bluuebunny/crossref_metadata_2025_split huggingface dataset:
P‐glycoprotein expression in immortalised rat brain endothelial cells: comparisons following exogenously applied hydrogen peroxide and after hypoxia–reoxygenation
Authors: Robertson Samantha J. , Kania Katarzyna D. , Hladky Stephen B. , Barrand Margery A. 
Published: July 2009

AbstractLevels of multidrug efflux transporter P‐glycoprotein (P‐gp) on endothelial cells lining brain blood vessels are important for limiting access of many compounds to the brain.In vivostudies have indicated that ischaemia–reperfusion that generates reactive oxygen species also increases P‐gp levels in brain endothelial cells. To investigate possible mechanisms,in vitrostudies were performed on immortalised (GPNT) and primary rat brain endothelial cells. Exposure to hydrogen peroxide (200 μM) resulted in intracellular oxidative stress as detected from higher levels of dichlorofluorescein fluorescence and raised levels of P‐gp protein, mdr1a and mdr1b transcripts and, in GPNT cells, increased mdr1a and mdr1b promoter activity. The P‐gp protein increases were abolished by pre‐treatment with polyethylene glycol‐catalase and were curtailed by co‐culture with primary rat astrocytes. Exposure of GPNT cells to 6 h hypoxia followed by 24 h reoxygenation produced less intracellular oxidative stress as judged from smaller increments in dichlorofluorescein fluorescence but still resulted in raised levels of P‐gp protein, an effect partially abolished by pre‐treatment with polyethylene glycol‐catalase. However, transcript levels and promoter activities were not significantly increased. These data suggest that hydrogen peroxide contributes to P‐gp up‐regulation following hypoxia–reoxygenation but the underlying mechanisms of its actions differ from those occurring after direct hydrogen peroxide application.
Modulation of indoleamine‐2,3‐dioxygenase expression and activity by HIV‐1 in human macrophages
Authors: Manéglier Benjamin , Malleret Benoit , Guillemin Gilles J. , Spreux‐Varoquaux Odile , Devillier Philippe , Rogez‐Kreuz Christine , Porcheray Fabrice , Thérond Patrice , Dormont Dominique , Clayette Pascal 
Published: July 2009

AbstractHuman immunodeficiency virus (HIV) infection is often complicated by the development of acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). Implications of kynurenine pathway (KP) are suggested in ADC and other inflammatories brain diseases. The first and regulatory enzyme of the KP is the indoleamine‐2,3‐dioxygenase (IDO). IDO activation is known to contribute to the modulation of the immune response during various infectious diseases particularly in AIDS. HIV and viral proteins can activate IDO in immune cells leading to an increase catabolism of tryptophan through the KP; the consequence being the production of immuno‐modulative and neuroactive metabolites. This mechanism is likely to favour HIV persistence. The present study analysed concomitantly several parameters involved in IDO regulation and activity associated with HIV‐1‐infection. We investigated relevant intracellular and extracellular mechanisms involved in the regulation of IDO expression and activity during the HIV infection and replication in human monocyte‐derived macrophages (MdM). Using a complementary set of in vitro experiments, we found that HIV‐1/Ba‐L infection induces IDO expression and increases its activity in MdM. We also showed that IDO activation by HIV‐1 is likely to be a direct effect of the infection and seems to be independent of IFN‐γ production.
Chronic cough: Stepwise application in primary care practice of the ACCP guidelines for diagnosis and management of cough
Authors: Gahbauer Mary , Keane Patricia 
Published: July 2009

AbstractPurpose: To illustrate decision points encountered when using evidence‐based guidelines for diagnosis and management of chronic cough by means of a composite case study based on primary care practice.Data source: The American College of Chest Physicians (ACCP) Evidence‐Based Clinical Practice Guidelines for Diagnosis and Management of Cough, and review of the scientific literature on cough and related topics.Conclusions: The ACCP guidelines offer a systematic approach that uses trials of empirical therapies to diagnose and resolve the very common, often perplexing complaint of chronic cough. The major diagnoses reached are upper airway cough syndrome, cough‐variant asthma, gastroesophageal reflux disease, and nonasthmatic eosinophilic bronchitis.Implications for practice: Cough is the most common reason that patients seek an office visit. Many coughs resolve spontaneously, but coughs that persist significantly impair the quality of life. Use of the ACCP guidelines allows a stepwise empirical approach to the problem of unexplained chronic cough. This approach greatly increases the percentage of chronic coughs that are accurately diagnosed and effectively treated, and avoids unnecessary diagnostic testing.
Influence of metyrapone treatment during pregnancy on the development and maturation of brain monoaminergic systems in the rat
Authors: Leret M. L. , Rua C. , Garcia‐Montojo M. , Lecumberri M. , González J. C. 
Published: July 2009

AbstractAim:  This study examines the effect of reducing the corticosterone levels of gestating rat dams on the postnatal development and maturation of monoaminergic systems in their offspring’s brains.Methods:  Metyrapone, an inhibitor of CORT synthesis, was administered to pregnant rats from E0 to E17 of gestation. Monoamine concentrations were determined in male and female offspring at postnatal days (PN) 23 and 90 in the hippocampus, hypothalamus and striatum.Results:  Reducing maternal corticosterone (mCORT) during gestation led to alterations in dopamine and serotonin levels in all three brain areas studied at PN 23. Alterations persisted until at least PN 90 in the serotonergic systems; the dopamine content of the hippocampus also remained modified. Reduced mCORT during gestation also led to alterations in the development and maturation of the hypothalamic noradrenergic systems. Sexually dimorphic responses were observed in all these monoaminergic systems at different times.Conclusion:  These results suggest that while they are still developing, brain monoaminergic systems are particularly sensitive to epigenetic influences. An adequate foetal level of CORT is required for the normal ontogeny of brain monoaminergic systems. The present data also provide that during the critical period of brain development, maternal CORT plays an important role in the sexual differentiation of monoaminergic systems, with particular influence on brain serotonergic neurones.
Electrophysiological determinants of hypokalaemia‐induced arrhythmogenicity in the guinea‐pig heart
Authors: Osadchii O. E. , Olesen S. P. 
Published: July 2009

AbstractAim: Hypokalaemia is an independent risk factor contributing to arrhythmic death in cardiac patients. In the present study, we explored the mechanisms of hypokalaemia‐induced tachyarrhythmias by measuring ventricular refractoriness, spatial repolarization gradients, and ventricular conduction time in isolated, perfused guinea‐pig heart preparations.Methods: Epicardial and endocardial monophasic action potentials from distinct left ventricular (LV) and right ventricular (RV) recording sites were monitored simultaneously with volume‐conducted electrocardiogram (ECG) during steady‐state pacing and following a premature extrastimulus application at progressively reducing coupling stimulation intervals in normokalaemic and hypokalaemic conditions.Results: Hypokalaemic perfusion (2.5 mmK+for 30 min) markedly increased the inducibility of tachyarrhythmias by programmed ventricular stimulation and rapid pacing, prolonged ventricular repolarization and shortened LV epicardial and endocardial effective refractory periods, thereby increasing the critical interval for LV re‐excitation. Hypokalaemia increased the RV‐to‐LV transepicardial repolarization gradients but had no effect on transmural dispersion of APD90and refractoriness across the LV wall. As determined by local activation time recordings, the LV‐to‐RV transepicardial conduction and the LV transmural (epicardial‐to‐endocardial) conduction were slowed in hypokalaemic heart preparations. This change was attributed to depressed diastolic excitability as evidenced by increased ventricular pacing thresholds.Conclusion: These findings suggest that hypokalaemia‐induced arrhythmogenicity is attributed to shortened LV refractoriness, increased critical intervals for LV re‐excitation, amplified RV‐to‐LV transepicardial repolarization gradients and slowed ventricular conduction in the guinea‐pig heart.
Structure and Composition of Aggregates in Two Large European Rivers, Based on Confocal Laser Scanning Microscopy and Image and Statistical Analyses
Authors: Luef Birgit , Neu Thomas R. , Zweimüller Irene , Peduzzi Peter 
Published: July 2009

ABSTRACT
Floating riverine aggregates are composed of a complex mixture of inorganic and organic components from their respective aquatic habitats. Their architecture and integrity are supplemented by the presence of extracellular polymeric substances of microbial origin. They are also a habitat for virus-like particles, bacteria, archaea, fungi, algae, and protozoa. In this study we present different confocal laser scanning microscopy strategies to examine aggregates collected from the Danube and Elbe Rivers. In order to collect multiple types of information, various approaches were necessary. Small aggregates were examined directly. To analyze large and dense aggregates, limitations of the technique were overcome by cryo-sectioning and poststaining of the samples. The staining procedure included positive staining (specific glycoconjugates and cellular nucleic acid signals) as well as negative staining (aggregate volume) and multichannel recording. Data sets of cellular nucleic acid signals (CNAS) and the structure of aggregates were visualized and quantified using digital image analysis. The Danube and Elbe Rivers differed in their aggregate composition and in the relative contribution of specific glycoconjugate and CNAS volume to the aggregate volume; these contributions also changed over time. We report different spatial patterns of CNAS inside riverine aggregates, depending on aggregate size and season. The spatial structure of CNAS inside riverine aggregates was more complex in the Elbe River than in the Danube River. Based on our samples, we discuss the strengths and challenges involved in scanning and quantifying riverine aggregates.
Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification
Authors: Yankson Kweku K. , Steck Todd R. 
Published: July 2009

ABSTRACT
We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.
Increased Malonyl Coenzyme A Biosynthesis by Tuning the
Escherichia coli
Metabolic Network and Its Application to Flavanone Production
Authors: Fowler Zachary L. , Gikandi William W. , Koffas Mattheos A. G. 
Published: July 2009

ABSTRACT

Identification of genetic targets able to bring about changes to the metabolite profiles of microorganisms continues to be a challenging task. We have independently developed a cipher of evolutionary design (CiED) to identify genetic perturbations, such as gene deletions and other network modifications, that result in optimal phenotypes for the production of end products, such as recombinant natural products. Coupled to an evolutionary search, our method demonstrates the utility of a purely stoichiometric network to predict improved
Escherichia coli
genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity. The engineered
E. coli
strains were constructed first by the targeted deletion of native genes predicted by CiED and then second by incorporating selected overexpressions, including those of genes required for the coexpression of the plant-derived flavanones, acetate assimilation, acetyl-CoA carboxylase, and the biosynthesis of coenzyme A. As a result, the specific flavanone production from our optimally engineered strains was increased by over 660% for naringenin (15 to 100 mg/liter/optical density unit [OD]) and by over 420% for eriodictyol (13 to 55 mg/liter/OD).
Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “
Bacteroidales
” Populations
Authors: Lamendella Regina , Santo Domingo Jorge W. , Yannarell Anthony C. , Ghosh Shreya , Di Giovanni George , Mackie Roderick I. , Oerther Daniel B. 
Published: July 2009

ABSTRACT

In this study, we evaluated the specificity, distribution, and sensitivity of
Prevotella
strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “
Bacteroidales
” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific
Prevotella-
based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the
Prevotella
markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the
Prevotella
assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal
Bacteroidales
species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.
Microbial Diversity and Host-Specific Sequences of Canada Goose Feces
Authors: Lu Jingrang , Santo Domingo Jorge W. , Hill Stephen , Edge Thomas A. 
Published: July 2009

ABSTRACTMethods to assess the impact of goose fecal contamination are needed as the result of the increasing number of Canada geese (Branta canadensis) near North American inland waters. However, there is little information on goose fecal microbial communities, and such data are important for the development of host-specific source-tracking methods. To address this issue, 16S rRNA gene clone libraries for Canada goose fecal samples from Ontario, Canada, and Ohio were analyzed. Analyses of fecal clones from Ontario (447) and Ohio (302) showed that goose fecal communities are dominated by the classes “Clostridia” (represented by 33.7% of clones) and “Bacilli” (38.1% of clones) and the phylum “Bacteroidetes” (10.1% of clones). Sequences not previously found in other avian fecal communities were used to develop host-specific assays. Fecal DNA extracts from sewage plants (10 samples) and different species of birds (11 samples) and mammals (18 samples) were used to test for host specificity. Of all the assays tested, one assay showed specificity for Canada goose fecal DNA. The PCR assay was positive for Canada goose fecal DNA extracts collected from three locations in North America (Ohio, Oregon, and Ontario, Canada). Additionally, of 48 DNA extracts from Lake Ontario waters presumed to be impacted by waterfowl feces, 19 tested positive by the assay, although 10 were positive only after a nested PCR approach was used. Due to the level of host specificity and the presence of signals in environmental waters, the assay is proposed as a part of the toolbox to detect Canada goose contamination in waterfowl-contaminated waters.
Role of Transporter Proteins in Bile Tolerance ofLactobacillus acidophilus
Authors: Pfeiler Erika A. , Klaenhammer Todd R. 
Published: July 2009

ABSTRACTLactobacillus acidophilusNCFM derivatives containing deletion mutations in the transporter genes LBA0552, LBA1429, LBA1446, and LBA1679 exhibited increased sensitivity to bile. These strains showed unique patterns of sensitivity to a variety of inhibitory compounds, as well as differential accumulations of ciprofloxacin and taurocholate.
Contributions of O Island 48 to Adherence of Enterohemorrhagic
Escherichia coli
O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops
Authors: Yin Xianhua , Wheatcroft Roger , Chambers James R. , Liu Bianfang , Zhu Jing , Gyles Carlton L. 
Published: July 2009

ABSTRACT

O island 48 (OI-48) of
Escherichia coli
consists of three functional gene clusters that encode urease, tellurite resistance (Te
r
), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic
E. coli
(EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Te
r
gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type
ter
genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Te
r
-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%;
P
= 0.168). The OI-48
aidA
gene deletion had no effect on adherence in vitro or in vivo. Deletion of the
iha
and
ureC
genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Te
r
, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.
Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in
Thiocapsa roseopersicina
Authors: Maróti Gergely , Tong Yingkai , Yooseph Shibu , Baden-Tillson Holly , Smith Hamilton O. , Kovács Kornél L. , Frazier Marvin , Venter J. Craig , Xu Qing 
Published: July 2009

ABSTRACT

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a
Thiocapsa roseopersicina
O
2
-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the
hyaA
and
hyaB
genes of an
Alteromonas macleodii
hydrogenase, indicating that it belongs to the
Alteromonas
clade. We were able to express our new
Alteromonas
hydrogenase in
T. roseopersicina
. Expression was accomplished by coexpressing only two accessory genes,
hyaD
and
hupH
, without the need to express any of the
hyp
accessory genes (
hypABCDEF
). These results suggest that the native accessory proteins in
T. roseopersicina
could substitute for the
Alteromonas
counterparts that are absent in the host to facilitate the assembly of a functional
Alteromonas
hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new
Alteromonas hyaD
gene into the
T. roseopersicina hynD
mutant. Interestingly,
Alteromonas
endopeptidase HyaD could complement
T. roseopersicina
HynD to cleave endoproteolytically the C-terminal end of the
T. roseopersicina
HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.
Cloning, Expression, and Characterization of a Peculiar Choline-Binding β-Galactosidase from
Streptococcus mitis
Authors: Campuzano Susana , Serra Beatriz , Llull Daniel , García José L. , García Pedro 
Published: July 2009

ABSTRACT

A
Streptococcus mitis
genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in
Escherichia coli.
This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In
S. mitis
, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.
U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism
Authors: Shelobolina Evgenya S. , Konishi Hiromi , Xu Huifang , Roden Eric E. 
Published: July 2009

ABSTRACTPrevious studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca5(PO4)3OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism.