bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic deconstruction of xylan for biofuel production
Authors: DODD DYLAN , CANN ISAAC K. O.
Published: February 2009

The combustion of fossil‐derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction.
Multiple, large sialoliths of the submandibular gland duct: a case report
Authors: Huang TC , Dalton JB , Monsour FN , Savage NW
Published: February 2009

This paper reviews the major clinical and radiographic features of sialoliths and illustrates these with an unusual case of multiple sialoliths within the submandibular gland duct. The differential diagnosis of other calcific structures both within and outside the salivary gland that may mimic a sialolith is also presented.
τ-function of discrete isomonodromy transformations and probability
Authors: Arinkin D. , Borodin A.
Published: February 2009

We introduce theτ-function of a difference rational connection (d-connection) and its isomonodromy transformations. We show that in a continuous limit ourτ-function agrees with the Jimbo–Miwa–Uenoτ-function. We compute theτ-function for the isomonodromy transformations leading to difference Painlevé V and difference Painlevé VI equations. We prove that the gap probability for a wide class of discrete random matrix type models can be viewed as theτ-function for an associated d-connection.
Vibration Suppression of Mistuned Coupled-Blade-Disk Systems Using Piezoelectric Circuitry Network
Authors: Yu Hongbiao , Wang K. W.
Published: February 2009

For bladed-disk assemblies in turbomachinery, the elements are often exposed to aerodynamic loadings, the so-called engine order excitations. It has been reported that such excitations could cause significant structural vibration. The vibration level could become even more excessive when the bladed disk is mistuned, and may cause fatigue damage to the engine components. To effectively suppress vibration in bladed disks, a piezoelectric transducer networking concept has been explored previously by the authors. While promising, the idea was developed based on a simplified bladed-disk model without considering the disk dynamics. To advance the state of the art, this research further extends the investigation with focus on new circuitry designs for a more sophisticated and realistic system model with the consideration of coupled-blade-disk dynamics. A novel multicircuit piezoelectric transducer network is synthesized and analyzed for multiple-harmonic vibration suppression of bladed disks. An optimal network is derived analytically. The performance of the network for bladed disks with random mistuning is examined through Monte Carlo simulation. The effects of variations (mistuning and detuning) in circuit parameters are also studied. A method to improve the system performance and robustness utilizing negative capacitance is discussed. Finally, experiments are carried out to demonstrate the vibration suppression capability of the proposed piezoelectric circuitry network.
Long-Term Microturbine Exposure of an Advanced Alloy for Microturbine Primary Surface Recuperators
Authors: Matthews Wendy J. , More Karren L. , Walker Larry R.
Published: February 2009

Haynes alloy HR-120 (Haynes and HR-120 are trademarks of Haynes International, Inc.) forms a protective oxide scale when exposed to the harsh operating environment of a microturbine primary surface recuperator. Primary surface recuperators manufactured from HR-120 are currently in use on the Capstone C65 MicroTurbine (MicroTurbine is a registered trademark of Capstone Turbine Corporation). Long-term microturbine tests of this alloy are currently being conducted at an elevated turbine exit temperature (∼100°F higher than that in a normal operation) at Capstone Turbine Corporation. Alloy samples that have been tested under steady-state microturbine operating conditions are removed after predetermined exposure intervals for characterization by Capstone Turbine Corporation in collaboration with Oak Ridge National Laboratory. Such evaluations include the characterization of surface oxide scales and the associated alloy compositional changes following a steady-state operation ranging from 1800 h to 14,500 h. Results from the microstructural and compositional analyses of these long-term steady-state engine-tested HR-120 samples are used to illustrate the progression of alloy oxidation in the microturbine operating environment.
Axial Fan Blade Tone Cancellation Using Optimally Tuned Quarter Wavelength Resonators
Authors: Gorny Lee , Koopmann Gary H.
Published: February 2009

Fan noise challenges noise control engineers in developing products ranging in scale from small ventilation systems to large turbomachines. The dominant noise source in many axial fans is the tonal noise due to rotor/stator interactions at the fundamental blade passing frequency. Flow-excited resonators have been used in the past for minimizing blade tone sound pressure levels (SPLs) generated by centrifugal fans through means of secondary source cancellation. The focus of this research is to extend that cancellation method to axial fans by attaching flow-driven quarter wavelength resonators fitted with optimal mouth perforations around the perimeter of the fan’s shroud. A ducted-fan test facility was developed to measure upstream and downstream noise radiated from a test fan. Resonators were mounted at specific locations around the fan’s shroud to obtain reductions in blade tone SPLs in both flow directions. They were driven into resonance via the unsteady pressure from the passing blades. An analytical model using transmission line theory was developed and validated experimentally to characterize the resonator’s behavior under various flow conditions and mouth geometries. This model was used to predict the resonator’s potential for reducing in-duct blade tones for specific flows and mouth perforation patterns. In a series of experiments to obtain the optimal resonator mouth perforations, it was observed that upstream and downstream SPL attenuations require different placement of the resonator mouth relative to the blade of the fan. With a single tuned resonator it was demonstrated that the fundamental blade tone SPLs can be reduced by as much as 20 dB in either the upstream or the downstream duct but not in both directions simultaneously. This behavior results when combining the resonator’s monopolelike sound field with the dipolelike sound field of the fan’s blades. Further studies are underway to extend the above method to higher pressure fans operating at speeds that generate higher order duct modes.
On-Condition Maintenance for Nonmodular Jet Engines: An Experience
Authors: Siladic Mato F. , Rasuo Bosko P.
Published: February 2009

Using updated knowledge and gained experience in engine control and maintenance, a specific on-condition maintenance concept of RD-33 engines installed on MiG-29s was developed. The engines had several built-in limitations: number of starts, number of hours at the maximum power and reheat, number of hours at the special regime of elevated temperatures, and time between overhauls (TBOs), that is, number of flight hours. During field data collection and analysis, it was found that engines worked with different working loads and different levels of life consumption. Hence, it was concluded that the limitation of TBO, expressed in terms of flight hours, do not represent actual engine health condition and that a new way of monitoring actual load needs to be introduced. An analysis of all flight profiles was carried out and a specific relation between flight hours and total accumulated cycles (TACs) was established. For this purpose, a distributed expert system in relation-operation unit—Air Force Technical Institute—overhaul depot was introduced. Each of the three participants has its own level of responsibility in the engine health monitoring, engine maintenance, and engine health condition decision-making process. Nondestructive inspection, remote visual inspection, spectral oil analysis, fault tolerant control techniques of hot engine parts, engine electronic control unit, airplane information-display system, engine performance trend monitoring, vibration monitoring, and postflight data analysis play key roles in the concept. It has been applied in practice since 1994; all faults were discovered right in time, and there were not any critical situations in flight. Detected faults were isolated and assessed for severity, so that the remaining useful life could be estimated. The original TBO was safely extended on the basis of TAC of up to more than 50% of the originally prescribed TBO hours, while maintaining the same safe margin.
Effect of In-Plane Forces on Sound Radiation From Convected, Fluid Loaded Plates
Authors: Schmidt Peter L. , Frampton Kenneth D.
Published: February 2009

The purpose of this work is to study the effects that plane stress has on the acoustic radiation from structures subjected to convected fluid loading. It is well established that fluid flow can have significant effects on structural acoustic behavior, along with the fact that induced coupling between discrete modes of vibration becomes significant as flow velocity increases. Work in this area has been confined to flows in air, over unloaded structures, with the effects on sound radiation efficiency, kinetic energy, and sound power radiation quantified and compared for various flow speeds. Theoretical development of the equations governing the vibration of a simply-supported plate subjected to in-plane forces in an infinite baffle and a semi-infinite flowing medium is presented along with a method for coupling these systems. Computational results are presented illustrating the effect of coupling on the sound power radiated from the plate in both subsonic and supersonic flows, for a variety of stress loading cases. It is shown that the state of stress in the plate affects the radiation efficiency of the plate, and that increasing stress eliminates a frequency shift in radiated sound power shown to exist for both subsonic and supersonic flow in previous work.
Nonlinear Vibration of a Magneto-Elastic Cantilever Beam With Tip Mass
Authors: Pratiher Barun , Dwivedy Santosha K.
Published: February 2009

In this work the effect of the application of an alternating magnetic field on the large transverse vibration of a cantilever beam with tip mass is investigated. The governing equation of motion is derived using D’Alembert’s principle, which is reduced to its nondimensional temporal form by using the generalized Galerkin method. The temporal equation of motion of the system contains nonlinearities of geometric and inertial types along with parametric excitation and nonlinear damping terms. Method of multiple scales is used to determine the instability region and frequency response curves of the system. The influences of the damping, tip mass, amplitude of magnetic field strength, permeability, and conductivity of the beam material on the frequency response curves are investigated. These perturbation results are found to be in good agreement with those obtained by numerically solving the temporal equation of motion and experimental results. This work will find extensive applications for controlling vibration in flexible structures using a magnetic field.
Friction-Induced Vibration in Lead Screw Systems: Mathematical Modeling and Experimental Studies
Authors: Vahid O. , Eslaminasab N. , Golnaraghi M. F.
Published: February 2009

Lead screw mechanisms are used to convert rotary to linear motion. The velocity-dependent coefficient of friction at the contact between lead screw and nut threads can lead to self-excited vibrations, which may result in excessive noise generated by the system. In this paper, based on a practical example of a powered automotive seat adjuster, the nonlinear dynamics of lead screw systems is studied. A test setup is developed to perform experiments on the horizontal motion drive. The experimental results are used in a novel two-step identification approach to estimate friction, damping, and stiffness parameters of the system. The identified parameters together with other known system parameters are used in the numerical simulations. The accuracy of the mathematical model is validated by comparing numerical simulation results with actual measurements in cases where limit cycles are developed. Using simulation results for a range of lead screw angular velocities and axial forces, regions of stability were found. Also, the effects of damping and stiffness parameters on the steady-state amplitude of vibration were investigated.
Simulation of Turbulent Lifted Flames Using a Partially Premixed Coherent Flame Model
Authors: Zhang Yongzhe , Rawat Rajesh
Published: February 2009

Partially premixed combustion occurs in many combustion devices of practical interest, such as gas-turbine combustors. Development of corresponding turbulent combustion models is important to improve the design of these systems in efforts to reduce fuel consumption and pollutant emissions. Turbulent lifted flames have been a canonical problem for testing models designed for partially premixed turbulent combustion. In this paper we propose modifications to the coherent flame model so that it can be brought to the simulation of partially premixed combustion. For the primary premixed flame, a transport equation for flame area density is solved in which the wrinkling effects of the flame stretch and flame annihilation are considered. For the subsequent nonpremixed zone, a laminar flamelet presumed probability density function (PPDF) methodology, which accounts for the nonequilibrium and finite-rate chemistry effects, is adopted. The model is validated against the experimental data on a lifted H2∕N2 jet flame issuing into a vitiated coflow. In general there is fairly good agreement between the calculations and measurements both in profile shapes and peak values. Based on the simulation results, the flame stabilization mechanism for lifted flames is investigated.
Development of an Improved Desiccant-Based Evaporative Cooling System for Gas Turbines
Authors: Zadpoor Amir Abbas , Nikooyan Ali Asadi
Published: February 2009

The evaporative inlet cooling systems do not work well in humid areas. However, desiccant wheels can be used to dehumidify the air before passing it through the evaporative cooler. A previous study of combined direct and indirect evaporative coolers showed that a single desiccant wheel does not offer much higher effectiveness compared with the multistage evaporative systems. In this paper, additional dehumidification and indirect evaporative cooling stages are added to increase the effectiveness of the desiccant-based inlet cooling. A typical gas turbine cycle along with an industrial gas turbine with actual performance curves are used to study the performance of the proposed system in three different climatic conditions. It is shown that the added stages substantially improve the effectiveness of the desiccant-based inlet cooling.
Recent Advances in Nuclear Based Hydrogen Production With the Thermochemical Copper-Chlorine Cycle
Authors: Naterer G. F. , Gabriel K. , Lu L. , Wang Z. , Zhang Y.
Published: February 2009

This paper presents a review of recent advances in nuclear-based hydrogen production with a thermochemical copper-chlorine cycle. Growing attention has focused on thermochemical water decomposition as a promising alternative to steam-methane reforming for a sustainable future method of large-scale hydrogen production. Recent advances in specific processes within the Cu–Cl cycle will be presented, particularly for overall heat requirements of the cycle, preferred configurations of the oxygen cell, disposal of molten salt, electrochemical process of copper electrowinning, and safety/reliability assessment of the systems. An energy balance for each individual process is formulated and results are presented for heat requirements of the processes.
What Scientists Caught from Kuru
Authors: Lindee M. Susan
Published: February 2009

The Collectors of Lost Souls
. Turning Kuru Scientists into Whitemen.
By Warwick Anderson
. Johns Hopkins University Press, Baltimore, 2008. 328 pp. $24.95. ISBN 9780801890406.

Anderson weaves together stories of kuru, the Fore people it afflicted, and the biomedical researchers and anthropologists the disease and its victims attracted to the highlands of New Guinea.
Gourmet Food, Served by Dogs
Authors: Bohannon John
Published: February 2009

The Gonzo Scientist goes to the dogs--truffle-hunting dogs, to be precise--to answer the question of whether gourmet food is really worth the price. Would you eat dog food if it were served like paté? You may not be so sure of your answer after this episode.
GRA2 and ROP1 Recombinant Antigens as Potential Markers for Detection of
Toxoplasma gondii
-Specific Immunoglobulin G in Humans with Acute Toxoplasmosis
Authors: Holec-Gąsior Lucyna , Kur Józef , Hiszczyńska-Sawicka Elżbieta
Published: February 2009

A goal of the current study was to evaluate serological applications of
Toxoplasma gondii
GRA2 and rhoptry protein 1 (ROP1) antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at the N and C terminals were obtained using an
Escherichia coli
expression system. Purification by one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per liter of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 serum samples from patients with acute toxoplasmosis was compared to a panel of 90 serum samples from individuals with past infection. The results indicated that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently in samples from individuals with acute infections (100% and 94.6%, respectively) than in samples from individuals with chronic infections (22.5% and 15.5%, respectively). These results suggest that immunoglobulin G antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.
Development of a Bovine Ileal Cannulation Model To Study the Immune Response and Mechanisms of Pathogenesis of Paratuberculosis
Authors: Allen Andrew J. , Park Kun Taek , Barrington George M. , Lahmers Kevin K. , Hamilton Mary Jo , Davis William C.
Published: February 2009

An ileal cannulation model was developed in conjunction with a flow cytometric assay to gain a better understanding of the mechanisms of immunopathogenesis of Johne's disease caused byMycobacterium aviumsubsp.paratuberculosis. Initial studies with calves showed thatM. aviumsubsp.paratuberculosisDNA is detectable by PCR in ileal biopsies during the first months following experimental infection. Inflammatory lesions were not detected on endoscopic evaluation up to 8 months postexperimental infection.M. aviumsubsp.paratuberculosisDNA was detected in multiple tissues at necropsy 8 months postinfection. Examination of the activation status of epithelial lymphocytes from the jejunum and ileum from infected and control animals at necropsy revealed that none of the major subsets of lymphocytes (NK, CD2+, and CD2−γδ T lymphocytes, or CD4 and CD8 αβ T lymphocytes) expressed activation molecules CD25, CD26, CD71, ACT1, or ACT16. Subsets of CD4 and CD8 T lymphocytes from control and infected animals expressed CD26. The majority of CD4 and CD8 T lymphocytes expressed CD45R0, the memory T-lymphocyte marker. An immune response toM. aviumsubsp.paratuberculosiswas detected by 3 months postinfection, dominated by a strong proliferative response of CD4 memory T lymphocytes. The findings indicate an immune response develops following initial exposure toM. aviumsubsp.paratuberculosisthat controls but does not eliminate the pathogen. This persistence ofM. aviumsubsp.paratuberculosispossibly leads to erosion and dysregulation of protective immunity at later time points postinfection. Continuous access to the ileum offers an opportunity to elucidate the cellular and molecular events leading to immune dysregulation and development of chronic inflammatory ileitis.
Nevirapine Resistance in Human Immunodeficiency Virus Type 1-Positive Infants Determined Using Dried Blood Spots Stored for Up to Six Years at Room Temperature
Authors: Nelson Julie A. E. , Loftis Amy M. , Kamwendo Deborah , Fawzi Wafaie W. , Taha Taha E. , Goldenberg Robert L. , Fiscus Susan A.
Published: February 2009

Dried blood spots that had been stored ambiently for 3 to 6 years lost approximately 1 log
10
of human immunodeficiency virus type 1 (HIV-1) RNA, but the majority could still be genotyped for resistance. Nevirapine resistance was found in 7/16 (43.5%) HIV-1-positive HIVNET 024 infants at 4 to 6 weeks, but no resistance was found at other time points.
Temporal Trends of Invasive
Streptococcus pneumoniae
Serotypes and Antimicrobial Resistance Patterns in Spain from 1979 to 2007
Authors: Fenoll A. , Granizo J. J. , Aguilar L. , Giménez M. J. , Aragoneses-Fenoll L. , Hanquet G. , Casal J. , Tarragó D.
Published: February 2009

Temporal trends of serotypes from invasive pneumococcal disease (IPD) in Spain from 1979 to September 2007 under antibiotic and vaccine pressure were analyzed. A significant trend in pneumococcal conjugate 7-valent vaccine (PCV7) serotypes (except serotype 4) was found, whereby the prevalence increased from the early 1980s and decreased in the 2000s for all but serotype 23F, which began decreasing in the late 1980s. Among the major non-PCV7 serotypes, a significant decrease was observed for serotypes 1, 5, and 7F in the 1980s. From the late 1990s, serotypes 1, 5, 6A, 7F, and 19A increased significantly, while serotypes 3 and 8 showed similar but nonsignificant trends over time. The incidence of IPD cases was 10.7/100,000 for the period 1996 to 2006, with reporting coverage ranging from 18% to 43%. A significant decrease in IPD incidence due to PCV7 serotypes was observed, while the incidence of non-PCV7 serotypes increased, with the consequence that there was no clear pattern in the overall incidence of IPD. Penicillin nonsusceptibility was correlated with the proportion of PCV7 serotypes. Erythromycin nonsusceptibility increased in association with long-half-life macrolide consumption and then decreased in 2004 to 2007. The increase in PCV7 serotypes and antibiotic nonsusceptibility related to antibiotic consumption in the 1980s and 1990s was reversed in the 2000s, probably as a result of PCV7 immunization. The decrease in IPD incidence due to PCV7 serotypes was mirrored by an increase in that of non-PCV7 serotypes. The impact of various preventive/therapeutic strategies on pneumococcal evolution is serotype dependent, and the dynamics remain unpredictable.
Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease
Authors: Ginevra Christophe , Lopez Marie , Forey Françoise , Reyrolle Monique , Meugnier Helene , Vandenesch François , Etienne Jerome , Jarraud Sophie , Molmeret Maëlle
Published: February 2009

Sequence-based typing (SBT) is a powerful method based on the sequencing of seven genes of
Legionella pneumophila
isolates. SBT performed directly on clinical samples has been used only in a limited number of cases. In our study, its efficiency was tested with 63 legionellosis respiratory samples. Sixty-three clinical samples, which included 23 samples from sporadic cases and 40 collected during four French outbreaks, confirmed by culture or urinary antigen testing and all positive by
L. pneumophila
quantitative PCR were subtyped by SBT according to the European Working Group for
Legionella
Infections standard scheme. Only 28.6% of the samples provided nucleotide sequences by SBT. Nested-PCR-based SBT (NPSBT) applied to the same respiratory samples was thus evaluated with new PCR primers surrounding the first set of primers used for the SBT. Sequencing results were obtained with 90.5% of the samples. Complete allelic profiles (seven genes sequenced) were obtained for 3.2% versus 53.9% of the samples by SBT and NPSBT, respectively. More importantly, of the 28 culture-negative samples, only 4 did not give any sequencing results. Taken together, NPSBT applied directly to clinical specimens significantly improved epidemiological typing compared to the initial SBT, in particular when no isolates are available.
The Putative Leucine Zipper of the U
L
6-Encoded Portal Protein of Herpes Simplex Virus 1 Is Necessary for Interaction with pU
L
15 and pU
L
28 and Their Association with Capsids
Authors: Yang Kui , Wills Elizabeth , Baines Joel D.
Published: February 2009

Herpes simplex virus (HSV) type 1 capsids contain a single portal vertex that is composed of 12 copies of the U
L
6 gene product (pU
L
6), which forms a pore through which DNA is inserted during packaging. This unique vertex is also believed to comprise the site with which a molecular motor, termed the terminase, associates during the DNA packaging reaction. In HSV, the terminase likely comprises the U
L
15, U
L
28, and U
L
33 proteins (pU
L
15, pU
L
28, and pU
L
33, respectively). The current study was undertaken to identify portal domains required for interaction with the terminase. Both the amino and carboxyl termini, as well as amino acids 422 to 443 of pU
L
6 forming a putative leucine zipper motif, were critical for coimmunoprecipitation with pU
L
15 in the absence of other viral proteins. Amino acids 422 to 443 were also necessary for interaction with pU
L
28 in the absence of other viral proteins. By using an engineered recombinant virus, it was further determined that although amino acids 422 to 443 were dispensable for interaction with scaffold protein and incorporation of portal protein into capsids, they were necessary for coimmunoprecipitation of pU
L
6 and pU
L
15 from infected cell lysates, association of optimal levels of pU
L
15, pU
L
28, and pU
L
33 with capsids, and DNA cleavage and packaging. These data identify a portal protein domain critical for terminase association with the capsid and suggest that both the pU
L
15- and pU
L
28-bearing terminase subunits mediate docking of the terminase with the portal vertex.
The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination
Authors: Gammon Don B. , Evans David H.
Published: February 2009

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3′-to-5′ proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3′ ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV
r
) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3′-to-5′ exonuclease domain of the viral polymerase. Viruses encoding a CDV
r
mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3′ ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3′-to-5′ exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.
Experimental Infection of Pigs with the Human 1918 Pandemic Influenza Virus
Authors: Weingartl Hana M. , Albrecht Randy A. , Lager Kelly M. , Babiuk Shawn , Marszal Peter , Neufeld James , Embury-Hyatt Carissa , Lekcharoensuk Porntippa , Tumpey Terrence M. , García-Sastre Adolfo , Richt Jürgen A.
Published: February 2009

Swine influenza was first recognized as a disease entity during the 1918 “Spanish flu” pandemic. The aim of this work was to determine the virulence of a plasmid-derived human 1918 pandemic H1N1 influenza virus (reconstructed 1918, or 1918/rec, virus) in swine using a plasmid-derived A/swine/Iowa/15/1930 H1N1 virus (1930/rec virus), representing the first isolated influenza virus, as a reference. Four-week-old piglets were inoculated intratracheally with either the 1930/rec or the 1918/rec virus or intranasally with the 1918/rec virus. A transient increase in temperature and mild respiratory signs developed postinoculation in all virus-inoculated groups. In contrast to other mammalian hosts (mice, ferrets, and macaques) where infection with the 1918/rec virus was lethal, the pigs did not develop severe respiratory distress or become moribund. Virus titers in the lower respiratory tract as well as macro- and microscopic lesions at 3 and 5 days postinfection (dpi) were comparable between the 1930/rec and 1918/rec virus-inoculated animals. In contrast to the 1930/rec virus-infected animals, at 7 dpi prominent lung lesions were present in only the 1918/rec virus-infected animals, and all the piglets developed antibodies at 7 dpi. Presented data support the hypothesis that the 1918 pandemic influenza virus was able to infect and replicate in swine, causing a respiratory disease, and that the virus was likely introduced into the pig population during the 1918 pandemic, resulting in the current lineage of the classical H1N1 swine influenza viruses.
Densovirus Infectious Pathway Requires Clathrin-Mediated Endocytosis Followed by Trafficking to the Nucleus
Authors: Vendeville Agnès , Ravallec Marc , Jousset Françoise-Xavière , Devise Micheline , Mutuel Doriane , López-Ferber Miguel , Fournier Philippe , Dupressoir Thierry , Ogliastro Mylène
Published: February 2009

Junonia coenia
densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest
Lymantria dispar
to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.
Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection
Authors: McNally Kristin L. , Ward Anne E. , Priola Suzette A.
Published: February 2009

The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.
Internalization of Swine Vesicular Disease Virus into Cultured Cells: a Comparative Study with Foot-and-Mouth Disease Virus
Authors: Martín-Acebes Miguel A. , González-Magaldi Mónica , Vázquez-Calvo Angela , Armas-Portela Rosario , Sobrino Francisco
Published: February 2009

We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses.
Reactions of β-aminovinyl bromodifluoromethyl ketones with alkyl phosphites: Perkow versus Arbuzov
Authors: Tarasenko Karen V. , Gerus Igor I. , Kukhar Valery P. , Polovinko Vitaly V.
Published: February 2009

New bromodifluoromethyl enaminones 1a–1f and γ-bromo-β-morpholinopropenyl fluoromethyl ketones 2a, 2b were synthesized. N-Substituted bromodifluoromethyl enaminones 1a–1d do not react with triethyl or diethyl phosphites, whereas N-acylated enaminones 1e, 1f gave difluorodienyl phosphates 4a, 4b as Perkow rearrangement products. Fluoroketone 2a reacts easily with triethyl phosphite according to the Arbuzov protocol and a perspective building block – trifluoromethyl-containing phosphonate 7a is formed.
Sequential effects of GSNO and H2O2 on the Ca2+ sensitivity of the contractile apparatus of fast- and slow-twitch skeletal muscle fibers from the rat
Authors: Spencer Timothy , Posterino Giuseppe S.
Published: February 2009

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and nitric oxide (NO), have been shown to differentially alter the Ca2+ sensitivity of the contractile apparatus of fast-twitch skeletal muscle, leading to the proposal that normal muscle function is controlled by perturbations in the amounts of these two groups of molecules ( 28 ). However, no previous studies have examined whether these opposing actions are retained when the contractile apparatus is subjected to both molecule types. Using mechanically skinned fast- and slow-twitch skeletal muscle fibers of the rat, we compared the effects of sequential addition of nitrosoglutathione (GSNO), a NO donor, and H2O2 on the Ca2+ sensitivity of the contractile apparatus. As expected from previous reports in fast-twitch fibers, when added separately, GSNO (1 mM) reduced the Ca2+ sensitivity of the contractile apparatus, whereas H2O2 (10 mM; added during contractions) increased the Ca2+ sensitivity of the contractile apparatus. When added sequentially to the same fiber, such that the oxidation by one molecule (e.g., GSNO) preceded the oxidation by the other (e.g., H2O2), and vice versa, the individual effects of both molecules on the Ca2+ sensitivity were retained. Interestingly, neither molecule had any effect on the Ca2+ sensitivity of slow-twitch skeletal muscle. The data show that H2O2 and GSNO retain the capacity to independently affect the contractile apparatus to modulate force. Furthermore, the absence of effects in slow-twitch muscle may further explain why this fiber type is relatively insensitive to fatigue.
Schlafen 3, a novel gene, regulates colonic mucosal growth during aging
Authors: Patel Bhaumik B. , Yu Yingjie , Du Jianhua , Rishi Arun K. , Sarkar Fazlul H. , Tarca Adi L. , Wali Anil , Majumdar Adhip P. N.
Published: February 2009

Although aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. Gene expression profiling (Illumina platform) was carried out in freshly isolated colonic mucosal cells from young (4–6 mo old) and aged (22–24 mo old) Fischer 344 rats. Sixty-six genes were differentially expressed in the colonic mucosa between young and old animals ( P < 0.05). In particular, the expression of schlafen 3, a negative regulator of proliferation, was decreased by 8- to 10-fold in the colonic mucosa of aged rats. Administration of wortmannin, which inhibited colonic mucosal proliferation in the colonic mucosa of aged rats, stimulated the expression of schlafen 3, indicating a growth regulatory role of this gene. To further determine the growth regulatory properties of schlafen 3 gene, schlafen 3 cDNA was transfected in colon cancer HCT-116 cells. This resulted in a 30–40% inhibition of cellular growth, accompanied by decreased expression of PCNA and cyclin D1 and reduced phosphorylation of retinoblastoma protein. In conclusion, our present study demonstrates that several genes involved in proliferation and apoptosis are differentially expressed in the colonic mucosa of young and aged rats. Schlafen 3, a novel negative regulator of growth, which is markedly downregulated in the colonic mucosa of the aged, may play a role in regulating colonic mucosal growth during aging.
Phosphodiesterase inhibitor-dependent inverse agonism of agouti-related protein on melanocortin 4 receptor in sea bass (Dicentrarchus labrax)
Authors: Sánchez Elisa , Rubio Vera Cruz , Thompson Darren , Metz Juriaan , Flik Gert , Millhauser Glenn L. , Cerdá-Reverter José Miguel
Published: February 2009

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor mainly expressed in the central nervous system of vertebrates. Activation of the MC4R leads to a decrease in food intake, whereas inactivating mutations are a genetic cause of obesity. The binding of agouti-related protein (AGRP) reduces not only agonist-stimulated cAMP production (competitive antagonist) but also the basal activity of the receptor, as an inverse agonist. Transgenic zebrafish overexpressing AGRP display increased food intake and linear growth, indicative of a physiological role for the melanocortin system in the control of the energy balance in fish. We report on the cloning, pharmacological characterization, tissue distribution, and detailed brain mapping of a sea bass ( Dicentrarchus labrax) MC4R ortholog. Sea bass MC4R is profusely expressed within food intake-controlling pathways of the fish brain. However, the activity of the melanocortin system during progressive fasting does not depend on the hypothalamic/pituitary proopiomelanocortin (POMC) and MC4R expression, which suggests that sea bass MC4R is constitutively activated and regulated by AGRP binding. We demonstrate that AGRP acts as competitive antagonist and reduces MTII-induced cAMP production. AGRP also decreases the basal activity of the receptor as an inverse agonist. This observation suggests that MC4R is constitutively active and supports the evolutionary conservation of the AGRP/MC4R interactions. The inverse agonism, but not the competitive antagonism, depends on the presence of a phosphodiesterase inhibitor (IBMX). This suggests that inverse agonism and competitive antagonism operate through different intracellular signaling pathways, a view that opens up new targets for the treatment of melanocortin-induced metabolic syndrome.
Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen
Authors: Sánchez-Lemus Enrique , Benicky Julius , Pavel Jaroslav , Larrayoz Ignacio M. , Zhou Jin , Baliova Martina , Nishioku Tsuyoshi , Saavedra Juan M.
Published: February 2009

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.
EGF receptor signaling is involved in expression of osmoprotective TonEBP target gene aldose reductase under hypertonic conditions
Authors: Küper Christoph , Steinert Daniela , Fraek Maria-Luisa , Beck Franz-X. , Neuhofer Wolfgang
Published: February 2009

Renal medullary cells adapt to their hyperosmotic environment by enhanced expression of various osmoprotective genes. Although it is clearly established that TonEBP contributes to the expression of these genes, neither the precise signaling mechanism by which hypertonicity activates TonEBP is completely understood, nor is it known whether a membrane-bound osmosenser, corresponding to yeast and bacteria, is present in mammalian cells. We found evidence that metalloproteinase (MMP)-dependent activation of the epidermal growth factor receptor (EGFR) signals to TonEBP and stimulates the expression of the TonEBP target gene aldose reductase (AR) under hypertonic conditions. Phosphorylation of EGFR and the downstream MAP kinases ERK1/2 and p38 was significantly enhanced by high NaCl in Madin-Darby canine kidney (MDCK) cells. Conversely, the broad-spectrum MMP inhibitor GM6001 or the EGFR inhibitor AG1478 diminished phosphorylation of EGFR, p38, and ERK1/2, the induction of AR mRNA and protein, and AR promoter reporter activity in response to hypertonicity. Accordingly, neutralizing antibodies against the putative EGFR ligand transforming growth factor-α (TGF-α) abolished AR induction during osmotic stress. Furthermore, tonicity-induced phosphorylation of p38 and ERK1/2 and expression of AR were reduced significantly in MDCK cells transfected with a dominant-negative Ras construct. These effects were not caused by reduced nuclear abundance of TonEBP during osmotic stress; however, inhibition of EGFR or p38 diminished TonEBP transactivation activity under hypertonic conditions. The contribution of MMP/EGFR signaling in vivo was confirmed in C57BL/6 mice, in which treatment with GM6001 was associated with reduced AR induction following dehydration. Taken together, these results indicate that osmotic stress induces MMP-dependent activation of EGFR, likely via shedding of TGF-α, and downstream activation of Ras and the MAP kinases p38 and ERK1/2, which stimulate TonEBP transactivation activity. This EGFR-Ras-MAPK pathway contributes to TonEBP transcriptional activation and targets gene expression during osmotic stress, thus establishing a membrane-associated signal input that contributes to the regulation of TonEBP activity.
mPGES-1 deletion impairs diuretic response to acute water loading
Authors: Soodvilai Sunhapas , Jia Zhanjun , Wang Mong-Heng , Dong Zheng , Yang Tianxin
Published: February 2009

PGE2 has an established role in renal water handling. The present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1) in the diuretic response to acute and chronic water loading. Compared with wild-type (+/+) controls, mPGES-1 −/− mice exhibited impaired ability to excrete an acute, but not chronic water load. In response to acute water loading, urinary PGE2 excretion in the +/+ mice increased at 2 h, in parallel with increased urine flow. In contrast, the −/− mice exhibited a delayed increase in urinary PGE2 excretion, coinciding with the stimulation of renal medullary mRNA expression of cytosolic prostaglandin E synthase but not mPGES-2. At baseline, renal aquaporin-2 (AQP2) expression in mPGES-1 −/− mice was enhanced compared with the +/+ control. In response to acute water loading, renal AQP2 expression in the +/+ mice was significantly reduced, and this reduction was blunted in the −/− mice. Despite striking changes in AQP2 protein expression, renal AQP2 mRNA in both genotypes largely remained unchanged. Overall, these data support an important role of mPGES-1 in provoking the diuretic response to acute water loading.