bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro
Authors: Bikson Marom , Inoue Masashi , Akiyama Hiroki , Deans Jackie K. , Fox John E. , Miyakawa Hiroyoshi , Jefferys John G. R.
Published: February 2004

The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage‐sensitive dye recordings. Small electric fields (<|40| mV mm−1), applied parallel to the somato‐dendritic axis, induced polarization of CA1 pyramidal cells; the relationship between applied field and induced polarization was linear (0.12 ± 0.05 mV per mV mm−1 average sensitivity at the soma). The peak amplitude and time constant (15–70 ms) of membrane polarization varied along the axis of neurons with the maximal polarization observed at the tips of basal and apical dendrites. The polarization was biphasic in the mid‐apical dendrites; there was a time‐dependent shift in the polarity reversal site. DC fields altered the thresholds of action potentials evoked by orthodromic stimulation, and shifted their initiation site along the apical dendrites. Large electric fields could trigger neuronal firing and epileptiform activity, and induce long‐term (>1 s) changes in neuronal excitability. Electric fields perpendicular to the apical–dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time‐dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non‐linear properties of the cell membrane, and effects on afferents.
Effect of hypothermia on baroreflex control of heart rate and renal sympathetic nerve activity in anaesthetized rats
Authors: Sabharwal R. , Coote J. H. , Johns E. J. , Egginton S.
Published: February 2004

The present study investigated the effect of acute hypothermia on baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) by generating baroreflex logistic function curves, using bolus doses of phenylephrine and sodium nitroprusside, in anaesthetized male Wistar rats at a core temperature (Tb) of 37°C, during acute severe hypothermia at Tb= 25°C and on rewarming to 37°C. Comparisons were made between rats without (euthermic, n= 6) and with (acclimated, n= 7) prior exposure to lower ambient temperatures and shorter photoperiod, simulating adaptation to winter conditions. In both groups of rats, acute hypothermia to Tb= 25°C shifted the baroreflex‐RSNA curve slightly leftwards and downwards with decreases in the setpoint pressure and maximal gain, whereas it markedly impaired the baroreflex‐HR curve characterized by decreases in response range by ∼90% (P < 0.001), minimum response by ∼10% (P < 0.05) and maximum gain by ∼95% (P < 0.001), from that at Tb= 37°C. All parameters were restored to precooling levels on rewarming. Electrical stimulation of cardiac vagal efferents induced a voltage‐related bradycardia, the magnitude of which was partially reduced during acute hypothermia, and there was a significant prolongation of the electrocardiogram intervals indicating a delay in cardiac conduction. Mild suppression of baroreflex control of RSNA could contribute to hypothermic hypotension and may primarily reflect an effect of Tb on central drive. The marked attenuation of the baroreflex control of HR during hypothermia was likely to be due to an impairment of both the central and peripheral components of the reflex arc. Baroreflex control of RSNA and HR was similar between both groups of rats, which implied that the control was non‐adaptive on chronic cold exposure.
P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes
Authors: Yamamoto‐Mizuma Shintaro , Wang Ge‐Xin , Hume Joseph R.
Published: February 2004

The intracellular signalling pathways and molecular mechanisms responsible for P2‐purinoceptor‐mediated chloride (Cl−) currents (ICl,ATP) were studied in mouse ventricular myocytes. In standard NaCl‐containing extracellular solutions, extracellular ATP (100 μm) activated two different currents, ICl,ATP with a linear I–V relationship in symmetrical Cl− solutions, and an inwardly rectifying cation conductance (cationic IATP). Cationic IATP was selectively inhibited by Gd3+ and Zn2+, or by replacement of extracellular NaCl by NMDG; ICl,ATP was Cl− selective, and inhibited by replacement of extracellular Cl− by Asp−; both currents were prevented by suramin or DIDS pretreatment. In GTPγS‐loaded cells, ICl,ATP was irreversibly activated by ATP, but cationic IATP was still regulated reversibly. GDPβS prevented activation of the ICl,ATP, even though pertussis toxin pretreatment did not modulate ICl,ATP. These results suggest that activation of ICl,ATP occurs via a G‐protein coupled P2Y purinergic receptor. The ICl,ATP persistently activated by GTPγS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. In ventricular cells of cftr−/− mice, extracellular ATP activated cationic IATP, but failed to activate any detectable ICl,ATP. These results provide compelling evidence that activation of CFTR Cl− channels in mouse heart are coupled to G‐protein coupled P2Y purinergic receptors.
Subtypes of vagal afferent C‐fibres in guinea‐pig lungs
Authors: Undem B. J. , Chuaychoo B. , Lee M.‐G. , Weinreich D. , Myers A. C. , Kollarik M.
Published: February 2004

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C‐fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C‐fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C‐fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C‐fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C‐fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C‐fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C‐fibre population responded with action potential discharge to the P2X selective receptor agonist α,β‐methylene‐ATP. Whole cell patch clamp recording of capsaicin‐sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C‐fibre neurones express functional P2X receptors, whereas lung specific jugular C‐fibres do not. The data support the hypothesis that both neural crest‐derived neurones (jugular ganglia) and placode‐derived neurones (nodose ganglia) project C‐fibres in the vagus, and that these two C‐fibre populations represent distinct phenotypes.
Dendritic relationship between starburst amacrine cells and direction‐selective ganglion cells in the rabbit retina
Authors: Dong Wei , Sun Wenzhi , Zhang Yingye , Chen Xiaorong , He Shigang
Published: February 2004

We investigated the dendritic relationship between starburst amacrine cells (SAs) and morphologically and physiologically characterized ON and ON‐OFF direction‐selective ganglion cells (DSGCs) in the rabbit retina. ON and ON‐OFF DSGCs were found to exhibit tight dendritic cofasciculation with the SA plexus, visualized by immunolabelling of the vesicular acetylcholine transporter (VAChT). The degree of cofasciculation of both types of DSGC dendrites and SA plexus was found to be significant, unlike the relationship between non‐DS cells and the SA plexus, which was close to chance distribution. No difference in the degree of cofasciculation in different regions of the DS dendritic field was observed. Individual SAs intracellularly injected both on the ‘preferred’ and ‘null’ side of the DSGCs showed the same degree of cofasciculation with the DSGCs. Therefore, the computation of motion direction is unlikely to result from apparent asymmetry in geometric proximity between SAs and DSGCs. Highly selective synaptic connections between SAs and DSGCs are necessary.
Synaptic dynamics control the timing of neuronal excitation in the activated neocortical microcircuit
Authors: Silberberg Gilad , Wu Caizhi , Markram Henry
Published: February 2004

It is well established that sensory stimulation results in the activity of multiple functional columns in the neocortex. The manner in which neurones within each column are active in relation to each other is, however, not known. Multiple whole‐cell recordings in activated neocortical slices from rat revealed diverse correlation profiles of excitatory synaptic input to different types of neurones. The specific correlation profile between any two neurones could be predicted by the settings of synaptic depression and facilitation at the input synapses. Simulations further showed that patterned activity is essential for synaptic dynamics to impose the temporal dispersion of excitatory input. We propose that synaptic dynamics choreograph neuronal activity within the neocortical microcircuit in a context‐dependent manner.
Basolateral anion transport mechanisms underlying fluid secretion by mouse, rat and guinea‐pig pancreatic ducts
Authors: Fernández‐Salazar M. Paz , Pascua Patricia , Calvo José Julián , López María A. , Case R. Maynard , Steward Martin C. , San Román José I.
Published: February 2004

Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO3− requirement for secretin‐evoked secretion in mouse, rat and guinea‐pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nm secretin and 5 μm forskolin in the absence of extracellular HCO3−. In guinea‐pig ducts, however, fluid secretion was totally dependent on HCO3−. Forskolin‐stimulated fluid secretion by mouse and rat ducts in the absence of HCO3− was dependent on extracellular Cl− and was completely inhibited by bumetanide (30 μm). It was therefore probably mediated by a basolateral Na+–K+–2Cl− cotransporter. In the presence of HCO3−, forskolin‐stimulated fluid secretion was reduced ∼40% by bumetanide, ∼50% by inhibitors of basolateral HCO3− uptake (3 μm EIPA and 500 μm H2DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin‐evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na+–H+ exchange and Na+–HCO3− cotransport mediating HCO3− uptake, and Na+–K+–2Cl− cotransport mediating Cl− uptake. The absence or inactivity of the Cl− uptake pathway in the guinea‐pig pancreatic ducts may help to account for the much higher concentrations of HCO3− secreted in this species.
In Vitro Evolution Predicts that the IMP-1 Metallo-β-Lactamase Does Not Have the Potential To Evolve Increased Activity against Imipenem
Authors: Hall Barry G.
Published: February 2004

In vitro evolution was used to predict whether the IMP-1 metallo-β-lactamase has the potential to evolve an increased ability to confer resistance to imipenem. Screening of eight libraries containing 9.8 × 10
6
± 1.4 × 10
6
(mean ± standard error) variants per library, with an average of 1.2 mutations per variant, detected no increased resistance to imipenem. The results predict, with >99.9% confidence, that even under intense selection the IMP-1 β-lactamase will not evolve to confer increased resistance to imipenem.
In Vitro Activities of New Quinolones and Oxazolidinones against
Actinomadura madurae
Authors: Vera-Cabrera Lucio , Ochoa-Felix Elsa Y. , Gonzalez Gloria , Tijerina Rolando , Choi Sung H. , Welsh Oliverio
Published: February 2004

In this work, we studied the in vitro sensitivity of 24 strains of
Actinomadura madurae
to a new oxazolidinone (DA-7867), gatifloxacin, moxifloxacin, and garenoxacin by using a broth microdilution method. We observed that the
A. madurae
strains had a high level of sensitivity to all the antimicrobials tested. The most active drug was DA-7867, with a MIC at which 90% of the strains are inhibited (MIC
90
) of 0.125 μg/ml and a MIC
50
of 0.06 μg/ml.
Inhibitors of Antibiotic Efflux in Resistant
Enterobacter aerogenes
and
Klebsiella pneumoniae
Strains
Authors: Chevalier Jacqueline , Bredin Jérôme , Mahamoud Abdallah , Malléa Monique , Barbe Jacques , Pagès Jean-Marie
Published: February 2004

In
Enterobacter aerogenes
and
Klebsiella pneumoniae
, efflux provides efficient extrusion of antibiotics and contributes to the multidrug resistance phenotype. One of the alkoxyquinoline derivatives studied here, 2,8-dimethyl-4-(2′-pyrrolidinoethyl)-oxyquinoline, restores noticeable drug susceptibility to resistant clinical strains. Analyses of energy-dependent chloramphenicol efflux indicate that this compound inhibits the efflux pump mechanism and improves the activity of structurally unrelated antibiotics on multidrug-resistant
E. aerogenes
and
K. pneumoniae
isolates.
Activities of Dalbavancin In Vitro and in a Rabbit Model of Experimental Endocarditis Due to
Staphylococcus aureus
with or without Reduced Susceptibility to Vancomycin and Teicoplanin
Authors: Lefort Agnès , Pavie Juliette , Garry Louis , Chau Françoise , Fantin Bruno
Published: February 2004

For the treatment of rabbit endocarditis, dalbavancin given once daily (10 mg/kg of body weight for 4 days) or as a single 40-mg/kg dose was active against
Staphylococcus aureus
with or without reduced susceptibility to glycopeptides, as expected from its good in vitro activity, even in broth supplemented with 90% serum and given its prolonged elimination half-life.
Resistance to Cefepime and Cefpirome Due to a 4-Amino-Acid Deletion in the Chromosome-Encoded AmpC β-Lactamase of a
Serratia marcescens
Clinical Isolate
Authors: Mammeri Hedi , Poirel Laurent , Bemer Pascal , Drugeon Henri , Nordmann Patrice
Published: February 2004

A multiresistant
Serratia marcescens
strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The
bla
AmpC
gene of the strain was amplified by PCR and cloned into
Escherichia coli
DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the
bla
AmpC
gene from
S. marcescens
HD had a 12-nucleotide deletion compared to the
bla
AmpC
gene from reference strain
S. marcescens
S3, leading to a 4-amino-acid deletion located in the H-10 helix of the β-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type β-lactamases.
A Second Tylosin Resistance Determinant, Erm B, inArcanobacterium pyogenes
Authors: Jost B. Helen , Trinh Hien T. , Songer J. Glenn , Billington Stephen J.
Published: February 2004

Arcanobacterium pyogenes, a common inhabitant of the mucosal surfaces of livestock, is also a pathogen associated with a variety of infections. In livestock,A. pyogenesis exposed to antimicrobial agents used for prophylaxis and therapy, notably tylosin, a macrolide used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Many, but not all, tylosin-resistantA. pyogenesisolates carryerm(X), suggesting the presence of other determinants of tylosin resistance. Oligonucleotide primers designed for conserved regions oferm(B),erm(C), anderm(T) were used to amplify a 404-bp fragment from a tylosin-resistantA. pyogenesisolate, OX-7. DNA sequencing revealed that the PCR product was 100% identical toerm(B) genes, and theerm(B) gene region was cloned inEscherichia coli. TheA. pyogenesErm B determinant had the most DNA identity with an Erm B determinant carried by theClostridium perfringensplasmid pIP402. However, theA. pyogenesdeterminant lacked direct repeat DR1 and contained a deletion in DR2. Flanking theA. pyogenes erm(B) gene were partial and entire genes similar to those found on theEnterococcus faecalismultiresistance plasmid pRE25. This novel architecture suggests that theerm(B) element may have arisen by recombination of two distinct genetic elements. Ten of 32 tylosin-resistant isolates carriederm(B), as determined by DNA hybridization, and all 10 isolates carried a similar element. Insertion of the element was site specific, as PCR and Southern blotting analysis revealed that theerm(B) element was inserted intoorfY, a gene of unknown function. However, in three strains, this insertion resulted in a partial duplication oforfY.
Mechanism of Action of the Mannopeptimycins, a Novel Class of Glycopeptide Antibiotics Active against Vancomycin-Resistant Gram-Positive Bacteria
Authors: Ruzin Alexey , Singh Guy , Severin Anatoly , Yang Youjun , Dushin Russell G. , Sutherland Alan G. , Minnick Albert , Greenstein Michael , May Michael K. , Shlaes David M. , Bradford Patricia A.
Published: February 2004

The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C
55
-MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.
Activity of Posaconazole Combined with Amphotericin B against
Aspergillus flavus
Infection in Mice: Comparative Studies in Two Laboratories
Authors: Najvar Laura K. , Cacciapuoti Anthony , Hernandez Steve , Halpern Judith , Bocanegra Rosie , Gurnani Maya , Menzel Frederick , Loebenberg David , Graybill John R.
Published: February 2004

Posaconazole and/or amphotericin B was given to mice pretreated with a steroid and then infected by inhalation of
Aspergillus flavus
conidia. Two laboratories conducted studies using almost identical protocols to evaluate both survival and lung tissue burdens 8 days after infection. The results of the in vivo studies performed at both laboratories were consistent. We found that (i) up to 5 mg of amphotericin B per kg of body weight was poorly effective in treating invasive aspergillosis; (ii) posaconazole at 2 or 10 mg/kg/dose prolonged survival and reduced lung tissue CFU; and (iii) there was generally no antagonistic interaction of the drugs in combination, even when the experiments were designed to maximize the likelihood of antagonism. These studies do not confirm the antagonistic interaction of triazoles and polyenes reported by others.
Activities of Garenoxacin against Quinolone-Resistant
Streptococcus pneumoniae
Strains In Vitro and in a Mouse Pneumonia Model
Authors: Azoulay-Dupuis E. , Bédos J. P. , Mohler J. , Peytavin G. , Isturiz R. , Moine P. , Rieux V. , Cherbuliez C. , Péchère J. C. , Fantin B. , Köhler T.
Published: February 2004

Garenoxacin is a novel des-F(6) quinolone with enhanced in vitro activities against both gram-positive and gram-negative bacteria. We compared the activity of garenoxacin with that of trovafloxacin (TVA) against
Streptococcus pneumoniae
, together with their efficacies and their capacities to select for resistant mutants, in a mouse model of acute pneumonia. In vitro, garenoxacin was more potent than TVA against wild-type
S. pneumoniae
and against a mutant with a single mutation (
parC
), a mutant with double mutations (
gyrA
and
parC
), and a mutant with triple mutations (
gyrA
,
parC
, and
parE
). Swiss mice were infected with 10
5
CFU of virulent, encapsulated
S. pneumoniae
strain P-4241 or its derived isogenic
parC
,
gyrA
,
gyrA parC
, and efflux mutants and 10
7
CFU of poorly virulent clinical strains carrying a
parE
mutation or
gyrA
,
parC
, and
parE
mutations. The drugs were administered six times, every 12 h, beginning at either 3 or 18 h postinfection. The pulmonary pharmacokinetic parameters in mice infected with strain P-4241 and treated with garenoxacin or TVA (25 mg/kg of body weight) were as follows: maximum concentration of drug in serum (
C
max
; 17.3 and 21.2 μg/ml, respectively),
C
max
/MIC ratio (288 and 170, respectively), area under the concentration-time curve (AUC; 48.5 and 250 μg · h/ml, respectively), and AUC/MIC ratio (808 and 2,000, respectively). Garenoxacin at 25 and 50 mg/kg was highly effective (survival rates, 85 to 100%) against the wild-type strain and mutants harboring a single mutation. TVA was as effective as garenoxacin against these strains. TVA at 200 mg/kg and garenoxacin at 50 mg/kg were ineffective against the mutant with the
parC
and
gyrA
double mutations and the mutant with the
gyrA
,
parC
, and
parE
triple mutations. The efficacy of garenoxacin was reduced only when strains bore several mutations for quinolone resistance.
Quantitative Evaluation of the Therapeutic Effects of Antibiotics Using Silkworms Infected with Human Pathogenic Microorganisms
Authors: Hamamoto Hiroshi , Kurokawa Kenji , Kaito Chikara , Kamura Koushirou , Manitra Razanajatovo Iony , Kusuhara Hiroyuki , Santa Tomofumi , Sekimizu Kazuhisa
Published: February 2004

The injection of bacteria (Staphylococcus aureus,Stenotrophomonas maltophilia) or true fungi (Candida albicans,Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.
Preclinical Pharmacology and Pharmacokinetics of GW433908, a Water-Soluble Prodrug of the Human Immunodeficiency Virus Protease Inhibitor Amprenavir
Authors: Furfine Eric S. , Baker Christopher T. , Hale Michael R. , Reynolds David J. , Salisbury Jo A. , Searle Andy D. , Studenberg Scott D. , Todd Dan , Tung Roger D. , Spaltenstein Andrew
Published: February 2004

GW433908 is the water-soluble, phosphate ester prodrug of the human immunodeficiency virus type 1 protease inhibitor amprenavir (APV). A high-yield synthesis of GW433908 is achieved by phosphorylation of the penultimate precursor of APV with phosphorous oxychloride (POCl
3
) in pyridine. A single-dose pharmacokinetic study of GW433908 sodium salt in dogs showed that APV exposure was similar to that achieved with an equivalent molar dose of the APV clinical formulation (Agenerase) and that systemic exposure to the prodrug was minimal (0.3% of the APV exposure). However, the sodium salt of GW433908 was a hygroscopic, amorphous solid and thus not suitable for pharmaceutical development. The calcium salt was a developable crystalline solid, but oral dosing afforded only 24% of the APV exposure in dogs compared with Agenerase. Acidification of the dog stomach by coadministration of HCl increased the bioavailability of the calcium salt to levels near those of the sodium salt. Single-dose administration of GW433908 calcium salt in dogs and rats produced portal vein GW433908 concentrations that were maximally 1.72 and 0.79% of those of APV concentrations, respectively. Furthermore, GW433908 had poor transepithelial flux and APV showed significant flux across human-derived Caco-2 cell monolayers (a model of intestinal permeability). Taken together, these results suggest that GW433908 is primarily metabolized to APV at or in the epithelial cells of the intestine and that the prodrug is not substantially absorbed. Based in part on these findings, GW433908 was advanced to clinical development.
Disposition of Caspofungin: Role of Distribution in Determining Pharmacokinetics in Plasma
Authors: Stone Julie A. , Xu Xin , Winchell Gregory A. , Deutsch Paul J. , Pearson Paul G. , Migoya Elizabeth M. , Mistry Goutam C. , Xi Liwen , Miller Alisha , Sandhu Punam , Singh Romi , deLuna Florencia , Dilzer Stacy C. , Lasseter Kenneth C.
Published: February 2004

The disposition of caspofungin, a parenteral antifungal drug, was investigated. Following a single, 1-h, intravenous infusion of 70 mg (200 μCi) of [
3
H]caspofungin to healthy men, plasma, urine, and feces were collected over 27 days in study A (
n
= 6) and plasma was collected over 26 weeks in study B (
n
= 7). Supportive data were obtained from a single-dose [
3
H]caspofungin tissue distribution study in rats (
n
= 3 animals/time point). Over 27 days in humans, 75.4% of radioactivity was recovered in urine (40.7%) and feces (34.4%). A long terminal phase (
t
1/2
= 14.6 days) characterized much of the plasma drug profile of radioactivity, which remained quantifiable to 22.3 weeks. Mass balance calculations indicated that radioactivity in tissues peaked at 1.5 to 2 days at ∼92% of the dose, and the rate of radioactivity excretion peaked at 6 to 7 days. Metabolism and excretion of caspofungin were very slow processes, and very little excretion or biotransformation occurred in the first 24 to 30 h postdose. Most of the area under the concentration-time curve of caspofungin was accounted for during this period, consistent with distribution-controlled clearance. The apparent distribution volume during this period indicated that this distribution process is uptake into tissue cells. Radioactivity was widely distributed in rats, with the highest concentrations in liver, kidney, lung, and spleen. Liver exhibited an extended uptake phase, peaking at 24 h with 35% of total dose in liver. The plasma profile of caspofungin is determined primarily by the rate of distribution of caspofungin from plasma into tissues.
Quality Assurance Program for Clinical Measurement of Antiretrovirals: AIDS Clinical Trials Group Proficiency Testing Program for Pediatric and Adult Pharmacology Laboratories
Authors: Holland Diane T. , DiFrancesco Robin , Stone Judith , Hamzeh Fayez , Connor James D. , Morse Gene D.
Published: February 2004

Clinical trials designed to compare antiretroviral regimens, investigate therapeutic drug monitoring, or measure pharmacometrics often include protease inhibitors (PIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and nucleoside reverse transcriptase inhibitors, requiring the measurement of these antiretrovirals in plasma. Within the adult and pediatric AIDS Clinical Trials Group (ACTG), a network of Pharmacology Support Laboratories (PSLs) is a component of the group laboratory infrastructure and conducts these types of pharmacologic assays. The adult ACTG has developed a comprehensive quality assurance program for the conduct of clinical pharmacology protocols, one component of which is the antiretroviral proficiency testing (PT) program that has been implemented between the adult and pediatric pharmacology laboratories of the ACTG. PT testing samples were prepared and distributed in July 2001, February 2002, and July 2002. High, medium, and low concentrations of PIs (indinavir, saquinavir, amprenavir, lopinavir, ritonavir, and nelfinavir) and NNRTIs (nevirapine and efavirenz) were added to drug-free EDTA plasma and distributed, on dry ice, to eight ACTG PSLs. One testing laboratory used liquid chromatography-tandem mass spectrometry, and seven used high-performance liquid chromatography-UV analysis. A result was considered acceptable if it was within 20% deviation of the assigned concentration. For all concentrations of PIs evaluated, 96% of samples tested (430 of 448 measurements) met the acceptance criteria. For both NNRTIs, 100% of samples tested (140 of 140 measurements) met the acceptance criteria. In conclusion, the PT program results presented demonstrate excellent interlaboratory agreement for all antiretrovirals tested and provide support for the merger of plasma concentration data among laboratories for large clinical trials.
Geldanamycin, a Ligand of Heat Shock Protein 90, Inhibits the Replication of Herpes Simplex Virus Type 1 In Vitro
Authors: Li Yu-Huan , Tao Pei-Zhen , Liu Yu-Zhen , Jiang Jian-Dong
Published: February 2004

Geldanamycin (GA) is an antibiotic targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90). In screening for anti-herpes simplex virus type 1 (HSV-1) candidates, we found GA active against HSV-1. HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093 μM and a concentration that inhibited cellular growth 50% in comparison with the results seen with untreated controls of 350 μM. The therapeutic index of GA was over 3,700 (comparable to the results seen with acyclovir). GA did not inhibit HSV-1 thymidine kinase. Cells infected with HSV-1 demonstrated cell cycle arrest at the G
1
/S transition; however, treatment with GA resulted in a cell cycle distribution pattern identical to that of untreated cells, indicating a restoration of cell growth in HSV-1-infected cells by GA treatment. Accordingly, HSV-1 DNA synthesis was suppressed in HSV-1
+
cells treated with GA. The antiviral mechanism of GA appears to be associated with Hsp90 inactivation and cell cycle restoration, which indicates that GA exhibits broad-spectrum antiviral activity. Indeed, GA exhibited activities in vitro against other viruses, including severe acute respiratory syndrome coronavirus. Since GA inhibits HSV-1 through a cellular mechanism unique among HSV-1 agents, we consider it a new candidate agent for HSV-1.
In Vitro Activities of 3-(Halogenated Phenyl)-5-Acyloxymethyl- 2,5-Dihydrofuran-2-ones against Common and Emerging Yeasts and Molds
Authors: Buchta Vladimír , Pour Milan , Kubanová Petra , Silva Luis , Votruba Ivan , Vopršálová Marie , Schiller Radan , Fáková Helena , Špulák Marcel
Published: February 2004

Three 3-(halogenated phenyl)-5-acyloxymethyl-2,5-dihydrofuran-2-ones were evaluated for activity against 191 strains of common and emerging yeasts andAspergillusspecies by the broth microdilution test performed according to NCCLS guidelines. The furanone derivatives displayed broad-spectrum in vitro activity against potentially pathogenic yeasts and molds, especiallyAspergillusspp. (MIC ≤ 2.0 μg/ml) and fluconazole-resistant yeast isolates, includingCandida glabrataandSaccharomyces cerevisiae. The 4-bromophenyl derivative was the most effective derivative against the majority of species tested, except for theCandida tropicalisandC. glabratastrains, which were more susceptible to the 3-chlorophenyl derivative. The 3,4-dichlorophenyl derivative possessed a lesser in vitro antifungal effect. The potential of further experiments on animal infection and clinical studies is supported by the relatively low cytotoxicity and acute toxicity of the 4-bromophenyl compound. Thus, the halogenated 3-phenyl-5-acyloxymethyl derivatives of 2,5-dihydrofuran-2-one represent a novel, promising group of compounds with significant activity against relevant opportunistic fungi that are pathogenic to humans.
Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY
Authors: Stachyra Thérèse , Dini Christophe , Ferrari Paul , Bouhss Ahmed , van Heijenoort Jean , Mengin-Lecreulx Dominique , Blanot Didier , Biton Jacques , Le Beller Dominique
Published: February 2004

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-

N
ε

-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an
Escherichia coli
overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.
Tolerability, Pharmacokinetics, and Serum Bactericidal Activity of Intravenous Dalbavancin in Healthy Volunteers
Authors: Leighton Anton , Gottlieb Alice Bendix , Dorr Mary Beth , Jabes Daniela , Mosconi Giorgio , VanSaders Claudia , Mroszczak Edward J. , Campbell Kathleen C. M. , Kelly Ellen
Published: February 2004

Fifty-two healthy adult male and female volunteers were enrolled in this double-blind study to determine the maximum tolerated dose, characterize the pharmacokinetics, and obtain serum bactericidal activity (SBA) data for intravenous dalbavancin. Subjects were assigned to single- or multiple-dose groups and randomized to receive dalbavancin or placebo intravenously over 30 min. Doses started at 140 mg in the single-dose group and with a 300-mg loading dose (LD), followed by six daily 30-mg maintenance doses (MDs), in the multiple-dose cohort and escalated to a 1,120-mg single dose and a 1,000-mg LD and 100-mg MD regimen. Plasma, urine, and skin blister fluid aspirate drug concentrations were measured, and pharmacokinetic parameters were determined via noncompartmental methods. SBA against methicillin-resistant
Staphylococcus aureus
(MRSA) was determined at several time points. Adverse events and changes from the baseline for laboratory data, electrocardiograms, audiologic assessments, physical examinations, and vital signs were assessed. Concentrations increased in proportion to the dose. Steady-state concentrations were achieved by day 3 with the 10:1 LD-MD regimen. The half-life averaged 181 h, and the mean volume of distribution and clearance were 9.75 liters and 0.0473 liters/h, respectively. Mean values were similar in all groups and in males and females. The portion of the dose excreted renally averaged 33.5%. Bactericidal activity was demonstrated in serum at 7 days in all subjects receiving single doses of ≥500 mg. All doses were well tolerated. Dose-limiting toxicity was not encountered. No changes in auditory or vestibular function occurred. The long half-life and maintenance of SBA against MRSA for 1 week suggest that weekly dosing may be feasible.
Interactions between Penicillin-Binding Proteins (PBPs) and Two Novel Classes of PBP Inhibitors, Arylalkylidene Rhodanines and Arylalkylidene Iminothiazolidin-4-ones
Authors: Zervosen Astrid , Lu Wei-Ping , Chen Zhouliang , White Ronald E. , Demuth Thomas P. , Frère Jean-Marie
Published: February 2004

Several non-β-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant
Staphylococcus aureus
MI339 and vancomycin-resistant
Enterococcus faecium
EF12. Several compounds were found to inhibit the cell wall synthesis of
S. aureus
and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of
Escherichia coli
or cell wall membrane preparations of
Bacillus megaterium
. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on
E. coli
PBP 3 and PBP 5,
Streptococcus pneumoniae
PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of
S. aureus
, and the
Actinomadura
sp. strain R39 and
Streptomyces
sp. strain R61
dd
-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 μM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (
Ki
= 10 μM) was not influenced by the presence of the substrate.
Diversity of ectomycorrhizas associated with Quercus garryana in southern Oregon
Authors: Valentine L L , Fiedler T L , Hart A N , Petersen C A , Berninghausen H K , Southworth D
Published: February 2004

We investigated diversity of ectomycorrhizas associated with Quercus garryana Dougl. ex Hook. (Oregon white oak, or Garry oak) at Whetstone Savanna Preserve in southern Oregon. Based on morphotyping and DNA restriction fragments, we described 39 ectomycorrhizas. The most common five morphotypes were found in 5% or more of 160 soil cores. Cenococcum geophilum, the most abundant morphotype, occurred in 75% of soil cores. Another common morphotype yielded a restriction fragment length polymorphism (RFLP) pattern similar to that of Tuber species. Uncommon morphotypes were responsible for the majority of ectomycorrhizal diversity on Q. garryana. Morphotype diversity of seedlings was more similar to that of their parent tree than to seedlings under other trees. Internal transcribed spacer (ITS)  RFLP patterns of ectomycorrhizas found beneath sporocarps did not match those of the sporocarps fruiting above ground. An understanding of the diversity of the ectomycorrhizal community on Q. garryana will enable us to compare ectomycorrhizas on other oak species and habitats; determine seasonality of ectomycorrhizal growth; evaluate treatments such as fire, grazing, invasion by exotic plants, and other anthropogenic disturbances; and aid restoration protocols.Key words: biocomplexity, biodiversity, ectomycorrhizas, hypogeous fungi, morphotypes, Peziza infossa, Tuber.
Developmental versus environmental control of early leaf phenology in juvenile Ohio buckeye (Aesculus glabra)
Authors: Augspurger Carol K
Published: February 2004

The spring phenology of juveniles of many canopy tree species in deciduous forests predates that of adult conspecifics. To determine whether the earlier phenology of seedlings of Aesculus glabra Willd. (Ohio buckeye) in Illinois, U.S.A., is developmentally or environmentally controlled, seedlings of five maternal parents were grown either in the understory or above a barn roof, simulating environmental conditions experienced at canopy height. Relative to canopy seedlings, understory seedlings had significantly earlier bud break (mean = 6 d), leaf expansion (8 d), leaf senescence (23 d), and leaf drop (18 d). Bud break and leaf expansion of canopy seedlings equalled that of canopy trees of Ohio buckeye, but senescence and leaf drop of canopy seedlings predated canopy trees by 45 and 67 d, respectively. Overall, results show evidence for environmental control over the spring phenology of juveniles. Thermal sums in spring accumulated more rapidly in the forest understory where nighttime temperatures were warmer than above the barn roof. Thus, the environmental control of spring phenology appears to be a non-stage-specific temperature cue that accumulates at different rates along the forest's vertical gradient. In contrast, senescence and leaf drop, while somewhat responsive to the environment, displayed strong developmental constraint.Key words: bud break, developmental constraint, leaf drop, leaf expansion, leaf senescence, thermal sums.
Receptor kinase signalling in plants
Authors: Haffani Yosr Z , Silva Nancy F , Goring Daphne R
Published: February 2004

Recent plant genome analyses have revealed a large family of plant receptor kinases with very divergent extracellular domains. While a large proportion of this family remains uncharacterized, emerging functions for several plant receptor kinases reveal roles in a variety of biological processes including growth, development, hormone perception, and plantmicrobe interactions. Significant progress has also been made in the understanding of four plant receptor kinase systems including their respective ligands and signalling pathways. Interestingly, a wide range of signalling proteins have been identified as functioning with these receptor kinases. In this review, an overview of plant receptor kinases, their biological functions, and their signalling pathways is presented.Key words: plants, Arabidopsis, receptor kinase, signal transduction.
Natural revegetation of hydrocarbon-contaminated soil in semi-arid grasslands
Authors: Robson Diana Bizecki , Knight J Diane , Farrell Richard E , Germida James J
Published: February 2004

One way to identify hydrocarbon-tolerant plant species for reclamation is to sample vegetation at contaminated sites allowed to recover naturally. We compared vegetation and soils of 14 hydrocarbon-contaminated plots in southern Saskatchewan to those of nearby uncontaminated plots to determine the impact on plant communities and soil properties. Contaminated plots had less vegetation and litter cover than uncontaminated plots, and significantly higher soil carbon to nitrogen ratios, pH, and hydrocarbon concentration, and lower nitrogen and phosphorus. Although species richness was not significantly different, Shannon's diversity was lower on contaminated plots. Mean compositional similarity of the plots, measured using Jaccard's index, was only 31%, and cover similarity, measured using Spatz's index, was only 22%. Vegetation composition differences occurred because mycorrhizal, woody and vegetatively reproducing species, and species using birds or unassisted means for seed dispersal were significantly less common on contaminated than uncontaminated plots. Self-pollinated species were significantly more common on contaminated plots. The most abundant species on contaminated soils were the annual forb Kochia scoparia and the native perennial grasses Hordeum jubatum, Distichlis stricta, Agropyron smithii, Agropyron trachycaulum, and Poa canbyi. This research shows that some plant species and functional groups are tolerant of the altered soil conditions at hydrocarbon-contaminated sites.Key words: functional groups, oil spills, phytoremediation, reclamation, succession, vegetation recovery.
Visual characterization of the extracellular matrix of Cochliobolus heterostrophus and a mutant strain with a modified matrix
Authors: Healy Rosaria A , Horner Harry T , Bronson Charlotte R
Published: February 2004

Two layers of extracellular matrix (ECM) and a film secreted outside the layers were visualized on germlings of Cochliobolus heterostrophus Drechsler grown on glass slides, cellophane membranes, and the surface of maize leaves. A mutant of C. heterostrophus, less virulent than the wild type, possessed the inner layer of ECM and the film, but not the outer layer. Using cytochemical and morphological methods, we explored the hypothesis that the reduced virulence of the mutant in leaves was due to the absence of the outer layer of the ECM. All ECMs were characterized using ruthenium red fixation, cryopreservation, immunocytochemistry, and colloidal gold labeling, before being examined with light and electron microscopy. With immunocytochemistry, antigens were localized in islands stained with ruthenium red within the scaffolding of the outer layer of the wild-type ECM on leaf surfaces and within the leaf. In the mutant, antigens were localized in the film on leaf surfaces. Comparisons between leaves infected by the two strains showed hyphae to be enclosed within material interpreted to be host response within intercellular spaces of leaves infected by the mutant, but not the wild type.Key words: Cochliobolus, cytochemistry, extracellular matrix, microscopy, mutant, virulence.
Reduction and uptake of methylene blue by human erythrocytes
Authors: May James M. , Qu Zhi-chao , Cobb Charles E.
Published: February 2004

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB+) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB+. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB+ <5 μM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB+-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB+-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB+, which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB+, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB+ reduction may correspond to MB+-dependent NAD(P)H reductase activity in erythrocyte ghosts.
BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury
Authors: Mousavi Kambiz , Parry David J. , Jasmin Bernard J.
Published: February 2004

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system.