bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones
Authors: Ahdut‐Hacohen Ronit , Duridanova Dessislava , Meiri Halina , Rahamimoff Rami
Published: February 2004

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non‐specific ion channel in the synaptic vesicle membrane, in the context of the post‐fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non‐specific ion channel was examined using the fused vesicle‐attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non‐specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.
Shorter latencies for motion trajectories than for flashes in population responses of cat primary visual cortex
Authors: Jancke Dirk , Erlhagen Wolfram , Schöner Gregor , Dinse Hubert R.
Published: February 2004

Psychophysical evidence in humans indicates that localization is different for stationary flashed and coherently moving objects. To address how the primary visual cortex represents object position we used a population approach that pools spiking activity of many neurones in cat area 17. In response to flashed stationary squares (0.4 deg) we obtained localized activity distributions in visual field coordinates, which we referred to as profiles across a ‘population receptive field’ (PRF). We here show how motion trajectories can be derived from activity across the PRF and how the representation of moving and flashed stimuli differs in position. We found that motion was represented by peaks of population activity that followed the stimulus with a speed‐dependent lag. However, time‐to‐peak latencies were shorter by ∼16 ms compared to the population responses to stationary flashes. In addition, motion representation showed a directional bias, as latencies were more reduced for peripheral‐to‐central motion compared to the opposite direction. We suggest that a moving stimulus provides ‘preactivation’ that allows more rapid processing than for a single flash event.
Quantitative analysis of cardiovascular modulation in respiratory neural activity
Authors: Dick Thomas E. , Morris Kendall F.
Published: February 2004

We propose the ‘δ2‐statistic’ for assessing the magnitude and statistical significance of arterial pulse‐modulated activity of single neurones and present the results of applying this tool to medullary respiratory‐modulated units. This analytical tool is a modification of the η2‐statistic and, consequently, based on the analysis of variance. The η2‐statistic reflects the consistency of respiratory‐modulated activity on a cycle‐by‐cycle basis. However, directly applying this test to activity during the cardiac cycle proved ineffective because subjects‐by‐treatments matrices did not contain enough ‘information’. We increased information by dividing the cardiac cycle into fewer bins, excluding cycles without activity and summing activity over multiple cycles. The analysed neuronal activity was an existing data set examining the neural control of respiration and cough. Neurones were recorded in the nuclei of the solitary tracts, and in the rostral and caudal ventral respiratory groups of decerebrate, neuromuscularly blocked, ventilated cats (n= 19). Two hundred of 246 spike trains were respiratory modulated; of these 53% were inspiratory (I), 36.5% expiratory (E), 6% IE phase spanning and 4.5% EI phase spanning and responsive to airway stimulation. Nearly half (n= 96/200) of the respiratory‐modulated units were significantly pulse modulated and 13 were highly modulated with δ2 values exceeding 0.3. In 10 of these highly modulated units, η2 values were greater than 0.3 and all 13 had, at least, a portion of their activity during expiration. We conclude that cardiorespiratory interaction is reciprocal; in addition to respiratory‐modulated activity in a subset of neuronal activity patterns controlling the cardiovascular system, pulse‐modulated activity exists in a subset of neuronal activity patterns controlling the respiratory system. Thus, cardio‐ventilatory coupling apparent in respiratory motor output is evident and, perhaps, derived from the neural substrate driving that output.
Carbachol regulation of rabbit ileal brush border Na+–H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent
Authors: Li Xuhang , Zhang Huiping , Cheong Alice , Yueping Chen Sharon Leu , Elowsky Christian G. , Donowitz Mark
Published: February 2004

The epithelial brush border membrane (BBM) Na+–H+ exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na+ absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na+ absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent‐soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent‐resistant membranes. The size of BBM NHE3 complexes increased in carbachol‐exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent‐soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent‐resistant fraction of ileal BBM. NHERF2, α‐actinin‐4 and protein kinase C were among those NHE3‐associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol‐mediated inhibition since: (1) c‐Src was rapidly activated in the detergent‐resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na+ absorption was completely abolished by  the Src family inhibitor 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine (PP2). Moreover, the carbachol‐induced increase in the size of NHE3‐containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent‐soluble fraction but not the detergent‐resistant membrane; and (3) the molecular level, at which NHE3 complex‐associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co‐IP of NHERF2 with α‐actinin‐4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol‐induced and Src‐mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.
Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA
Authors: Diebold Sandra S. , Kaisho Tsuneyasu , Hemmi Hiroaki , Akira Shizuo , Reis e Sousa Caetano
Published: February 2004

Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-α in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.
Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8
Authors: Heil Florian , Hemmi Hiroaki , Hochrein Hubertus , Ampenberger Franziska , Kirschning Carsten , Akira Shizuo , Lipford Grayson , Wagner Hermann , Bauer Stefan
Published: February 2004

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus–1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-α and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)–deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.
Nanos
Maintains Germline Stem Cell Self-Renewal by Preventing Differentiation
Authors: Wang Zhong , Lin Haifan
Published: February 2004

Despite much progress in understanding how extrinsic signaling regulates stem cell self-renewal, little is known about how cell-autonomous gene regulation controls this process. In
Drosophila
ovaries, germline stem cells (GSCs) divide asymmetrically to produce daughter GSCs and cystoblasts, the latter of which develop into germline cysts. Here, we show that removing the translational repressor Nanos from either GSCs or their precursors, the primordial germ cells (PGCs), causes both cell types to differentiate into germline cysts. Thus, Nanos is essential for both establishing and maintaining GSCs by preventing their precocious entry into oogenesis. These functions are likely achieved by repressing the translation of differentiation factors in PGCs and GSCs.
Evaluation of Acute Hematologic and Long-Term Pulmonary Toxicities of Radioimmunotherapy of
Cryptococcus neoformans
Infection in Murine Models
Authors: Dadachova E. , Bryan R. A. , Frenkel A. , Zhang T. , Apostolidis C. , Nosanchuk J. S. , Nosanchuk J. D. , Casadevall A.
Published: February 2004

We evaluated acute hematological and long-term pulmonary toxicity of radioimmunotherapy in murine models of
Cryptococcus neoformans
infection. Activities up to 250 μCi were well tolerated by healthy A/JCr mice for
213
Bi-18B7 and
188
Re-18B7 monoclonal antibodies. In infected mice, doses up to 150 μCi produced only transient toxicity. The lungs of treated mice had no evidence of radiation fibrosis.
In Vitro Activities of Moxifloxacin against 900 Aerobic and Anaerobic Surgical Isolates from Patients with Intra-Abdominal and Diabetic Foot Infections
Authors: Edmiston Charles E. , Krepel Candace J. , Seabrook Gary R. , Somberg Lewis R. , Nakeeb Atilla , Cambria Robert A. , Towne Jonathan B.
Published: February 2004

The in vitro activities of moxifloxacin, ciprofloxacin, levofloxacin, gatifloxacin, imipenem, piperacillin-tazobactam, clindamycin, and metronidazole against 900 surgical isolates were determined using NCCLS testing methods. Moxifloxacin exhibited good to excellent antimicrobial activity against most aerobic (90.8%) and anaerobic (97.1%) microorganisms, suggesting that it may be effective for the treatment of polymicrobial surgical infections.
Ribosomal Mutations in
Arcanobacterium pyogenes
Confer a Unique Spectrum of Macrolide Resistance
Authors: Jost B. Helen , Trinh Hien T. , Songer J. Glenn , Billington Stephen J.
Published: February 2004

Four macrolide-resistant
Arcanobacterium pyogenes
isolates contained A2058T, A2058G, or C2611G (
Escherichia coli
numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin.
Selection during Cefepime Treatment of a New Cephalosporinase Variant with Extended-Spectrum Resistance to Cefepime in an
Enterobacter aerogenes
Clinical Isolate
Authors: Barnaud G. , Benzerara Y. , Gravisse J. , Raskine L. , Sanson-Le Pors M. J. , Labia R. , Arlet G.
Published: February 2004

Enterobacter aerogenes
resistant to cefepime (MIC, 32 μg/ml) was isolated from a patient treated with cefepime for an infection caused by a strain of
E. aerogenes
overproducing its AmpC β-lactamase (MIC of cefepime, 0.5 μg/ml). The AmpC β-lactamase of the resistant strain had an L-293-P amino acid substitution and a high
k
cat
/
K
m
ratio for cefepime. Both of these modifications were necessary for resistance to cefepime.
Comparative In Vitro Activities of AC98-6446, a Novel Semisynthetic Glycopeptide Derivative of the Natural Product Mannopeptimycin α, and Other Antimicrobial Agents against Gram-Positive Clinical Isolates
Authors: Petersen Peter J. , Wang T. Z. , Dushin Russell G. , Bradford Patricia A.
Published: February 2004

AC98-6446 is a novel semisynthetic cyclic glycopeptide antibiotic related to the natural product mannopeptimycin α (AC98-1). In the present study the activity of AC98-6446 was evaluated against a variety of recent clinical gram-positive pathogens including multiply resistant strains. AC98-6446 demonstrated similar potent activities against methicillin-susceptible and methicillin-resistant staphylococci and glycopeptide-intermediate staphylococcal isolates (MICs at which 90% of isolates are inhibited [MIC
90
s], 0.03 to 0.06 μg/ml). AC98-6446 also demonstrated good activities against both vancomycin-resistant and -susceptible strains of enterococci (MIC
90
s, 0.12 and 0.25 μg/ml, respectively) as well as against streptococcal strains (MIC
90
s, ≤ 0.008 to 0.03 μg/ml). AC98-6446 demonstrated bactericidal activity in terms of the reduction in the viable counts (>3 log
10
CFU/ml) of staphylococcal and streptococcal isolates and a marked decrease in the viable counts of most enterococcal strains (from 0.2 to 2.5 log
10
CFU/ml). Unlike vancomycin, which demonstrates time-dependent killing, AC98-6446 demonstrated concentration-dependent killing. The potent activity, novel structure, and bactericidal activity demonstrated by AC98-6446 make it an attractive candidate for further development.
In Vitro Double and Triple Synergistic Activities of Polymyxin B, Imipenem, and Rifampin against Multidrug-Resistant
Acinetobacter baumannii
Authors: Yoon Jimmy , Urban Carl , Terzian Christian , Mariano Noriel , Rahal James J.
Published: February 2004

Eight unrelated clinical
Acinetobacter baumannii
isolates resistant to all commonly used antibiotics were subjected to three-dimensional checkerboard microtiter plate dilution and time-kill studies at one-fourth of their MICs of polymyxin B, imipenem, and rifampin. Synergy was demonstrated with combinations of polymyxin B and imipenem, polymyxin B and rifampin, and polymyxin B, imipenem, and rifampin. Double combinations of polymyxin B and imipenem and of polymyxin B and rifampin were bactericidal for seven of eight isolates, and triple combinations were bactericidal for all isolates within 24 h. Future clinical studies using double and triple therapy with these antibacterials may provide an effective option against potentially lethal infection due to multiresistant
Acinetobacter baumannii
.
Early Bactericidal Activity of Moxifloxacin in Treatment of Pulmonary Tuberculosis: a Prospective, Randomized Study
Authors: Pletz Mathias W. R. , De Roux Andres , Roth Andreas , Neumann Karl-Heinz , Mauch Harald , Lode Hartmut
Published: February 2004

Moxifloxacin is the most active fluoroquinolone against
Mycobacterium tuberculosis
in vitro. However, data about the efficacy in patients are not available. We enrolled 17 patients with tuberculosis in a prospective, randomized study. After 5 days of monotherapy with either moxifloxacin or isoniazid, we detected significant decreases in mean CFU per milliliter in sputum in both groups. The calculated early bactericidal activities for isoniazid and moxifloxacin were 0.209 and 0.273 log
10
CFU per ml of sputum per day, respectively. According to the data from our study, moxifloxacin exhibits an early bactericidal activity that is comparable to that of isoniazid.
Effect of Zinc Salts on Respiratory Syncytial Virus Replication
Authors: Suara Rahaman O. , Crowe James E.
Published: February 2004

Zinc supplementation decreases the morbidity of lower respiratory tract infection in pediatric patients in the developing world. We sought to determine if zinc mediates a specific inhibitory effect against the major cause of pediatric lower respiratory tract disease, respiratory syncytial virus (RSV). We determined the in vitro inhibitory effect of three zinc salts (zinc acetate, lactate, and sulfate) on the replication of RSV at various concentrations of 10 and 1 mM and 100 and 10 μM. The degree of inhibition of RSV replication was examined in the presence of zinc during preincubation, adsorption, or penetration and was compared with that caused by salts of other divalent cations. Complete inhibition of RSV plaque formation was observed at 1 and 10 mM, representing reductions that were ≥106-fold. At the lowest concentration tested, 10 μM, we observed ≥1,000-fold reductions in RSV yield when zinc was present during preincubation, adsorption, penetration, or egress of virus. The therapeutic indices, determined as ratios of 50% toxicity concentration to 50% inhibitory concentration, were 100, 150, and 120 for zinc acetate, zinc lactate, and zinc sulfate, respectively. The inhibitory effect of zinc salts on RSV was concentration dependent and was not observed with other salts containing divalent cations such as calcium, magnesium, and manganese. RSV plaque formation was prevented by pretreatment of HEp-2 cell monolayer cultures with zinc or by addition of zinc to methylcellulose overlay media after infection. The results of this study suggest that zinc mediates antiviral activity on RSV by altering the ability of the cell to support RSV replication.
New Findings on the Structure-Phototoxicity Relationship and Photostability of Fluoroquinolones with Various Substituents at Position 1
Authors: Hayashi Norihiro , Nakata Yoshihiro , Yazaki Akira
Published: February 2004

The present study examined the phototoxicities of a series of 7-(3-aminopyrrolidinyl) quinolones containing various substituents at position 1 (in which the substituent at R8 is a hydrogen or a halogen) by use of a mouse model. For the 7-(3-aminopyrrolidinyl) quinolones with a halogen atom at position 8, well-known substituent groups such as a cyclopropyl, an ethyl, or a difluorophenyl at position 1 were found to be responsible for severe phototoxicity. However, when an aminodifluorophenyl or an isoxazolyl group was placed at position 1, even 8-halogeno quinolones were found to be mildly phototoxic. This is the first report of 8-halogeno quinolones that are not severely phototoxic. Two structurally similar 8-chloro quinolones (the 1-aminodifluorophenyl 8-chloro quinolone and the 1-difluorophenyl 8-chloro quinolone) were investigated further. The former was mildly phototoxic; the latter was severely phototoxic. We demonstrate that these two 8-chloro quinolones have practically the same areas under the concentration-time curves from 0 to 4 h in auricular tissue, suggesting that the mild phototoxicity is not due to pharmacokinetic instability. The rates of UV photodegradation of these compounds were also measured. We found that these two quinolones photodegrade at similar rates, suggesting that the mild phototoxicity is not attained through increased photostability. In conclusion, the phototoxic potentials of fluoroquinolones are influenced not only by the substituent at position 8 but also by that at position 1 (a new finding from this study). We also discovered a mildly phototoxic 8-chloro quinolone which did not have increased photostability.
Pharmacokinetics of Posaconazole Coadministered with Antacid in Fasting or Nonfasting Healthy Men
Authors: Courtney Rachel , Radwanski Elaine , Lim Josephine , Laughlin Mark
Published: February 2004

Posaconazole is a potent broad-spectrum azole antifungal agent in clinical development for the treatment of invasive fungal infections. This study evaluated the potential for a pH-dependent pharmacokinetic interaction between posaconazole and an antacid (Mylanta), under fasting and nonfasting conditions. Twelve men completed this randomized, four-period crossover, single-dose study. Subjects received 200 mg of posaconazole following a 10-h fast, with 20 ml of Mylanta and a 10-h fast, with 20 ml of Mylanta and a high-fat breakfast, and with a high-fat breakfast alone. Antacid coadministration had no statistically significant effects on posaconazole bioavailability under fasting or nonfasting conditions. In the fasting state, antacid slightly increased the relative oral bioavailability of posaconazole by 15% (P= 0.296); in the nonfasting state, antacid decreased the relative bioavailability of posaconazole by 12% (P= 0.352). Food increased the relative oral bioavailability of posaconazole by 400% (P= 0.001). In conclusion, the effect of antacid on posaconazole exposure in the fasting or nonfasting state was small and is not considered clinically significant.
Possible Involvement of the Drug Transporters P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2 in Disposition of Azithromycin
Authors: Sugie Masami , Asakura Emiko , Zhao Ying Lan , Torita Shoko , Nadai Masayuki , Baba Kenji , Kitaichi Kiyoyuki , Takagi Kenji , Takagi Kenzo , Hasegawa Takaaki
Published: February 2004

P glycoprotein and multidrug resistance-associated protein 2 (Mrp2), ATP-dependent membrane transporters, exist in a variety of normal tissues and play important roles in the disposition of various drugs. The present study seeks to clarify the contribution of P glycoprotein and/or Mrp2 to the disposition of azithromycin in rats. The disappearance of azithromycin from plasma after intravenous administration was significantly delayed in rats treated with intravenous injection of cyclosporine, a P-glycoprotein inhibitor, but was normal in rats pretreated with intraperitoneal injection erythromycin, a CYP3A4 inhibitor. When rats received an infusion of azithromycin, cyclosporine and probenecid, a validated Mrp2 inhibitor, significantly decreased the steady-state biliary clearance of azithromycin to 5 and 40% of the corresponding control values, respectively. However, both inhibitors did not alter the renal clearance of azithromycin, suggesting the lack of renal tubular secretion of azithromycin. Tissue distribution experiments showed that azithromycin is distributed largely into the liver, kidney, and lung, whereas both inhibitors did not alter the tissue-to-plasma concentration ratio of azithromycin. Significant reduction in the biliary excretion of azithromycin was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. An in situ closed-loop experiment showed that azithromycin was excreted from the blood into the gut lumen, and the intestinal clearance of azithromycin was significantly decreased by the presence of cyclosporine in the loop. These results suggest that azithromycin is a substrate for both P glycoprotein and Mrp2 and that the biliary and intestinal excretion of azithromycin is mediated via these two drug transporters.
In Vitro and In Vivo Activities of Antimicrobials against
Nocardia brasiliensis
Authors: Gomez-Flores Alejandra , Welsh Oliverio , Said-Fernández Salvador , Lozano-Garza Gerardo , Tavarez-Alejandro Roman Erick , Vera-Cabrera Lucio
Published: February 2004

In Mexico mycetomas are mostly produced by
Nocardia brasiliensis
, which can be isolated from about 86% of cases. In the present work, we determined the sensitivities of 30
N. brasiliensis
strains isolated from patients with mycetoma to several groups of antimicrobials. As a first screening step we carried out disk diffusion assays with 44 antimicrobials, including aminoglycosides, cephalosporins, penicillins, quinolones, macrolides, and some others. In these assays we observed that some antimicrobials have an effect on more than 66% of the strains: linezolid, amikacin, gentamicin, isepamicin, netilmicin, tobramycin, minocycline, amoxicillin-clavulanic acid, piperacillin-tazobactam, nitroxolin, and spiramycin. Drug activity was confirmed quantitatively by the broth microdilution method. Amoxicillin-clavulanic acid, linezolid, and amikacin, which have been used to treat patients, were tested in an experimental model of mycetoma in BALB/c mice in order to validate the in vitro results. Linezolid showed the highest activity in vivo, followed by the combination amoxicillin-clavulanic acid and amikacin.
Isolation and Characterization of Integron-Containing Bacteria without Antibiotic Selection
Authors: Barlow Robert S. , Pemberton John M. , Desmarchelier Patricia M. , Gobius Kari S.
Published: February 2004

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1andintI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such asEscherichia coli,Aeromonasspp.,Proteusspp.,Morganella morganii,Shewanellaspp., and urea-positiveProvidencia stuartiiisolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.
Selection of an Antibiotic-Hypersusceptible Mutant of
Pseudomonas aeruginosa
: Identification of the GlmR Transcriptional Regulator
Authors: Ramos-Aires Julio , Plésiat Patrick , Kocjancic-Curty Lasta , Köhler Thilo
Published: February 2004

Tn
501
random mutagenesis was applied to the
Pseudomonas aeruginosa
wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents. One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, β-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides. Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells. The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures. The insertion site of the Tn
501
transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors. The gene, which we called
glmR
, is located between the
glmS
(PA5549) and
glmU
(PA5552) homologues of
E. coli
, responsible for the synthesis of UDP-
N
-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan. We showed that GlmR represses the transcription of the adjacent
glmS
homologue (PA5549) in
P. aeruginosa
, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis. To our knowledge GlmR is the first regulator in
P. aeruginosa
that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents.
Miltefosine Induces Apoptosis-Like Death in
Leishmania donovani
Promastigotes
Authors: Paris Caroline , Loiseau Philippe M. , Bories Christian , Bréard Jaqueline
Published: February 2004

Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to
Leishmania donovani
. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of
Leishmania donovani
HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill
L. donovani
promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors,
z
-Val-Ala-
dl
-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.
Effect of Antibiotic Treatment Delay on Therapeutic Outcome of Experimental Acute Otitis Media Caused by
Streptococcus pneumoniae
Strains with Different Susceptibilities to Amoxicillin
Authors: Parra Araceli , Ponte Carmen , Cenjor Carlos , Martínez-Marín Carmen , Soriano Francisco
Published: February 2004

The effect of delayed administration of amoxicillin on the course of acute otitis media (AOM) caused by two
Streptococcus pneumoniae
strains with different susceptibilities to amoxicillin (MICs of 0.016 and 1 μg/ml for strains A and B, respectively) was evaluated in the gerbil model. The organisms were inoculated by transbullar challenge into the middle ear, and antibiotic treatment was administered at various times thereafter. The bacteriological and clinical efficacies of treatment diminished significantly with the delay of antibiotic administration. The bacterial eradication rates when antibiotic treatment was started at 2, 5, 8, 18, and 21 h post-bacterial inoculation were different for both strains (95, 95, 90, 55, and 55% for strain A and 95, 95, 65, 10, and 0% for strain B). Results of further experiments using strain B with higher antibiotic doses and numbers of administrations and different follow-up times indicate that the failures observed with the delayed administration were not related to the bacterial burden, selection of antibiotic-resistant mutants, or inadequate pharmacodynamic parameters. Such failures may be related to the metabolic bacterial status. The delayed amoxicillin treatment of AOM caused by
S. pneumoniae
may lead to therapeutic failures, mainly when organisms with diminished antibiotic susceptibility are involved.
Plasmodium falciparum
Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance
Authors: Ahmed Anwar , Bararia Deepak , Vinayak Sumiti , Yameen Mohammed , Biswas Sukla , Dev Vas , Kumar Ashwani , Ansari Musharraf A. , Sharma Yagya D.
Published: February 2004

The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant
Plasmodium falciparum
malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of
P. falciparum
collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double
dhfr
mutations and single
dhps
mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.
Killing by Ampicillin and Ofloxacin Induces Overlapping Changes in
Escherichia coli
Transcription Profile
Authors: Kaldalu Niilo , Mei Rui , Lewis Kim
Published: February 2004

The basis of bactericidal versus bacteriostatic action of antibiotics and the mechanism of bacterial cell death are largely unknown. Related to this important issue is the essential invulnerability to killing of persisters: cells forming a small subpopulation largely responsible for the recalcitrance of biofilms to chemotherapy. To learn whether death is accompanied by changes in expression of particular genes, we compared transcription profiles of log-phase
Escherichia coli
treated with bactericidal concentrations of two unrelated antibiotics: ampicillin and ofloxacin. Massive changes in transcription profile were observed in response to either agent, and there was a significant overlap in genes whose transcription was affected. A small group of mostly uncharacterized genes was induced and a much larger set was transcriptionally repressed by both antibiotics. Among the repressed genes were those required for flagellar synthesis, energy metabolism, transport of small molecules, and protein synthesis.
Tetracycline and Streptomycin Resistance Genes, Transposons, and Plasmids in
Salmonella enterica
Isolates from Animals in Italy
Authors: Pezzella Cristina , Ricci Antonia , DiGiannatale Elisabetta , Luzzi Ida , Carattoli Alessandra
Published: February 2004

Fifty-eight multidrug-resistant
Salmonella enterica
strains of 20 serotypes, isolated from animal sources in Italy, were analyzed for
tet
(A) and
strA
-
strB
, conferring tetracycline and streptomycin resistance, respectively. The
strA
and
strB
genes were highly prevalent in
Salmonella
strains of our collection, being detected in 84% of the streptomycin-resistant strains. In many strains, the
strA
and
strB
genes were linked to a particular Tn
5393-
derivative transposon characterized by the presence of the insertion sequence IS
1133
, previously identified only in the plant pathogen
Erwinia amylovora.
Sixty-eight percent of the tetracycline-resistant strains were
tet
(A) positive, indicating that this gene is widely diffused in
Salmonella
strains circulating in animals in Italy. Most of the
tet
(A) genes were localized within a deleted Tn
1721
transposon variant. Two prevalent repN and repI1 resistance plasmids were identified in
Salmonella
isolates of our collection.
Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in
Staphylococcus aureus
Authors: Huang Jianzhong , O'Toole Paul W. , Shen Wei , Amrine-Madsen Heather , Jiang Xinhe , Lobo Neethan , Palmer Leslie M. , Voelker LeRoy , Fan Frank , Gwynn Michael N. , McDevitt Damien
Published: February 2004

Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in
Staphylococcus aureus
, MdeA (multidrug efflux A). MdeA was identified from screening an
S. aureus
open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on
S. aureus
to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of
Bacillus subtilis
and EmrB of
Escherichia coli
. Overexpression of
mdeA
in
S. aureus
reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The
mdeA
promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for
mdeA
, and their
mdeA
transcription levels were increased by as much as 15-fold. The
mdeA
gene was present in the genomes of all six strains of
S. aureus
examined. Uncharacterized homologs of MdeA were present elsewhere in the
S. aureus
genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including
Bacillus anthracis
, some of which were shown to be functional orthologs of MdeA.
Quercetin Induces Apoptosis of
Trypanosoma brucei gambiense
and Decreases the Proinflammatory Response of Human Macrophages
Authors: Mamani-Matsuda Maria , Rambert Jérôme , Malvy Denis , Lejoly-Boisseau Hélène , Daulouède Sylvie , Thiolat Denis , Coves Sara , Courtois Pierrette , Vincendeau Philippe , Mossalayi M. Djavad
Published: February 2004

In addition to parasite spread, the severity of disease observed in cases of human African trypanosomiasis (HAT), or sleeping sickness, is associated with increased levels of inflammatory mediators, including tumor necrosis factor (TNF)-α and nitric oxide derivatives. In the present study, quercetin (3,3′,4′,5,7-pentahydroxyflavone), a potent immunomodulating flavonoid, was shown to directly induce the death of
Trypanosoma brucei gambiense
, the causative agent of HAT, without affecting normal human cell viability. Quercetin directly promoted
T. b. gambiense
death by apoptosis as shown by Annexin V binding. In addition to microbicidal activity, quercetin induced dose-dependent decreases in the levels of TNF-α and nitric oxide produced by activated human macrophages. These results highlight the potential use of quercetin as an antimicrobial and anti-inflammatory agent for the treatment of African trypanomiasis.
Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in
Streptomyces clavuligerus
Authors: Tahlan Kapil , Park Hyeon Ung , Wong Annie , Beatty Perrin H. , Jensen Susan E.
Published: February 2004

Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (
pah1
), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in
Streptomyces clavuligerus
, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (
ceaS
) and β-lactam synthetase (
bls
) enzymes. These new paralogues are given the gene designations
ceaS1
and
bls1
and are located immediately upstream of
pah1
on the chromosome. Furthermore, sequence analysis of the region downstream of
pah1
revealed a second copy of a gene encoding ornithine acetyltransferase (
oat1
), thus indicating the presence of a cluster of paralogue genes.
ceaS1
,
bls1
, and
oat1
display 73, 60, and 63% identities, respectively, at the nucleotide level to the original
ceaS2
,
bls2
, and
oat2
genes from the clavulanic acid gene cluster. Single mutants defective in
ceaS1
,
bls1
, or
oat1
were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The
ceaS1
/
ceaS2
and the
bls1
/
bls2
mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand,
oat1
/
oat2
double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the
argJ
gene in
S. clavuligerus
, which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants.
Activities of Ciprofloxacin and Moxifloxacin against
Stenotrophomonas maltophilia
and Emergence of Resistant Mutants in an In Vitro Pharmacokinetic-Pharmacodynamic Model
Authors: Ba Boubakar B. , Feghali Hala , Arpin Corinne , Saux Marie-Claude , Quentin Claudine
Published: February 2004

A two-compartment in vitro pharmacokinetic-pharmacodynamic model, with full computer-controlled devices, was used to accurately simulate human plasma pharmacokinetic profiles after multidose oral regimens of ciprofloxacin (750 mg every 12 h) and moxifloxacin (400 mg every 24 h) during 48 h. Pharmacodynamics of these drugs was investigated against three quinolone-susceptible strains of
Stenotrophomonas maltophilia
(MICs of ciprofloxacin and moxifloxacin of 0.5 to 2 and 0.0625 to 0.5 μg/ml, respectively). The first dose of ciprofloxacin and moxifloxacin reduced the bacterial count by 1 and 2 log CFU/ml, respectively, prior to a bacterial regrowth that reached the plateau value of the growth control curve at 13 to 24 h versus 24 to 36 h and persisted despite repeated administration of both drugs. The surviving bacterial cells were quinolone-resistant mutants (2 to 128 times the MIC) that exhibited cross-resistance to unrelated antibiotics. Their antibiotic resistance probably resulted from the overproduction of different multidrug resistance efflux system(s).
C
max
/MIC and area under the concentration-time curve from 0 to 24 h (AUC
0-24
)/MIC values were at least threefold higher for moxifloxacin than for ciprofloxacin. Moreover, integral parameters of ciprofloxacin and moxifloxacin, in particular the area under the killing and regrowth curve from 0 to 48 h (AUBC
0-48
, 342.3 to 401.3 versus 295.2 to 378.7 h × log CFU/ml, respectively) and the area between the control growth curve and the killing and regrowth curve from 0 to 48 h (ABBC
0-48
, 40.4 to 101.1 versus 72.9 to 144.7 h × log CFU/ml, respectively), demonstrated a better antibacterial effect of moxifloxacin than ciprofloxacin on
S. maltophilia
. However, selection of resistant mutants by both fluoroquinolones, although delayed with moxifloxacin, emphasizes the need to use maximal dosages and combined therapy in the treatment of systemic
S. maltophilia
infections.
In Vitro Evaluation of Double and Triple Combinations of Antifungal Drugs against
Aspergillus fumigatus
and
Aspergillus terreus
Authors: Dannaoui Eric , Lortholary Olivier , Dromer Françoise
Published: February 2004

Microdilution broth checkerboard techniques based on the National Committee for Clinical Laboratory Standards methodology were used to study double and triple antifungal combinations against clinical isolates of
Aspergillus fumigatus
and
A. terreus
. The influences of the end-point definition (partial or complete inhibition) and the mode of reading (visually or spectrophotometrically) were determined. Interactions between antifungal drugs were also evaluated by agar diffusion tests. Combinations of caspofungin with either amphotericin B or voriconazole were additive for all the isolates, and antagonism was not observed. The interaction between caspofungin and flucytosine was synergistic for 62% of the isolates. In contrast, the interaction between voriconazole and flucytosine was never synergistic and antagonism was noted for 93% of the isolates. The triple combination of caspofungin with flucytosine and amphotericin B was synergistic for all the isolates tested. The triple combination of caspofungin with flucytosine and voriconazole was also mostly synergistic; but complex interactions were obtained for some isolates, with synergy or antagonism depending on the concentrations of caspofungin and voriconazole. Analysis of the influence of the reading technique on the results showed that spectrophotometric reading was a good alternative to the recommended visual reading. The results of these in vitro tests suggest that the activity of flucytosine as part of a double combination with caspofungin and as part of a triple combination with caspofungin and amphotericin B against
Aspergillus
spp. warrants further investigations. Animal studies are needed to evaluate the in vivo efficacies of these combinations.
Amphotericin B and Fluconazole, a Potent Combination Therapy for Cryptococcal Meningitis
Authors: Larsen Robert A. , Bauer Madeline , Thomas Ann M. , Graybill J. Richard
Published: February 2004

We evaluated the antifungal activities of amphotericin B, fluconazole, and flucytosine, alone and in combination, in a murine model of cryptococcal meningitis. The objectives were to determine the greatest antifungal effects achievable with these drugs alone or in combination. Meningitis was established in male BALB/c mice weighing 23 to 25 g by intracerebral injection of
Cryptococcus neoformans
. Treatment was started on day 2. Amphotericin B was tested at 0.3 to 1.3 mg/kg of body weight/day by slow intravenous injection. Fluconazole at 10 to 40 mg/kg/day and flucytosine at 20 to 105 mg/kg/day were administered in the sole source of drinking water. The mice were killed at 16 days, and the numbers of fungal colonies in the brain were quantified. The association between the response and the dose combination was evaluated by local nonparametric response surface methods; 99% confidence intervals were used to evaluate the antifungal effects. Ninety-five percent of the mice treated with amphotericin B at 0.5 mg/kg survived to the end of the experiment, regardless of the fluconazole or flucytosine dose used. The greatest activity was seen with amphotericin B plus fluconazole with or without flucytosine. However, the addition of flucytosine did not increase the antifungal activity. Given the widespread availability of amphotericin B and fluconazole and the relative safety profile of fluconazole compared to that of flucytosine, the full potential of this two-drug combination deserves further evaluation.