bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiovascular responses to human calf muscle stretch during varying levels of muscle metaboreflex activation
Authors: Fisher James P. , Bell Martin P. D. , White Michael J.
Published: July 2005

The purpose of the present study was to investigate the cardiovascular responses to muscle metaboreflex‐ and concurrent muscle stretch‐induced mechanoreflex activation. Eight subjects (7 males, 1 female) performed 90 s of isometric calf plantarflexion at 0, 30, 50 and 70% of maximum voluntary contraction. During exercise and for 3.5 min postexercise, circulatory occlusion (PECO) was ensured by inflation of a thigh cuff. After 90 s of PECO the calf muscle was stretched for 60 s (Stretch). Heart rate (HR; assessed from ECG), blood pressure (BP; Finapres) and phase of respiratory cycle were recorded. Exercise increased diastolic BP (DBP) from rest by 1 ± 0.8, 14 ± 2.5, 29 ± 3.9 and 35 ± 3.6 mmHg, during the 0, 30, 50 and 70% conditions, respectively (ANOVA rest versus exercise, P < 0.05). During PECO DBP remained elevated, by 2 ± 0.4, 8 ± 0.3, 12 ± 0.3 and 13 ± 0.9 mmHg, respectively. Stretch produced a further increase in DBP that was not different between conditions (3 ± 1.4, 2 ± 0.8, 3 ± 1.0 and 3 ± 0.9 mmHg, for the 0, 30, 50 and 70%, respectively). HR increased during exercise but returned to baseline during PECO. HR increased at Stretch onset in all conditions. No EMG was detected from the gastrocnemius and soleus during Stretch. Our data show that the cardiovascular responses to human calf Stretch are independent of the level of concurrent muscle metaboreflex activation.
Performance and Development of a Miniature Rotary Shaft Pump
Authors: Blanchard Danny , Ligrani Phil , Gale Bruce
Published: July 2005

The development and performance of a novel miniature pump called the rotary shaft pump (RSP) is described. The impeller is made by boring a 1.168 mm hole in one end of a 2.38 mm dia shaft and cutting slots in the side of the shaft at the bottom of the bored hole such that the metal between the slots defines the impeller blades. The impeller blades and slots are 0.38 mm tall. Several impeller designs are tested over a range of operating conditions. Pump performance characteristics, including pressure rise, hydraulic efficiency, slip factor, and flow rate, are presented for several different pump configurations, with maximum flow rate and pressure rise of 64.9ml∕min and 2.1 kPa, respectively, when the working fluid is water. Potential applications include transport of biomedical fluids, drug delivery, total analysis systems, and electronics cooling.
Steady and Dynamic Models of Fuel and Air Flow in Carburetors for Small Engines
Authors: Arias Diego A. , Shedd Timothy A.
Published: July 2005

This work presents the mathematical model of a complex flow network containing short metering orifices, compressible flow, and two-phase flow in small diameter pipes. It has been developed to study the steady and dynamic flows in a carburetor for small engines. It extends the previously published models by incorporating a detailed review of two-phase flow pressure drop, the effect of the fuel well on the control of air-bleed flow, and dynamic flow. The homogeneous two-phase flow model, which is commonly used in previous models, was compared to an empirical correlation derived from experiments in small pipes and found to be in poor agreement. In order to assess dynamic flow conditions, the model was extended by solving instantaneous one-dimensional Navier-Stokes equations in single-phase pipes. This strategy proved successful in explaining the mixture enrichment seen under pulsating flow conditions. The model was also used to derive a sensitivity analysis of geometries and physical properties of air and fuel.
Fast and Accurate Solutions of Steady Stokes Flows Using Multilevel Boundary Element Methods
Authors: Dargush G. F. , Grigoriev M. M.
Published: July 2005

Most recently, we have developed a novel multilevel boundary element method (MLBEM) for steady Stokes flows in irregular two-dimensional domains (Grigoriev, M.M., and Dargush, G.F., Comput. Methods. Appl. Mech. Eng., 2005). The multilevel algorithm permitted boundary element solutions with slightly over 16,000 degrees of freedom, for which approximately 40-fold speedups were demonstrated for the fast MLBEM algorithm compared to a conventional Gauss elimination approach. Meanwhile, the sevenfold memory savings were attained for the fast algorithm. This paper extends the MLBEM methodology to dramatically improve the performance of the original multilevel formulation for the steady Stokes flows. For a model problem in an irregular pentagon, we demonstrate that the new MLBEM formulation reduces the CPU times by a factor of nearly 700,000. Meanwhile, the memory requirements are reduced more than 16,000 times. These superior run-time and memory reductions compared to regular boundary element methods are achieved while preserving the accuracy of the boundary element solution.
Adaptive Wavelet Method for Incompressible Flows in Complex Domains
Authors: Wirasaet Damrongsak , Paolucci Samuel
Published: July 2005

An adaptive wavelet-based method provides an alternative means to refine grids according to local demands of the physical solution. One of the prominent challenges of such a method is the application to problems defined on complex domains. In the case of incompressible flow, the application to problems with complicated domains is made possible by the use of the Navier-Stokes–Brinkman equations. These equations take into account solid obstacles by adding a penalized velocity term in the momentum equation. In this study, an adaptive wavelet collocation method, based on interpolating wavelets, is first applied to a benchmark problem defined on a simple domain to demonstrate the accuracy and efficiency of the method. Then the penalty technique is used to simulate flows over obstacles. The numerical results are compared to those obtained by other computational approaches as well as to experiments.
A Meshless Local Petrov-Galerkin Method for Fluid Dynamics and Heat Transfer Applications
Authors: Arefmanesh Ali , Najafi Mohammad , Abdi Hooman
Published: July 2005

The meshless local Petrov-Galerkin method has been modified to develop a meshless numerical technique to solve computational fluid dynamics and heat transfer problems. The theory behind the proposed technique, hereafter called “the meshless control volume method,” is explained and a number of examples illustrating the implementation of the method is presented. In this study, the technique is applied for one- and two-dimensional transient heat conduction as well as one- and two-dimensional advection-diffusion problems. Compared to other methods, including the exact solution, the results appear to be highly accurate for the considered cases. Being a meshless technique, the control volumes are arbitrarily chosen and possess simple shapes, which, contrary to the existing control volume methods, can overlap. The number of points within each control volume and, therefore, the degree of interpolation, can be different throughout the considered computational domain. Since the control volumes have simple shapes, the integrals can be readily evaluated.
Numerical Simulation of Vortex Cavitation in a Three-Dimensional Submerged Transitional Jet
Authors: Xing Tao , Li Zhenyin , Frankel Steven H.
Published: July 2005

Vortex cavitation in a submerged transitional jet is studied with unsteady three-dimensional direct numerical simulations. A locally homogeneous cavitation model that accounts for non-linear bubble dynamics and bubble/bubble interactions within spherical bubble clusters is employed. The velocity, vorticity, and pressure fields are compared for both cavitating and noncavitating jets. It is found that cavitation occurs in the cores of the primary vortical structures, distorting and breaking up the vortex ring into several sections. The velocity and transverse vorticity in the cavitating regions are intensified due to vapor formation, while the streamwise vorticity is weakened. An analysis of the vorticity transport equation reveals the influence of cavitation on the relative importance of the vortex stretching, baroclinic torque, and dilatation terms. Statistical analysis shows that cavitation suppresses jet growth and decreases velocity fluctuations within the vaporous regions of the jet.
A Framework for Coupling Reynolds-Averaged With Large-Eddy Simulations for Gas Turbine Applications
Authors: Schlüter J. U. , Wu X. , Kim S. , Shankaran S. , Alonso J. J. , Pitsch H.
Published: July 2005

Full-scale numerical prediction of the aerothermal flow in gas turbine engines are currently limited by high computational costs. The approach presented here intends the use of different specialized flow solvers based on the Reynolds-averaged Navier-Stokes equations as well as large-eddy simulations for different parts of the flow domain, running simultaneously and exchanging information at the interfaces. This study documents the development of the interface and proves its accuracy and efficiency with simple test cases. Furthermore, its application to a turbomachinery application is demonstrated.
Effect of Perforated Plate Open Area on Gas Holdup in Rayon Fiber Suspensions
Authors: Su Xuefeng , Heindel Theodore J.
Published: July 2005

Three different aeration plates are used to study their effect on gas holdup and flow regime transition in fiber suspensions. The aeration plates differ by their open-area ratios (A=0.57%, 0.99%, and 2.14%), where the hole diameter remains the same while the number of holes increase. Experiments are performed using three different Rayon fiber lengths (L=3, 6, and 12mm) over a range of superficial gas velocities (Ug⩽18cm∕s) and fiber mass fractions (0⩽C⩽1.8%) in a 15.24cm dia semi-batch bubble column. Experimental results show that the aeration plate with A=0.99% produces the highest gas holdup in an air-water system and low fiber mass fraction suspensions, and the plate with A=2.14% yields the lowest gas holdup in these systems. In medium fiber mass fraction suspensions, the plate with A=0.57% produces slightly higher gas holdup values, while the other two plates yield similar results. The effect of the aeration plate open area on gas holdup diminishes at high fiber mass fractions (C⩾1.2%). All aeration plates generate homogeneous, transitional, and heterogeneous flow regimes over the range of superficial gas velocities for air-water and low fiber mass fraction suspensions. However, the aeration plate with A=2.14% enhances the flow regime transition, i.e., the superficial gas velocity at which transitional flow appears is lower. Additionally, the fiber mass fraction at which pure heterogeneous flow is observed is lower when A=2.14%.
Permittivity measurements in solids, powders, and liquids
Authors: Thompson Frank
Published: July 2005

A microwave waveguide cell has been constructed to facilitate the measurement of permittivity on solids, powders, and liquids. By employing this cell and a vector network analyzer, permittivity values have been obtained, at room temperature, for samples of perspex, PTFE, sand, and sunflower oil. These values are in agreement with published data. It is hoped that this experiment will provide students with an opportunity to use modern microwave instrumentation and gain a greater appreciation of the physical properties of materials.
Digital video microscopy in the Millikan oil-drop experiment
Authors: Silva Kenneth J. , Mahendra Jacquelyn C.
Published: July 2005

We report on the ease and efficacy of using digital video microscopy techniques and computer software for analyzing data from the Millikan oil-drop experiment in an introductory physics laboratory course setting with applications for more advanced laboratories.
The optical mouse for harmonic oscillator experimentation
Authors: Ng T. W. , Ang K. T.
Published: July 2005

The optical mouse is an extremely cost-effective displacement sensor. Here we describe the use of an optical mouse in an experiment to record position as a function of time for a simple mechanical oscillator. The recorded data correspond closely to that expected for damped harmonic oscillations. These measurements can be made without the use of cumbersome attachments. The optical mouse is also advantageous in obviating concern about the integrity of electrical contact points and the deleterious effects of wear and of dirt accumulation on moving parts.
Teaching the concepts of measurement: An example of a concept-based laboratory course
Authors: Kung Rebecca Lippmann
Published: July 2005

For students to successfully complete an experiment, they must have an understanding of measurement and its related uncertainty. We argue for teaching the concepts of measurement and not only the calculations. An example of a concepts-based laboratory course is given, outlining the concepts presented, the design of the laboratory time, and the laboratory tasks. The concepts are briefly described and two often-overlooked concepts, predictive versus descriptive questions and internal versus external variation, are explained. Our survey results show that the fraction of students using range and not just average when comparing two data sets approximately doubled after instruction.
Master equation approach to the non-equilibrium negative specific heat at the glass transition
Authors: Bisquert Juan
Published: July 2005

Although glasses are disordered and complex systems, we show that important characteristics of the glass transition, such as a negative measured specific heat in the transition region, can be understood using a simple model of thermally activated hopping in a two-level system. The dynamics of the heating of a nonequilibrium state is modeled by a master equation. The calculation illustrates the connection between equilibrium and nonequilibrium, in particular, the determination of transition rates using the principle of detailed balance.
Application of the effective interaction method to the relativistic Coulomb problem
Authors: Adkins Gregory S. , McGovern Nathan M.
Published: July 2005

Effective theories are widely used to describe low-energy behavior in situations where a full description including high-energy dynamics is either unavailable or excessively complicated. As a simple example, we obtain the effective quantum mechanics of the low-energy behavior of a Dirac electron in an attractive Coulomb potential. We use this effective theory to evaluate the hydrogen fine structure through terms of order mα6.
Tipping time of a quantum rod
Authors: Shegelski Mark R. A. , Lundeberg Mark , Goodvin Glen L.
Published: July 2005

We study the tipping time of a quantum mechanical rod that is constrained to move in a plane in a gravitational potential. The initial state of the center of mass of the rod is localized within the base of support of the rod. The tipping time is found to increase exponentially with the height H of the rod as C1t0 exp[C2(H/H0)9], where C1 and C2 are dimensionless constants of order one, t0 is the time scale of the motion, and H0 is the length scale of the rod. We show that the tipping time cannot be obtained using the uncertainty principle alone, and compare our result to that obtained using the Wentzel–Kramers–Brillouin approximation.
On the v2/c2 expansion of the Dirac equation with external potentials
Authors: Zawadzki Wlodek
Published: July 2005

The v2/c2 expansion of the Dirac equation with external potentials is reexamined. A complete, gauge invariant form of the expansion to order (1/c)2 is established that contains two additional terms, in contrast to various published results. It is shown that the additional terms describe the relativistic decrease of the electron spin magnetic moment with increasing electron energy.
The physics of gauge transformations
Authors: Yang Kuo-Ho
Published: July 2005

A novel approach to classical electromagnetic gauge transformations with an emphasis on the propagation of potentials at finite speeds from charge and current densities is discussed. We consider a new velocity gauge, where the scalar potential propagates at any chosen speed, and the vector potential has a component that propagates at the same speed and another that travels at the finite speed c. Our emphasis on the propagation of the potentials is different from the vector field decomposition of Helmholtz. We discuss two defects of the Helmholtz method and show that our approach corrects them. Our approach enables us to visualize how the potentials propagate in space and see the link between the violation of gauge invariance and the violation of causality and relativity.
Resource Letter AER-1: Astronomy education research
Authors: Bailey Janelle M. , Slater Timothy F.
Published: July 2005

This Resource Letter guides the reader, whom we assume to be interested in the teaching and learning of astronomy, to the literature on astronomy education research. Journal articles and books are cited for the following topics: astronomy teaching resources; student understanding of astronomy topics, such as the nature of science in the context of astronomy, diurnal motion, seasons, lunar phases, Earth’s shape and gravity, the solar system, stars, galaxies, cosmology, and astrobiology; and the effectiveness of instructional interventions.
A Comparative Look at Governing Science
Authors: Kinderlerer Julian
Published: July 2005

Designs on Nature
. Science and Democracy in Europe and the United States.
By Sheila Jasanoff
. Princeton University Press, Princeton, NJ, 2005. 380 pp. $35.00, £22.95. ISBN 0-691-11811-6.

Examining the development of biotechnology in the United States, Britain, Germany, and the European Union over the past quarter century, the author examines how the public and private actors in each have interacted to produce the current range of approaches to the political management of biotechnology.
Embryos of an Early Jurassic Prosauropod Dinosaur and Their Evolutionary Significance
Authors: Reisz Robert R. , Scott Diane , Sues Hans-Dieter , Evans David C. , Raath Michael A.
Published: July 2005

Articulated embryos from the Lower Jurassic Elliot Formation of South Africa are referable to the prosauropod
Massospondylus carinatus
and, together with other material, provide substantial insights into the ontogenetic development in this early dinosaur. The large forelimbs and head and the horizontally held neck indicate that the hatchlings were obligate quadrupeds. In contrast, adult
Massospondylus
were at least facultatively bipedal. This suggests that the quadrupedal gait of giant sauropods may have evolved by retardation of postnatal negative allometry of the forelimbs. Embryonic body proportions and an absence of well-developed teeth suggest that hatchlings of this dinosaur may have required parental care.
Human Immunodeficiency Virus Type 1envClones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies
Authors: Li Ming , Gao Feng , Mascola John R. , Stamatatos Leonidas , Polonis Victoria R. , Koutsoukos Marguerite , Voss Gerald , Goepfert Paul , Gilbert Peter , Greene Kelli M. , Bilska Miroslawa , Kothe Denise L. , Salazar-Gonzalez Jesus F. , Wei Xiping , Decker Julie M. , Hahn Beatrice H. , Montefiori David C.
Published: July 2005

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functionalenvclones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.
Hepatitis C Virus Internal Ribosome Entry Site-Dependent Translation in
Saccharomyces cerevisiae
Is Independent of Polypyrimidine Tract-Binding Protein, Poly(rC)-Binding Protein 2, and La Protein
Authors: Rosenfeld Amy B. , Racaniello Vincent R.
Published: July 2005

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in
Saccharomyces cerevisiae
was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5′-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the
S. cerevisiae
genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the
S. cerevisiae
genome. HCV IRES-dependent translation in
S. cerevisiae
was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of
S. cerevisiae
encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.
Envelope Targeting: Hemagglutinin Attachment Specificity Rather than Fusion Protein Cleavage-Activation Restricts
Tupaia Paramyxovirus
Tropism
Authors: Springfeld Christoph , von Messling Veronika , Tidona Christian A. , Darai Gholamreza , Cattaneo Roberto
Published: July 2005

To engineer a targeting envelope for gene and oncolytic vector delivery, we characterized and modified the envelope proteins of
Tupaia paramyxovirus
(TPMV), a relative of the morbilli- and henipaviruses that neither infects humans nor has cross-reactive relatives that infect humans. We completed the TPMV genomic sequence and noted that the predicted fusion (F) protein cleavage-activation site is not preceded by a canonical furin cleavage sequence. Coexpression of the TPMV F and hemagglutinin (H) proteins induced fusion of
Tupaia
baby fibroblasts but not of human cells, a finding consistent with the restricted TPMV host range. To identify the factors restricting fusion of non-
Tupaia
cells, we initially analyzed F protein cleavage. Even without an oligo- or monobasic protease cleavage sequence, TPMV F was cleaved in F1 and F2 subunits in human cells. Edman degradation of the F1 subunit yielded the sequence IFWGAIIA, placing the conserved phenylalanine in position 2, a novelty for paramyxoviruses but not the cause of fusion restriction. We then verified whether the lack of a TPMV H receptor limits fusion. Toward this end, we displayed a single-chain antibody (scFv) specific for the designated receptor human carcinoembryonic antigen on the TPMV H ectodomain. The H-scFv hybrid protein coexpressed with TPMV F mediated fusion of cells expressing the designated receptor, proving that the lack of a receptor limits fusion and that TPMV H can be retargeted. Targeting competence and the absence of antibodies in humans define the TPMV envelope as a module to be adapted for ferrying ribonucleocapsids of oncolytic viruses and gene delivery vectors.
Identification of an Essential Molecular Contact Point on the Duck Hepatitis B Virus Reverse Transcriptase
Authors: Cao Feng , Badtke Matthew P. , Metzger Lisa M. , Yao Ermei , Adeyemo Babatunde , Gong Yunhao , Tavis John E.
Published: July 2005

The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.
Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections
Authors: Guerrero-Plata Antonieta , Baron Samuel , Poast Joyce S. , Adegboyega Patrick A. , Casola Antonella , Garofalo Roberto P.
Published: July 2005

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the
Paramyxoviridae
family,
Pneumovirinae
subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-α/β) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-α in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-α, or directly with recombinant IFN-α showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-α than hMPV. Similarly, when the ability to directly induce IFN-α production was assessed, RSV was clearly a weaker inducer of IFN-α than hMPV, as shown by both kinetics and the absolute amount of IFN-α secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-α production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-α production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-α in the lung during infection by members of the
Pneumovirinae
family.
Formaldehyde-Treated, Heat-Inactivated Virions with Increased Human Immunodeficiency Virus Type 1 Env Can Be Used To Induce High-Titer Neutralizing Antibody Responses
Authors: Poon B. , Hsu J. F. , Gudeman V. , Chen I. S. Y. , Grovit-Ferbas K.
Published: July 2005

The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses.
Encephalomyocarditis Virus Induces PKR-Independent Mitogen-Activated Protein Kinase Activation in Macrophages
Authors: Moran Jason M. , Moxley Michael A. , Buller R. Mark L. , Corbett John A.
Published: July 2005

In this study, we provide evidence that the double-stranded RNA-dependent protein kinase (PKR) is not required for virus-induced expression of inducible nitric oxide synthase (iNOS) or the activation of specific signaling pathways in macrophages. The infection of RAW264.7 cells with encephalomyocarditis virus (EMCV) induces iNOS expression and nitric oxide production, which are unaffected by a dominant-negative mutant of PKR. EMCV infection also activates the mitogen-activated protein kinase, cyclic AMP response element binding protein, and nuclear factor κB (NF-κB) signaling cascades at 15 to 30 min postinfection in PKR+/+and PKR−/−macrophages. Activation of these signaling cascades does not temporally correlate with PKR activity or the accumulation of EMCV RNA, suggesting that an interaction between a structural component of the virion and the cell surface may activate macrophages. Consistent with this hypothesis, empty EMCV capsids induced comparable levels of iNOS expression, nitrite production, and activation of these signaling cascades to those induced by intact virions. These findings support the hypothesis that virion-host cell interactions are primary mediators of the PKR-independent activation of signaling pathways that participate in the macrophage antiviral response of inflammatory gene expression.
Determinants of Human Immunodeficiency Virus Type 1 Resistance to Membrane-Anchored gp41-Derived Peptides
Authors: Lohrengel Sabine , Hermann Felix , Hagmann Isabel , Oberwinkler Heike , Scrivano Laura , Hoffmann Caroline , von Laer Dorothee , Dittmar Matthias T.
Published: July 2005

The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol.75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1NL4-3and HIV-1BaLby serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1NL4-3a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1BaLthe majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1BaLresults in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1NL4-3did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol.78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.
Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å
Authors: Xu Ting , Sampath Aruna , Chao Alex , Wen Daying , Nanao Max , Chene Patrick , Vasudevan Subhash G. , Lescar Julien
Published: July 2005

Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the
Flaviviridae
replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3′ single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.
Ebola Virus VP40 Late Domains Are Not Essential for Viral Replication in Cell Culture
Authors: Neumann Gabriele , Ebihara Hideki , Takada Ayato , Noda Takeshi , Kobasa Darwyn , Jasenosky Luke D. , Watanabe Shinji , Kim Jin H. , Feldmann Heinz , Kawaoka Yoshihiro
Published: July 2005

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.
Gamma Interferon Can Block Herpes Simplex Virus Type 1 Reactivation from Latency, Even in the Presence of Late Gene Expression
Authors: Decman Vilma , Kinchington Paul R. , Harvey Stephen A. K. , Hendricks Robert L.
Published: July 2005

Herpes simplex virus type 1 (HSV-1)-specific CD8+T cells and the cytokine gamma interferon (IFN-γ) are persistently present in trigeminal ganglia (TG) harboring latent HSV-1. We define “latency” as the retention of functional viral genomes in sensory neurons without the production of infectious virions and “reactivation” as a multistep process leading from latency to virion assembly. CD8+T cells can block HSV-1 reactivation in ex vivo mouse TG cultures and appear to be the sole source of IFN-γ in these cultures. Here we demonstrate that IFN-γ alone can block HSV-1 reactivation in some latently infected neurons, and we identify points of intervention in the life cycle of the reactivating virus. Cell suspensions of TG that were latently infected with recombinant RE HSV-1 expressing enhanced green fluorescent protein from the promoter for infected cell protein 0 (ICP0) or glycoprotein C (gC) were depleted of endogenous CD8+or CD45+cells and cultured in the presence or absence of IFN-γ. Our results demonstrate that IFN-γ acts on latently infected neurons to inhibit (i) HSV-1 reactivation, (ii) ICP0 promoter activity, (iii) gC promoter activity, and (iv) reactivation in neurons in which the ICP0 or gC promoter is active. Interestingly, we detected transcripts for ICP0, ICP4, and gH in neurons that expressed the ICP0 promoter but were prevented by IFN-γ from reactivation and virion formation. Thus, the IFN-γ blockade of HSV-1 reactivation from latency in neurons is associated with an inhibition of the expression of the ICP0 gene (required for reactivation) and a blockade of a step that occurs after the expression of at least some viral structural genes.
Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model
Authors: Mori Kazuyasu , Sugimoto Chie , Ohgimoto Shinji , Nakayama Emi E. , Shioda Tatsuo , Kusagawa Shigeru , Takebe Yutaka , Kano Munehide , Matano Tetsuro , Yuasa Takae , Kitaguchi Daisuke , Miyazawa Masaaki , Takahashi Yumiko , Yasunami Michio , Kimura Akinori , Yamamoto Naoki , Suzuki Yasuo , Nagai Yoshiyuki
Published: July 2005

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (Δ5G). In Δ5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and Δ5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in Δ5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in Δ5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.
Intrinsic Functional Dysregulation of CD4 T Cells Occurs Rapidly following Persistent Viral Infection
Authors: Brooks David G. , Teyton Luc , Oldstone Michael B. A. , McGavern Dorian B.
Published: July 2005

Effective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.
Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo
Authors: Kurg Reet , Sild Kristiina , Ilves Aigi , Sepp Mari , Ustav Mart
Published: July 2005

Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.
Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly
Authors: Newcomb William W. , Homa Fred L. , Brown Jay C.
Published: July 2005

DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.
The
Lymantria dispar
Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus
Authors: Slavicek James M. , Popham Holly J. R.
Published: July 2005

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two
enhancin
genes (E1 and E2) in the
Lymantria dispar
multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV
enhancin
genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of ∼84 kDa and 90 kDa, respectively, on Western blots of extracts from
L. dispar
652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the
enhancin
genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.