bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation by melatonin of glutamatergic synaptic transmission in the carp retina
Authors: Huang Hai , Lee Shu‐Chen , Yang Xiong‐Li
Published: October 2005

Melatonin is involved in a variety of physiological functions through activating specific receptors coupled to GTP‐binding protein. Melatonin and its receptors are abundant in the retina. Here we show for the first time that melatonin modulates glutamatergic synaptic transmission from cones to horizontal cells (HCs) in carp retina. Immunocytochemical data revealed the expression of the MT1 receptor on carp HCs. Whole‐cell recordings further showed that melatonin of physiological concentrations potentiated glutamate‐induced currents from isolated cone‐driven HCs (H1 cells) in a dose‐dependent manner, by increasing the efficacy and apparent affinity of the glutamate receptor. The effects of melatonin were reversed by luzindole, but not by K 185, indicating the involvement of the MT1 receptor. Like melatonin, methylene blue (MB), a guanylate cyclase inhibitor, also potentiated the glutamate currents, but internal infusion of cGMP suppressed them. The effects of melatonin were not observed in cGMP‐filled and MB‐incubated HCs. These results suggest that the melatonin effects may be mediated by decreasing the intracellular concentration of cGMP. Consistent with these observations, melatonin depolarized the membrane potential of H1 cells and reduced their light responses, which could also be blocked by luzindole. These effects of melatonin persisted in the presence of the antagonists of receptors for dopamine, GABA and glycine, indicating a direct action of melatonin on H1 cells. Such modulation by melatonin of glutamatergic transmission from cones to HCs is thought to be in part responsible for circadian changes in light responsiveness of cone HCs in teleost retina.
Two‐pore‐domain potassium channels in smooth muscles: new components of myogenic regulation
Authors: Sanders Kenton M. , Don Koh Sang
Published: October 2005

Gastrointestinal (GI) smooth muscles are influenced by many levels of regulation, including those provided by enteric motor neurones, hormones and paracrine substances. The integrated contractile responses to these regulatory mechanisms depend heavily on the state of excitability of smooth muscle cells. Resting ionic conductances and myogenic responses to agonists and physical parameters, such as stretch, are important in establishing basal excitability. This review discusses the role of 2‐pore‐domain K+ channels in contributing to background conductances and in mediating responses of GI muscles to enteric inhibitory nerve stimulation and stretch. Murine GI muscles express TREK‐1 channels and display a stretch‐dependent K+ (SDK) conductance that is also activated by nitric oxide via a cGMP‐dependent mechanism. Cloning and expression of mTREK‐1 produced an SDK conductance that was activated by cGMP‐dependent phosphorylation at serine‐351. GI muscle cells also express TASK‐1 and TASK‐2 channels that are inhibited by lidocaine and external acidification. These conductances appear to provide significant background K+ permeability that contributes to the negative resting potentials of GI muscles.
MaxiK channel partners: physiological impact
Authors: Lu Rong , Alioua Abderrahmane , Kumar Yogesh , Eghbali Mansoureh , Stefani Enrico , Toro Ligia
Published: October 2005

The basic functional unit of the large‐conductance, voltage‐ and Ca2+‐activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore‐forming α‐subunit (MaxiKα) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKα can associate with modulatory β‐subunits (β1–β4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKα‐related disorder caused by a gain‐of‐function C‐terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKα interaction with its surrounding signalling partners, and its targeting to cell‐specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C‐terminus of MaxiKα are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C‐terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue‐specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.
The cellular mechanisms by which adenosine evokes release of nitric oxide from rat aortic endothelium
Authors: Ray Clare J. , Marshall Janice M.
Published: October 2005

Adenosine and nitric oxide (NO) are important local mediators of vasodilatation. The aim of this study was to elucidate the mechanisms underlying adenosine receptor‐mediated NO release from the endothelium. In studies on freshly excised rat aorta, second‐messenger systems were pharmacologically modulated by appropriate antagonists while a NO‐sensitive electrode was used to measure adenosine‐evoked NO release from the endothelium. We showed that A1‐mediated NO release requires extracellular Ca2+, phospholipase A2(PLA2) and ATP‐sensitive K+(KATP) channel activation whereas A2A‐mediated NO release requires extracellular Ca2+and Ca2+‐activated K+(KCa) channels. Since our previous study showed that A1‐ and A2A‐receptor‐mediated NO release requires activation of adenylate cyclase (AC), we propose the following novel pathways. The K+efflux resulting from A1‐receptor‐coupled KATP‐channel activation facilitates Ca2+influx which may cause some stimulation of endothelial NO synthase (eNOS). However, the increase in [Ca2+]ialso stimulates PLA2to liberate arachidonic acid and stimulate cyclooxygenase to generate prostacyclin (PGI2). PGI2acts on its endothelial receptors to increase cAMP, so activating protein kinase A (PKA) to phosphorylate and activate eNOS resulting in NO release. By contrast, the K+efflux resulting from A2A‐coupled KCachannels facilitates Ca2+influx, thereby activating eNOS and NO release. This process may be facilitated by phosphorylation of eNOS by PKA via the action of A2A‐receptor‐mediated stimulation of AC increasing cAMP. These pathways may be important in mediating vasodilatation during exercise and systemic hypoxia when adenosine acting in an endothelium‐ and NO‐dependent manner has been shown to be important.
Olfactoretinal centrifugal input modulates zebrafish retinal ganglion cell activity: a possible role for dopamine‐mediated Ca2+ signalling pathways
Authors: Huang Luoxiu , Maaswinkel Hans , Li Lei
Published: October 2005

The vertebrate retina receives centrifugal input from the brain. In zebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA‐IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine‐mediated Ca2+ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca2+ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. This model of olfacto‐visual integration was directly tested by recording single‐unit RGC activity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage‐activated Ca2+ current and lowered Ca2+ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor‐coupled Ca2+ signalling pathways.
Interlinked Fast and Slow Positive Feedback Loops Drive Reliable Cell Decisions
Authors: Brandman Onn , Ferrell James E. , Li Rong , Meyer Tobias
Published: October 2005

Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling,
Xenopus
oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a “dual-time” switch that is both rapidly inducible and resistant to noise in the upstream signaling system.
The ß-Glucuronidase Klotho Hydrolyzes and Activates the TRPV5 Channel
Authors: Chang Q. , Hoefs S. , van der Kemp A. W. , Topala C. N. , Bindels R. J. , Hoenderop J. G.
Published: October 2005

Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a β-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.
The Biochemical Architecture of an Ancient Adaptive Landscape
Authors: Lunzer Mark , Miller Stephen P. , Felsheim Roderick , Dean Antony M.
Published: October 2005

Molecular evolution is moving from statistical descriptions of adaptive molecular changes toward predicting the fitness effects of mutations. Here, we characterize the fitness landscape of the six amino acids controlling coenzyme use in isopropylmalate dehydrogenase (IMDH). Although all natural IMDHs use nicotinamide adenine dinucleotide (NAD) as a coenzyme, they can be engineered to use nicotinamide adenine dinucleotide phosphate (NADP) instead. Intermediates between these two phenotypic extremes show that each amino acid contributes additively to enzyme function, with epistatic contributions confined to fitness. The genotype-phenotype-fitness map shows that NAD use is a global optimum.
Geographic Control of Titan's Mid-Latitude Clouds
Authors: Roe Henry G. , Brown Michael E. , Schaller Emily L. , Bouchez Antonin H. , Trujillo Chadwick A.
Published: October 2005

Observations of Titan's mid-latitude clouds from the W. M. Keck and Gemini Observatories show that they cluster near 350°W longitude, 40°S latitude. These clouds cannot be explained by a seasonal shift in global circulation and thus presumably reflect a mechanism on Titan such as geysering or cryovolcanism in this region. The rate of volatile release necessary to trigger cloud formation could easily supply enough methane to balance the loss to photolysis in the upper atmosphere.
Visualization of the Molecular Jahn-Teller Effect in an Insulating K
4
C
60
Monolayer
Authors: Wachowiak A. , Yamachika R. , Khoo K. H. , Wang Y. , Grobis M. , Lee D.-H. , Louie Steven G. , Crommie M. F.
Published: October 2005

We present a low-temperature scanning tunneling microscopy (STM) study of K

x

C
60
monolayers on Au(111) for 3 ≤
x
≤ 4. The STM spectrum evolves from one that is characteristic of a metal at
x
= 3 to one that is characteristic of an insulator at
x
= 4. This electronic transition is accompanied by a dramatic structural rearrangement of the C
60
molecules. The Jahn-Teller effect, a charge-induced mechanical deformation of molecular structure, is directly visualized in the K
4
C
60
monolayer at the single-molecule level. These results, along with theoretical analyses, provide strong evidence that the transition from metal to insulator in K

x

C
60
monolayers is caused by the Jahn-Teller effect.
Escaping an Epidemic
Authors: Anderson Larry J.
Published: October 2005

SARS
. A Case Study in Emerging Infections.
Angela R. McLean, Robert M. May, John Pattison, and Robin A. Weiss, Eds.
. Oxford University Press, New York, 2005. 141 pp. $99.50, £55. ISBN 0-19-856818-5. Paper, $39.50, £24.95. ISBN 0-19-856819-3.

Twenty-First Century Plague
. The Story of SARS.
By Thomas Abraham
. Johns Hopkins University Press, Baltimore, MD, 2005. 173 pp. $24.95. ISBN 0-8018-8124-2.

Two distinct and interesting perspectives on the sudden appearance and rapid spread of severe acute respiratory syndrome in 2003. The contributors to the McLean
et al
. volume approach the disease from academia and the laboratory. Abraham explores the biology and epidemiology of SARS, the political responses to the threat of the disease, and their interactions.
The Evolution of Titan's Mid-Latitude Clouds
Authors: Griffith C. A. , Penteado P. , Baines K. , Drossart P. , Barnes J. , Bellucci G. , Bibring J. , Brown R. , Buratti B. , Capaccioni F. , Cerroni P. , Clark R. , Combes M. , Coradini A. , Cruikshank D. , Formisano V. , Jaumann R. , Langevin Y. , Matson D. , McCord T. , Mennella V. , Nelson R. , Nicholson P. , Sicardy B. , Sotin C. , Soderblom L. A. , Kursinski R.
Published: October 2005

Spectra from Cassini's Visual and Infrared Mapping Spectrometer reveal that the horizontal structure, height, and optical depth of Titan's clouds are highly dynamic. Vigorous cloud centers are seen to rise from the middle to the upper troposphere within 30 minutes and dissipate within the next hour. Their development indicates that Titan's clouds evolve convectively; dissipate through rain; and, over the next several hours, waft downwind to achieve their great longitude extents. These and other characteristics suggest that temperate clouds originate from circulation-induced convergence, in addition to a forcing at the surface associated with Saturn's tides, geology, and/or surface composition.
Air-Stable All-Inorganic Nanocrystal Solar Cells Processed from Solution
Authors: Gur Ilan , Fromer Neil A. , Geier Michael L. , Alivisatos A. Paul
Published: October 2005

We introduce an ultrathin donor-acceptor solar cell composed entirely of inorganic nanocrystals spin-cast from solution. These devices are stable in air, and post-fabrication processing allows for power conversion efficiencies approaching 3% in initial tests. This demonstration elucidates a class of photovoltaic devices with potential for stable, low-cost power generation.
Selective Logging in the Brazilian Amazon
Authors: Asner Gregory P. , Knapp David E. , Broadbent Eben N. , Oliveira Paulo J. C. , Keller Michael , Silva Jose N.
Published: October 2005

Amazon deforestation has been measured by remote sensing for three decades. In comparison, selective logging has been mostly invisible to satellites. We developed a large-scale, high-resolution, automated remote-sensing analysis of selective logging in the top five timber-producing states of the Brazilian Amazon. Logged areas ranged from 12,075 to 19,823 square kilometers per year (±14%) between 1999 and 2002, equivalent to 60 to 123% of previously reported deforestation area. Up to 1200 square kilometers per year of logging were observed on conservation lands. Each year, 27 million to 50 million cubic meters of wood were extracted, and a gross flux of ∼0.1 billion metric tons of carbon was destined for release to the atmosphere by logging.
Population Pharmacokinetics of Rifapentine and Its Primary Desacetyl Metabolite in South African Tuberculosis Patients
Authors: Langdon Grant , Wilkins Justin , McFadyen Lynn , McIlleron Helen , Smith Peter , Simonsson Ulrika S. H.
Published: October 2005

This study was designed to describe the population pharmacokinetics of rifapentine (RFP) and 25-desacetyl RFP in a South African pulmonary tuberculosis patient population. Special reference was made to studying the influence of previous exposure to rifampin (RIF) and the variability in pharmacokinetic parameters between patients and between occasions and the influence of different covariates. Patients were included in the study if they had been receiving first-line antimycobacterial therapy (rifampin, isoniazid, pyrazinamide, and ethambutol) for not less than 4 weeks and not more than 6 weeks and were divided into three RFP dosage groups based on weight: 600 mg, <45 kg; 750 mg, 46 to 55 kg; and 900 mg, >55 kg. Participants received a single oral dose of RFP together with concomitant antimycobacterial agents, excluding RIF, on study days 1 and 5 after they ingested a soup-based meal. The RFP and 25-desacetyl RFP concentration-time data were analyzed by nonlinear mixed-effect modeling using NONMEM. The pharmacokinetics of the parent drug were modeled separately, and the individual pharmacokinetic parameters were used as inputs for the 25-desacetyl RFP pharmacokinetic model. A one-compartment disposition model was found to best describe the data for both the parent and the metabolite, and the metabolite was assumed to be formed only from the central compartment of the parent drug. Prior treatment with RIF did not alter the pharmacokinetics of RFP but appeared to increase the excretion of 25-desacetyl RFP in a nonlinear fashion. The RFP oral clearance and volume of distribution were found to increase by 0.049 liter/h and 0.691 liter, respectively, with a 1-kg increase from the median weight of 50 kg. The oral clearance of 25-desacetyl RFP was found to be 35% lower in female patients. The model developed here describes the population pharmacokinetics of RFP and its primary metabolite in tuberculosis patients and includes the effects of prior administration with RIF and covariate factors.
Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen
Cryptosporidium parvum
In Vitro
Authors: Cai Xiaomin , Woods Keith M. , Upton Steve J. , Zhu Guan
Published: October 2005

We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen
Cryptosporidium parvum
. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (

C
T

[P18S]
) and host cells (

C
T

[H18S]
), and evaluates the relative expression between parasite and host rRNA levels (i.e., Δ

C
T

=

C
T

[P18S]
−

C
T

[H18S]
) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[
P
]) and the normalized relative level of rRNA (ΔΔ

C
T

) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of
C. parvum
(IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC
50
values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit
C. parvum
development in vitro (MIC
50
= 15.8 mM), suggesting that the recently discovered
Cryptosporidium
alcohol dehydrogenases may be explored as new drug targets.
TEM-109 (CMT-5), a Natural Complex Mutant of TEM-1 β-Lactamase Combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRT-5)
Authors: Robin F. , Delmas J. , Chanal C. , Sirot D. , Sirot J. , Bonnet R.
Published: October 2005

Escherichia coli
CF349 exhibited a complex β-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a β-lactamase of pI 5.9. The corresponding
bla
gene was identified by PCR and sequencing as a
bla
TEM
gene. The deduced protein sequence revealed a new complex mutant of TEM-1 β-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum β-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (
k
cat
, 56 s
−1
and 105 s
−1
, respectively;

K
m

values, 226 and 247 μM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 μM and 0.27 μM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 μM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.
Anti-Human Immunodeficiency Virus Type 1 Activity of the Nonnucleoside Reverse Transcriptase Inhibitor GW678248 in Combination with Other Antiretrovirals against Clinical Isolate Viruses and In Vitro Selection for Resistance
Authors: Hazen Richard J. , Harvey Robert J. , St. Clair Marty H. , Ferris Robert G. , Freeman George A. , Tidwell Jeffrey H. , Schaller Lee T. , Cowan Jill R. , Short Steven A. , Romines Karen R. , Chan Joseph H. , Boone Lawrence R.
Published: October 2005

GW678248, a novel nonnucleoside reverse transcriptase inhibitor, has been evaluated for anti-human immunodeficiency virus activity in a variety of in vitro assays against laboratory strains and clinical isolates. When GW678248 was tested in combination with approved drugs in the nucleoside and nucleotide reverse transcriptase inhibitor classes or the protease inhibitor class, the antiviral activities were either synergistic or additive. When GW678248 was tested in combination with approved drugs in the nonnucleoside reverse transcriptase inhibitor class, the antiviral activities were either additive or slightly antagonistic. Clinical isolates from antiretroviral drug-experienced patients were selected for evaluation of sensitivity to GW678248 in a recombinant virus assay. Efavirenz (EFV) and nevirapine (NVP) had ≥10-fold increases in their 50% inhibitory concentrations (IC
50
s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a ≥10-fold increase in the IC
50
for only 17% of these isolates. Thus, 81 to 83% of the EFV- and/or NVP-resistant viruses from this data set were susceptible to GW678248. Virus populations resistant to GW678248 were selected by in vitro dose-escalating serial passage. Resistant progeny viruses recovered after eight passages had amino acid substitutions V106I, E138K, and P236L in the reverse transcriptase-coding region in one passage series and amino acid substitutions K102E, V106A, and P236L in a second passage series.
Photodynamic Sensitization ofLeishmania amazonensisin both Extracellular and Intracellular Stages with Aluminum Phthalocyanine Chloride for Photolysis In Vitro
Authors: Dutta Sujoy , Ray Debalina , Kolli Bala K. , Chang Kwang-Poo
Published: October 2005

Leishmania amazonensis, a causative agent of cutaneous leishmaniasis, is susceptible in vitro to light-mediated cytolysis in the presence of or after pretreatment with the photosensitizer aluminum phthalocyanine chloride. Cytolysis of both promastigotes and axenic amastigotes required less photosensitizer (e.g., one μg · ml−1) and a lower light dose (e.g., 1.5 J · cm−2) than did the mammalian cells examined for comparison. Exposure ofLeishmaniacells to the photosensitizer alone had little effect on their viability, as judged from their motility, growth, and/or retention of green fluorescent proteins genetically engineered for episomal expression. Fluorimetric assays for cell-associated and released green fluorescence proteins proved to be even more sensitive for the evaluation of cell viability than microscopy for the evaluation of motility and/or integrity. Axenic amastigotes pretreated with the photosensitizer infected macrophages of the J774 line but were lysed intracellularly when the infected cells were exposed to light. Addition of the photosensitizer to the already infected cells produced no effect on their intracellular parasites. However, light irradiation lysed these macrophages and also those infected with parasites preincubated with the photosensitizer at a concentration of 5 μg · ml−1or higher. PhotosensitizedLeishmaniacells are highly susceptible to cytolysis, apparently due to the generation of reactive oxidative species on light illumination, suggestive of inefficiency of their antioxidant mechanisms. Efficient delivery of photosensitizers to intracellularLeishmaniais expected to increase their therapeutic potentials against leishmaniasis.
Identification of a Dithiazoline Inhibitor of
Escherichia coli
l
,
d
-Carboxypeptidase A
Authors: Baum Ellen Z. , Crespo-Carbone Steven M. , Foleno Barbara , Peng Sean , Hilliard Jamese J. , Abbanat Darren , Goldschmidt Raul , Bush Karen
Published: October 2005

The enzyme
l
,
d
-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in
Escherichia coli
during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 μM. The inhibitor appeared to cause lysis of
E. coli
during stationary phase, behavior that is similar to a previously described deletion mutant of
l
,
d
-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje, EMBO J.
18:
4108-4117, 1999). As much as a one-log drop in CFU in stationary phase was observed upon treatment of
E. coli
with the inhibitor, and the amount of intracellular tetrapeptide substrate increased by approximately 33%, consistent with inhibition of the enzyme within bacterial cells. Stationary-phase targets such as
l
,
d
-carboxypeptidase A are largely underrepresented as targets of the antibiotic armamentarium but provide potential opportunities to interfere with bacterial growth and persistence.
Susceptibilities of Antiviral-Resistant Influenza Viruses to Novel Neuraminidase Inhibitors
Authors: Mishin Vasiliy P. , Hayden Frederick G. , Gubareva Larisa V.
Published: October 2005

The susceptibilities of five zanamivir-resistant and six oseltamivir-resistant influenza viruses were assessed against four neuraminidase (NA) inhibitors, including peramivir and A-315675, by a fluorometric NA activity inhibition assay. The enzyme activity of a majority of the variants was effectively inhibited by either A-315675 or both peramivir and A-315675 (50% inhibitory concentration, <10 nM). A novel oseltamivir-resistant influenza virus B variant carrying substitution at residue 198 (Asp→Asn) (N2 numbering) retained susceptibility to peramivir and A-315675. In vivo, the Asn198 variant showed no apparent fitness impairment as judged by its recovery on day 5 from the nasal washes of ferrets coinfected with equal doses of the wild-type virus and the Asn198 variant. Based on the sequence analysis of the virus in the nasal washes, oseltamivir treatment (5 mg/kg twice daily for 5 days) did not provide growth advantage to the Asn198 variant. Nevertheless, treatment with A-315675 (prodrug A-322278) reduced the number of the animals (two of seven) shedding the Asn198 variant. These studies indicate that different patterns of susceptibility and cross-resistance between NA inhibitors may prove important if antiviral resistance to zanamivir and oseltamivir were to emerge.
Evaluation of Antibiotic Treatment against “
Candidatus
Helicobacter suis” in a Mouse Model
Authors: Hellemans A. , Decostere A. , Haesebrouck F. , Ducatelle R.
Published: October 2005

“
Helicobacter
heilmannii
” (proposed name) type 1 colonizes
the human stomach. It has been shown to be identical to“
Candidatus
Helicobacter suis,” a
Helicobacter
species colonizing the stomachs of >60%
of slaughter pigs. This bacterium has not been isolated in vitro until
now. Antibiotic susceptibility testing of “
Candidatus
Helicobacter suis” has not been carried out so far. For the
present study, a mouse model was adopted to evaluate the antibiotic
susceptibility of this organism. Mice infected with“
Candidatus
Helicobacter suis” were treated
with amoxicillin and omeprazole, a therapy which is used to treat
H. heilmannii
infections in humans. Two different isolates of“
Candidatus
Helicobacter suis” were tested.
The excretion of bacterial DNA was assessed during treatment, using PCR
on fecal samples. At the end of the experiment, 8 days after the
cessation of treatment, the presence of infection was evaluated using a
urease test and a PCR test on stomach samples. A marked decrease in the
excretion of bacterial DNA was observed a few days after the onset of
treatment, and the level remained low until the end of the experiment.
A difference in susceptibility between the two“
Candidatus
Helicobacter suis” isolates was
pointed out. The in vivo mouse model infected with“
Candidatus
Helicobacter suis” will be useful
for further screening of potential therapeutic
regimens.
Pharmacokinetics, Safety, and Tolerability of Caspofungin in Children and Adolescents
Authors: Walsh Thomas J. , Adamson Peter C. , Seibel Nita L. , Flynn Patricia M. , Neely Michael N. , Schwartz Cindy , Shad Aziza , Kaplan Sheldon L. , Roden Maureen M. , Stone Julie A. , Miller Alisha , Bradshaw Susan K. , Li Susan X. , Sable Carole A. , Kartsonis Nicholas A.
Published: October 2005

Caspofungin is a parenteral antifungal that inhibits beta-1,3-
d
-glucan synthesis. Although licensed for adult use, the appropriate caspofungin dosing regimen in pediatric patients is not yet known. We therefore investigated the pharmacokinetics and safety of caspofungin in pediatric patients. Thirty-nine children (ages 2 to 11 years) and adolescents (ages 12 to 17 years) with neutropenia were administered caspofungin using either a weight-based regimen (1 mg/kg of body weight/day) or a body surface area regimen (50 mg/m
2
/day or 70 mg/m
2
/day). Plasma samples for caspofungin profiles were collected on days 1 and 4. These results were compared to those from adults treated with either 50 or 70 mg/day for mucosal candidiasis. In children receiving 1 mg/kg/day (maximum, 50 mg/day), the area under the concentration-time curve over 24 h (AUC
0-24
) was significantly smaller (46% after multiple doses) than that observed in adults receiving 50 mg/day (
P
< 0.001). In children and adolescents receiving 50 mg/m
2
/day (maximum, 70 mg/day), the AUC
0-24
following multiple doses was similar to that for the exposure in adults receiving 50 mg/day. The AUC
0-24
and concentration trough (at 24 h) in pediatric patients receiving the 50-mg/m
2
daily regimen were consistent across the range of ages. Caspofungin was generally well tolerated in this study. None of the patients developed a serious drug-related adverse event or were discontinued for toxicity. These results demonstrate that caspofungin at 1 mg/kg/day in pediatric patients is suboptimal. Caspofungin administration at 50 mg/m
2
/day provides a comparable exposure to that of adult patients treated with 50 mg/day.
High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme
Authors: Cancio Reynel , Silvestri Romano , Ragno Rino , Artico Marino , De Martino Gabriella , La Regina Giuseppe , Crespan Emmanuele , Zanoli Samantha , Hübscher Ulrich , Spadari Silvio , Maga Giovanni
Published: October 2005

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.
Prior Antimicrobial Therapy and Risk for Hospital-Acquired
Candida glabrata
and
Candida krusei
Fungemia: a Case-Case-Control Study
Authors: Lin Michael Y. , Carmeli Yehuda , Zumsteg Jennifer , Flores Ernesto L. , Tolentino Jocelyn , Sreeramoju Pranavi , Weber Stephen G.
Published: October 2005

The incidence of infections caused by
Candida glabrata
and
Candida krusei
, which are generally more resistant to fluconazole than
Candida albicans
, is increasing in hospitalized patients. However, the extent to which prior exposure to specific antimicrobial agents increases the risk of subsequent
C. glabrata
or
C. krusei
candidemia has not been closely studied. A retrospective case-case-control study was performed at a university hospital. From 1998 to 2003, 60 patients were identified with hospital-acquired non-
C. albicans
candidemia (
C. glabrata
or
C. krusei
; case group 1). For comparison, 68 patients with
C. albicans
candidemia (case group 2) and a common control group of 121 patients without candidemia were studied. Models were adjusted for demographic and clinical risk factors, and the risk for candidemia associated with exposure to specific antimicrobial agents was assessed. After adjusting for both nonantimicrobial risk factors and receipt of other antimicrobial agents, piperacillin-tazobactam (odds ratio [OR], 4.15; 95% confidence interval [CI], 1.04 to 16.50) and vancomycin (OR, 6.48; CI, 2.20 to 19.13) were significant risk factors for
C. glabrata
or
C. krusei
candidemia. For
C. albicans
candidemia, no specific antibiotics remained a significant risk after adjusted analysis. Prior fluconazole use was not significantly associated with either
C. albicans
or non-
C. albicans
(
C. glabrata
or
C. krusei
) candidemia. In this single-center study, exposure to antibacterial agents, specifically vancomycin or piperacillin-tazobactam, but not fluconazole, was associated with subsequent hospital-acquired
C. glabrata
or
C. krusei
candidemia. Further studies are needed to prospectively analyze specific antimicrobial risks for nosocomial candidemia across multiple hospital centers.
Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines
Authors: Varkey Jobin , Nagaraj Ramakrishnan
Published: October 2005

The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1ΔC) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1ΔC18) or the G was replaced with A (HNP-1ΔC18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1ΔC and HNP-1ΔC18 in the presence of 100 mM NaCl, except in the case ofPseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active againstP. aeruginosaeven in the presence of 100 mM NaCl. The antibacterial activity of HNP-1ΔC, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.
Pseudomonas aeruginosaAmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors
Authors: Kong Kok-Fai , Jayawardena Suriya Ravi , Indulkar Shalaka Dayaram , del Puerto Aimee , Koh Chong-Lek , Høiby Niels , Mathee Kalai
Published: October 2005

In members of the familyEnterobacteriaceae,ampC, which encodes a β-lactamase, is regulated by an upstream, divergently transcribed gene,ampR. However, inPseudomonas aeruginosa, the regulation ofampCis not understood. In this study, we compared the characteristics of aP. aeruginosa ampRmutant, PAOampR, with that of an isogenicampR+parent. TheampRmutation greatly altered AmpC production. In the absence of antibiotic, PAOampRexpressed increased basal β-lactamase levels. However, this increase was not followed by a concomitant increase in the PampCpromoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated β-lactamase, PoxB. We found that the expression ofP. aeruginosa ampRgreatly altered the β-lactamase production fromampCandpoxBinEscherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive PampRpromoter activity in PAOampRindicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying theampRmutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the PlasA, PlasI, and PlasRexpression. The reduction in the LasB activity was positively correlated with the PrhlRexpression. Thus, AmpR plays a dual role, positively regulating theampC,lasB, andrhlRexpression levels and negatively regulating thepoxB,lasA,lasI, andlasRexpression levels.
Nystatin Biosynthesis and Transport:
nysH
and
nysG
Genes Encoding a Putative ABC Transporter System in
Streptomyces noursei
ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin
Authors: Sletta Håvard , Borgos Sven E. F. , Bruheim Per , Sekurova Olga N. , Grasdalen Hans , Aune Randi , Ellingsen Trond E. , Zotchev Sergey B.
Published: October 2005

The genes
nysH
and
nysG
, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in
Streptomyces noursei
ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the
nysH
and
nysG
deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium
o
-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
In Vitro Assessment of Methylene Blue on Chloroquine-Sensitive and -Resistant
Plasmodium falciparum
Strains Reveals Synergistic Action with Artemisinins
Authors: Akoachere Monique , Buchholz Kathrin , Fischer Elisabeth , Burhenne Jürgen , Haefeli Walter E. , Schirmer R. Heiner , Becker Katja
Published: October 2005

Methylene blue (MB) represents a promising antimalarial drug candidate for combination therapies against drug-resistant parasite strains. To support and facilitate the application of MB in future field trials, we studied its antiparasitic effects in vitro. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant
P. falciparum
strains with 50% inhibitory concentration (IC
50
) values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high-performance liquid chromatography-tandem mass spectrometry on different cell culture compartments, MB is accumulated in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine, MB showed additive effects. In contrast, we observed synergistic effects of MB with artemisinin, artesunate, and artemether for all tested parasite strains. Artemisinin/MB combination concentration ratios of 3:1 were found to be advantageous, demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. Our in vitro data indicate that combinations of MB with artemisinin and related endoperoxides might be a promising option for treating drug-resistant malaria and should be studied in future field trials. Resistance development under this drug combination is unlikely to occur.
Phosphinothricin Tripeptide Synthetases in
Streptomyces viridochromogenes
Tü494
Authors: Schwartz Dirk , Grammel Nicolas , Heinzelmann Eva , Keller Ullrich , Wohlleben Wolfgang
Published: October 2005

The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from
Streptomyces viridochromogenes
, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor
N
-acetyl-demethylphosphinothricin (
N
-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both
phsB
and
phsC
appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene
phsA
, encoding PhsA, in the PTT biosynthesis gene cluster of
S. viridochromogenes
. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from
S. viridochromogenes
confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (
the1
and
the2
) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.
Mechanisms of Azole Resistance in a Clinical Isolate of
Candida tropicalis
Authors: Vandeputte Patrick , Larcher Gérald , Bergès Thierry , Renier Gilles , Chabasse Dominique , Bouchara Jean-Philippe
Published: October 2005

Azole resistance has been insufficiently investigated in the yeast
Candida tropicalis
. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of
C. tropicalis MDR1
(Ct
MDR1
), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the Ct
ERG11
gene coding for lanosterol 14α-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of Ct
ERG11
showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of Ct
ERG11
associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.
Amino Acid Mutations Essential to Production of an Altered PBP 2X Conferring High-Level β-Lactam Resistance in a Clinical Isolate of
Streptococcus pneumoniae
Authors: Smith Anthony M. , Klugman Keith P.
Published: October 2005

Altered penicillin-binding protein 2X (PBP 2X) is a primary β-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). Strain R6
2X/2B/1A/mur
(for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R6
2X/2B/1A/mur
. Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R6
2X/2B/1A/mur
. Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 μg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R6
2X/2B/1A/mur
to 0.06 μg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.