bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activities of Naphthylisoquinoline Alkaloids and Synthetic Analogs againstLeishmania major
Authors: Ponte-Sucre Alicia , Faber Johan H. , Gulder Tanja , Kajahn Inga , Pedersen Sarah E. H. , Schultheis Martina , Bringmann Gerhard , Moll Heidrun 
Published: December 2006

ABSTRACTThe current treatments for leishmaniasis are unsatisfactory due to their toxic side effects, high costs, and increasing problems with drug resistance. Thus, there is an urgent need for alternative drugs against leishmaniasis. Different approaches have been used to identify novel pharmacophores againstLeishmaniasp. parasites, and one strategy has been the analysis of naturally occurring plant-derived compounds, including naphthylisoquinoline alkaloids. In the present study, we examined the abilities of these alkaloids to inhibit the growth ofLeishmania majorpromastigotes and evaluated their effects on macrophages, dendritic cells, and fibroblasts. Furthermore, we determined the efficacy of selected compounds in decreasing the infection rate of macrophages and regulating their production of cytokines and nitric oxide. Our results demonstrate that the naphthylisoquinoline alkaloids ancistrocladiniums A and B (compounds 10 and 11) and the synthetic isoquinolinium salt (compound 14) were effective against intracellular amastigotes in the low submicromolar range, while toxicity against mammalian cells was observed at concentrations that were significantly higher than those needed to impair parasite replication. The activities of compounds 11 and 14 were mainly directed against the amastigote stage ofL. major. This effect was not associated with the stimulation of host macrophages to produce nitric oxide or secrete cytokines relevant for the leishmanicidal function. In conclusion, our data suggest that ancistrocladiniums A and B (compounds 10 and 11) and the synthetically prepared isoquinolinium salt (compound 14) are promising candidates to be considered as lead compounds for leishmanicidal drugs.
Variability of Voriconazole Plasma Levels Measured by New High-Performance Liquid Chromatography and Bioassay Methods
Authors: Pascual Andres , Nieth Valérie , Calandra Thierry , Bille Jacques , Bolay Saskia , Decosterd Laurent A. , Buclin Thierry , Majcherczyk Paul A. , Sanglard Dominique , Marchetti Oscar 
Published: December 2006

ABSTRACT

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible
Candida albicans
mutant lacking multidrug efflux transporters (
cdr1
Δ/
cdr1
Δ,
cdr2
Δ/
cdr2
Δ,
flu1
Δ/
flu1
Δ, and
mdr1
Δ/
mdr1
Δ) and calcineurin subunit A (
cna
Δ/
cna
Δ) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% ± 5.0%/96.5% ± 2.4% (HPLC) and 94.9% ± 6.1%/94.7% ± 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% ± 1.5%/5.4% ± 0.9% and 6.5% ± 2.5%/4.0% ± 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg · h/liter (range, 12.9 to 71.1) on the first and 27.4 mg · h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg · h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg · h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.
Inhibition of Anthrax Protective Antigen Outside and Inside the Cell
Authors: Backer Marina V. , Patel Vimal , Jehning Brian T. , Claffey Kevin P. , Karginov Vladimir A. , Backer Joseph M. 
Published: December 2006

ABSTRACT

In the course of
Bacillus anthracis
infection,
B. anthracis
lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.
Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants
Authors: Ruckenstuhl Christoph , Lang Silvia , Poschenel Andrea , Eidenberger Armin , Baral Pravas Kumar , Kohút Peter , Hapala Ivan , Gruber Karl , Turnowsky Friederike 
Published: December 2006

ABSTRACTSqualene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies top-hydroxybenzoate hydroxylase (PHBH) fromPseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of theERG1gene, which codes for theSaccharomyces cerevisiaeSE, we isolatederg1alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed inerg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase inerg1mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.
Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening
Authors: Hao Weidong , Herlihy Koleen J. , Zhang Noelle Jie , Fuhrman Shella A. , Doan Chau , Patick Amy K. , Duggal Rohit 
Published: December 2006

ABSTRACT

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized
Renilla
luciferase (hRLuc) gene separated from the selectable Neo
r
marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated
Z
′ value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 μM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals ≥50 and ≤40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.
Quinupristin-Dalfopristin Nonsusceptibility in Pneumococci from Sickle Cell Disease Patients
Authors: Obert Caroline A. , Miller Martha L. , Montgomery Jeremy , Adamkiewicz Thomas , Tuomanen Elaine I. 
Published: December 2006

ABSTRACT
Sickle cell disease (SCD) is a risk factor for fatal pneumococcal infection. Nonsusceptibilty to quinupristin-dalfopristin (Q-D) was absent from 105 non-SCD-associated pneumococcal isolates but was present in 33/148 (22%) SCD-associated isolates. One-third of the isolates harbored a known resistance mechanism. Q-D is not optimal for use for the treatment of pneumococcal infection in SCD patients.
Phenotypic and Molecular Analysis of Penicillin-Nonsusceptible
Streptococcus pneumoniae
Isolates in Poland
Authors: Sadowy Ewa , Izdebski Radosław , Skoczyńska Anna , Grzesiowski Paweł , Gniadkowski Marek , Hryniewicz Waleria 
Published: December 2006

ABSTRACT

β-Lactams are the drugs of choice for the treatment of infections caused by the important bacterial pathogen
Streptococcus pneumoniae
. The recent growth of resistance of this organism to penicillin observed worldwide is of the highest concern. In this study, using 887 surveillance pneumococcal isolates recovered in Poland from 1998 to 2002, we observed the increase in penicillin nonsusceptibility from 8.7% to 20.3%. All of the 109 penicillin-nonsusceptible
S. pneumoniae
(PNSP) isolates identified, together with 22 archival PNSP isolates from 1995 to 1997, were subsequently analyzed by susceptibility testing, serotyping, profiling of
pbp
genes, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Four predominant serotypes, serotypes 6B, 9V, 14, and 23F, characterized 85.5% of the isolates. MLST revealed the presence of 34 sequence types, 15 of which were novel types. Representatives of seven multiresistant international clones (Spain
23F
-1, Spain
6B
-2, Spain
9V
-3, Taiwan
23F
-15, Poland
23F
-16, Poland
6B
-20, and Sweden
15A
-25) or their closely related variants comprised the majority of the study isolates. The spread of Spain
9V
-3 and its related clone of serotype 14/ST143 has remarkably contributed to the recent increase in penicillin resistance in pneumococci in the country.
SGI1-K, a Variant of the SGI1 Genomic Island Carrying a Mercury Resistance Region, in
Salmonella enterica
Serovar Kentucky
Authors: Levings Renee S. , Partridge Sally R. , Djordjevic Steven P. , Hall Ruth M. 
Published: December 2006

ABSTRACT

A multiple-antibiotic-resistant
Salmonella enterica
serovar Kentucky strain was found to contain SGI1-K, a variant form of the
Salmonella
genomic island 1 (SGI1) with an In4-type class 1 integron that contains only one cassette array,
aacCA5-aadA7
, and an adjacent mercury resistance module. Part of the 3′-conserved segment (3′-CS) of the integron, together with the inverted short segment from the right-hand end of the integron transposition module normally found between the 3′-CS and IS
6100
in In4 family integrons, has been removed by an IS
6100
-mediated deletion. IRt, the right-hand inverted repeat found at the outer end of the integron, abuts a mercury resistance region instead of the usual SGI1 backbone segment. The
mer
module is a hybrid of those found in Tn
501
and Tn
21
. This
mer
region and a further uncharacterized segment of at least 10 kb appear to have been incorporated between IRt and the SGI1 backbone. These findings demonstrate that the multidrug resistance region in SGI1 can incorporate new DNA segments in the same way as multiple antibiotic resistance regions in plasmids.
Identification of theActinobacillus pleuropneumoniaeLeucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes
Authors: Wagner Trevor K. , Mulks Martha H. 
Published: December 2006

ABSTRACTActinobacillus pleuropneumoniaeis a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset ofA. pleuropneumoniaegenes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown inEscherichia colito regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized thatA. pleuropneumoniaecontains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of anA. pleuropneumoniaeserotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purifiedA. pleuropneumoniaeHis6-Lrp binds in vitro to theA. pleuropneumoniaepromoter regions forilvI, antisensecps1AB,lrp, andnqr. A genetically definedA. pleuropneumoniae lrpmutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from theilvIand antisensecps1ABpromoters in wild-type,lrpmutant, and complementedlrpmutant strains indicated that Lrp is required for induction of expression ofilvIunder BCAA limitation.
Essential Role of CCL2 in Clustering of Splenic ERTR-9+Macrophages during Infection of BALB/c Mice byListeria monocytogenes
Authors: Jablonska Jadwiga , Dittmar Kurt E. , Kleinke Tanja , Buer Jan , Weiss Siegfried 
Published: December 2006

ABSTRACTEarly interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection byListeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early afterListeriainfection in spleens of BALB/c mice. ERTR-9+macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+cells. Metallophilic MOMA-1+marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
The YfgL Lipoprotein Is Essential for Type III Secretion System Expression and Virulence of
Salmonella enterica
Serovar Enteritidis
Authors: Fardini Yann , Chettab Kamel , Grépinet Olivier , Rochereau Sandrine , Trotereau Jérôme , Harvey Philippa , Amy Maïté , Bottreau Elisabeth , Bumstead Nat , Barrow Paul A. , Virlogeux-Payant Isabelle 
Published: December 2006

ABSTRACT

Salmonella enterica
, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of
Salmonella
, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the
yfgL
,
engA
, and
yfgJ
open reading frames in
S. enterica
serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in
S. enterica
serovar Enteritidis of
yfgL
, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most
Salmonella
pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the Δ
yfgL
mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Δ
yfgL
mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all
Salmonella
TTSS and, thus, in the virulence of
Salmonella
. Therefore, this outer membrane protein seems to be a privileged target for fighting
Salmonella
.
Generation of Cross-Protective Antibodies against
Plasmodium falciparum
Sequestration by Immunization with an Erythrocyte Membrane Protein 1-Duffy Binding-Like 1α Domain
Authors: Moll Kirsten , Pettersson Fredrik , Vogt Anna M. , Jonsson Cathrine , Rasti Niloofar , Ahuja Sanjay , Spångberg Mats , Mercereau-Puijalon Odile , Arnot David E. , Wahlgren Mats , Chen Qijun 
Published: December 2006

ABSTRACT

The
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor on the surface of infected erythrocytes. Naturally acquired antibodies to PfEMP1 expressed by parasites causing severe malaria are suggested to be protective and of major interest for the development of a vaccine against severe disease. In this study, the PfEMP1 expressed by a parasite clone displaying a multiadhesive phenotype associated with severe malaria was well recognized by sera of malaria semi-immune children. The efficiency of the Duffy binding-like 1α (DBL1α) domain of this PfEMP1 was therefore, alone or in combination with two additional DBL1α domains, evaluated as a potential vaccine candidate using both a rodent model and a primate model. Antibodies against the DBL1α domain were generated by immunization with recombinant DBL1α-Semliki Forest virus particles and recombinant protein and analyzed in vitro. The immunized animals were challenged in vivo with various parasite strains or clones. Immunization with the PfEMP1-DBL1α domain abolished the PfEMP1-dependent sequestration of the homologous strain in immunized rats and substantially inhibited parasite adhesion in immunized monkeys. Protection against sequestration of heterologous parasite strains was also confirmed by direct or indirect challenge in the rat model. These results strongly support the use of the DBL1α domain in the development of a vaccine targeting severe malaria.
Absence of Toll-Like Receptor 4 Signaling Results in Delayed
Yersinia enterocolitica
YopP-Induced Cell Death of Dendritic Cells
Authors: Gröbner Sabine , Schulz Sebastian , Soldanova Irena , Gunst Dani S. J. , Waibel Michaela , Wesselborg Sebastian , Borgmann Stefan , Autenrieth Ingo B. 
Published: December 2006

ABSTRACT

In an initial period (≤4 h) Toll-like receptor 4 (TLR4) signaling is required for
Yersinia enterocolitica
YopP-induced dendritic cell (DC) death. Later (>4 h), DC die independent of TLR4 signaling. In TLR4-deficient DC caspase 8 cleavage is delayed, indicating that TLR4 signaling accelerates caspase 8 activation, leading to DC death.
Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection
Authors: Ryan Anthony A. , Spratt Joanne M. , Britton Warwick J. , Triccas James A. 
Published: December 2006

ABSTRACTA strain ofMycobacterium bovisBCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCGMCP-3) was developed. Mice vaccinated with BCGMCP-3displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCGMCP-3was equivalent to that with control BCG; however, immunodeficient mice infected with BCGMCP-3survived significantly longer than mice infected with the control BCG strain. Therefore, BCGMCP-3may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.