

K
d

, ≤1 μM) and a weaker affinity for amikacin (

K
d

, ≈60 μM). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6′)-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6′)-I family members. AAC(6′)-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6′)-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.
Aminoglycoside Resistance Gene
ant(4′)-IIb
of
Pseudomonas aeruginosa
BM4492, a Clinical Isolate from Bulgaria
Authors: Sabtcheva Stefana , Galimand Marc , Gerbaud Guy , Courvalin Patrice , Lambert Thierry
Published: April 2003

The
ant(4′)-IIb
gene of
Pseudomonas aeruginosa
BM4492, which encodes an aminoglycoside 4′-
O
-adenylyltransferase, was identified as a coding sequence of 756 bp corresponding to a protein with a calculated mass of 27,219 Da. Analysis of the deduced sequence indicated that the protein was related to aminoglycoside 4′-
O
-adenylyltransferases IIa and Ia found in
P. aeruginosa
and gram-positive bacteria, respectively. The enzyme conferred resistance to amikacin and tobramycin but not to dibekacin, gentamicin, or netilmicin. The
ant(4′)-IIb
gene had a chromosomal location in five of six clinical isolates of
P. aeruginosa
tested and was plasmid borne in the remaining strain. The
ant(4′)-IIb
gene was detected by PCR in some clinical strains of
P. aeruginosa
from the same hospital but not in members of other bacterial genera.