bluuebunny/crossref_metadata_2025 huggingface dataset:
Gain, Preservation, and Loss of a Group 1a Coronavirus Accessory Glycoprotein
Authors: Lorusso Alessio , Decaro Nicola , Schellen Pepijn , Rottier Peter J. M. , Buonavoglia Canio , Haijema Bert-Jan , de Groot Raoul J.
Published: July 2008

Coronaviruses are positive-strand RNA viruses of extraordinary genetic complexity and diversity. In addition to a common set of genes for replicase and structural proteins, each coronavirus may carry multiple group-specific genes apparently acquired through relatively recent heterologous recombination events. Here we describe an accessory gene, ORF3, unique to canine coronavirus type I (CCoV-I) and characterize its product, glycoprotein gp3. Whereas ORF3 is conserved in CCoV-I, only remnants remain in CCoV-II and CCoV-II-derived porcine and feline coronaviruses. Our findings provide insight into the evolutionary history of coronavirus group 1a and into the dynamics of gain and loss of accessory genes.
Sequestering of the Prehairpin Intermediate of gp41 by Peptide N36
Mut(e,g)
Potentiates the Human Immunodeficiency Virus Type 1 Neutralizing Activity of Monoclonal Antibodies Directed against the N-Terminal Helical Repeat of gp41
Authors: Gustchina Elena , Bewley Carole A , Clore G. Marius
Published: July 2008

Human immunodeficiency virus type 1 (HIV-1) neutralization can be effected by several classes of inhibitors that target distinct regions of gp41 that are accessible in the prehairpin intermediate (PHI) state and block the formation of the six-helix bundle (6-HB) conformation of gp41. The N-heptad repeat (N-HR) of gp41 is the site of action of two classes of inhibitors. One class binds to the trimeric N-HR coiled coil, while the other, exemplified by the peptide N36
Mut(e,g)
, disrupts the trimer and sequesters the PHI through the formation of heterotrimers. We recently reported a neutralizing Fab (Fab 3674), selected from a nonimmune phage library, that binds to the trimeric N-HR coiled coil through an epitope that remains exposed in the 6-HB and is also present in heterotrimers of the N-HR and N36
Mut(e,g)
peptide. Here we show that N36
Mut(e,g)
prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab 3674 and that bivalent Fab 3674 and N36
Mut(e,g)
neutralize HXB2 and SF162 strains of HIV-1, as well as isolates of diverse primary B and C HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36
Mut(e,g)
also rescues neutralizing activity of Fab 3674 against resistant virus strains and renders a series of related nonneutralizing Fabs neutralizing. Moreover, N36
Mut(e,g)
exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane-proximal extended region of gp41. The mechanistic implications of these findings are discussed.
Impaired Hepatitis C Virus (HCV)-Specific Effector CD8
+
T Cells Undergo Massive Apoptosis in the Peripheral Blood during Acute HCV Infection and in the Liver during the Chronic Phase of Infection
Authors: Radziewicz Henry , Ibegbu Chris C. , Hon Huiming , Osborn Melissa K. , Obideen Kamil , Wehbi Mohammad , Freeman Gordon J. , Lennox Jeffrey L. , Workowski Kimberly A. , Hanson Holly L. , Grakoui Arash
Published: July 2008

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8
+
T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8
+
T cells during the acute and chronic stages of infection. Although HCV-specific CD8
+
T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8
+
T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8
+
T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8
+
T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.
Early Interferon Therapy for Hepatitis C Virus Infection Rescues Polyfunctional, Long-Lived CD8
+
Memory T Cells
Authors: Badr Gamal , Bédard Nathalie , Abdel-Hakeem Mohamed S. , Trautmann Lydie , Willems Bernard , Villeneuve Jean-Pierre , Haddad Elias K. , Sékaly Rafick P. , Bruneau Julie , Shoukry Naglaa H.
Published: July 2008

The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-α) antiviral therapy achieves the highest rate of success when IFN-α is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-γ- and IL-2-producing and CD107a
+
) virus-specific CD8
+
T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8
+
T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-α rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.
Effector CD4+T-Cell Involvement in Clearance of Infectious Herpes Simplex Virus Type 1 from Sensory Ganglia and Spinal Cords
Authors: Johnson Alison J. , Chu Chin-Fun , Milligan Gregg N.
Published: July 2008

In primary infection, CD8+T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4+T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4+T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4+T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8+T-cell-deficient and CD8+T-cell-depleted mice, suggesting that CD4+T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4+T cells resolved neural infection, CD8+T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4+T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.
Remodeling of Endothelial Adherens Junctions by Kaposi's Sarcoma-Associated Herpesvirus
Authors: Mansouri Mandana , Rose Patrick P. , Moses Ashlee V. , Früh Klaus
Published: July 2008

Vascular endothelial cadherin (VE-cadherin) connects neighboring endothelial cells (ECs) via interendothelial junctions and regulates EC proliferation and adhesion during vasculogenesis and angiogenesis. The cytoplasmic domain of VE-cadherin recruits α- and β-catenins and γ-catenin, which interact with the actin cytoskeleton, thus modulating cell morphology. Dysregulation of the adherens junction/cytoskeletal axis is a hallmark of invasive tumors. We now demonstrate that the transmembrane ubiquitin ligase K5/MIR-2 of Kaposi's sarcoma-associated herpesvirus targets VE-cadherin for ubiquitin-mediated destruction, thus disturbing EC adhesion. In contrast, N-cadherin levels in K5-expressing cells were increased compared to those in control cells. Steady-state levels of α- and β-catenins and γ-catenin in K5-expressing ECs were drastically reduced due to proteasomal destruction. Moreover, the actin cytoskeleton was rearranged, resulting in the dysregulation of EC barrier function as measured by electric cell-substrate impedance sensing. Our data represent the first example of a viral protein targeting adherens junction proteins and suggest that K5 contributes to EC proliferation, vascular leakage, and the reprogramming of the EC proteome during Kaposi's sarcoma tumorigenesis.
Spontaneous Heteromerization of Gammaretrovirus Envelope Proteins: a Possible Novel Mechanism of Retrovirus Restriction
Authors: Dewannieux Marie , Collins Mary K.
Published: July 2008

The
env
gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral
env
genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.
Production and engineering application of C60 high-performance pump pebble concrete containing ultra-fine fly ash
Authors: Li Gao Ying , Guo Ma Bao , Zhou Shi Qiong
Published: July 2008

C60 grade high-performance pump concrete incorporating river pebbles and ultra-fine fly ash (UFA) was successfully prepared, with UFA replacing 30% of the cement, for the second-stage of construction of the Hunan Xiangtan Power Plant. Some of the properties of this pump concrete were studied using both laboratory and on-site testing. The results indicated that workability of UFA concrete was excellent. The initial slump surpassed 180 mm, and the slump loss was small. The 28 d strength of the concrete met the engineering requirement of conforming to C60 strength grade. It is shown that the resistance–chloride ion penetration of UFA pump concrete is better than that of ordinary concrete in terms of Association for Testing and Materials test method C1202-94. Dry shrinkage of concrete slightly reduced with the addition of UFA, and UFA concrete shows better crack resistance compared with ordinary concrete. The production technology of UFA pump pebble concrete is summarized based on test and engineering applications.
Seismic performance of a full-scale, reinforced high-performance concrete building. Part I: Experimental study
Authors: Mousseau Sébastien , Paultre Patrick
Published: July 2008

Full-scale tests provide valuable information on the characteristics of building structures that can be used to evaluate design methods, to calibrate modelling techniques, and to determine damage corresponding to loading levels. These tests are scarce due to the enormous requirements in testing space and specialized testing equipment. The seismic behaviour of a full-scale, two-storey, reinforced high-performance concrete building designed with moderate ductility detailing is evaluated by pseudo-dynamic testing, during which increasing seismic loads are applied, resulting in increasing levels of permanent damage to the structure. To monitor the level of damage, a series of successive forced-vibration tests are also carried out at each step of the process and are used to track changes in the key dynamic properties of the building. The paper presents the design of the test structure according to the new edition of the CSA A23.3-04 Design of concrete structures standard, the series of pseudo-dynamic tests simulating different levels of earthquake excitation consistent with the 2005 edition of the National building code of Canada, and the evaluation of the performance of the building. It is shown that the detailing requirements of CSA A23.3-04 are more than adequate to provide the ductility and overstrength expected.
Soft computing approach to construction performance prediction and diagnosis
Authors: Dissanayake Manjula , Robinson Fayek Aminah
Published: July 2008

Increasing project complexity and greater levels of fast-tracking on construction projects make the need for quickly detecting and diagnosing a deviation in a construction performance measure from its planned value a challenging task. In such a rapidly changing environment, timely detection of deviations is critical so that the most effective corrective actions can be taken. This paper presents an integrated model that is capable of predicting and diagnosing construction performance deviations based on a combination of field measurements and subjective assessments of performance-related factors. The proposed system is based on the synergistic integration of the soft computing approaches of fuzzy set theory, neural networks, and genetic algorithms. A systematic methodology to elicit and represent qualitative construction performance knowledge from a group of experts is presented. The essential features of the model are described in detail and are implemented in a computer system called XCOPE (explaining construction performance).
Dynamic risk analysis in construction projects
Authors: Nasirzadeh Farnad , Afshar Abbas , Khanzadi Mostafa
Published: July 2008

The occurrence of one risk may exacerbate other risks or portfolios of risks due to their highly complex interrelated structures and existing interactions. Hence, the cumulative impact of a chain of risks may be greater than the sum of their individual impacts. Commonly practiced risk analysis approaches do not account for these interactions and face deficiency in providing reliable information regarding the actual impact of the identified risks. This paper presents a new approach to construction risk analysis where the interrelated structure of risks and their interactions have been modeled through the governing feedback loops. The proposed methodology is a system dynamics-based approach for risk analysis and assessment. The full impact of a risk or a set of risks may efficiently be modeled, simulated, and quantified in terms of time and cost by the proposed object-oriented simulation methodology. To evaluate the performance of the proposed methodology, it has been incorporated into a bridge construction project. The interrelated structures of the identified risks have been modeled and their cumulative consequences simulated and quantified as an illustrative example.
Follower forces: Leipholz's early researches in elastic stability
Authors: Gladwell G. M. L.
Published: July 2008

This paper provides an historical account of Leipholz's research into elastic stability. Emphasis is placed on divergence and flutter instability of follower force systems, the derivation of lower bounds for the critical load for divergence, and estimates for critical loads for flutter. Key words: elastic stability, divergence, flutter, lower bounds, nonconservative systems, symmetrisable matrix.
Hydrodynamic coefficients of a vertical circular cylinder
Authors: Isaacson Michael , Mathai Thomas , Mihelcic Carol
Published: July 2008

The added mass and the damping coefficient of a large surface-piercing circular cylinder extending to the seabed and undergoing horizontal oscillations are described. A closed-form solution to the corresponding linear radiation problem is obtained by the use of eigenfunction expansions. Attention is given to the vertical distribution of these coefficients and to their high-frequency asymptotic behaviour. Comparisons are made with experimental measurements. The application to typical offshore structures is discussed. Key words: added mass, cylinders, damping, hydrodynamics, ocean engineering.
Post-cracking analysis of reinforced concrete panels including tension stiffening
Authors: Barzegar Fariborz , Schnobrich William C.
Published: July 2008

In finite element analysis of reinforced concrete structures, the effect of bond forces between concrete and reinforcement, referred to as tension stiffening, is discussed. To account for this phenomenon, the post-cracking constitutive model for concrete is modified by assigning a linear strain softening branch to its stress–strain curve in the tensile stress direction. For analyzing orthogonally reinforced concrete panels, a simple procedure for determining the termination strain on the softening branch is then developed. Appropriate constitutive models for steel and uncracked concrete along with the post-cracking model to simulate the behavior of cracked reinforced concrete are implemented in a finite element program. Three orthogonally reinforced concrete test panels subjected to pure shear loadings causing inclined cracking are analyzed. It is shown that the adopted numerical procedures are capable of predicting the post-cracking responses, ultimate capacities, and modes of failure for the analyzed panels with good accuracy. The capabilities of the employed post-cracking model to simulate the crack shifting behavior observed during testing of a highly anisotropically reinforced panel is also demonstrated. Key words: reinforced concrete panels, finite element, post-cracking, tension stiffening, crack shifting, ultimate load.
Damage caused by the November 25, 1988, Saguenay earthquake
Authors: Mitchell Denis , Tinawi René , Law Tim
Published: July 2008

The November 25, 1988, Saguenay earthquake prompted a site visit by a team representing the Canadian National Committee on Earthquake Engineering. This paper contains selected ground motion records in the form of acceleration-time histories, obtained from the Geological Survey of Canada, and corresponding response spectra. The horizontal acceleration spectrum obtained for Chicoutimi is compared with the design base shear coefficients from the 1980, 1985, and 1990 National Building Codes of Canada. Failures of natural slopes and embankments as well as both architectural and structural damage are discussed. The significant role played by the presence of soft subsoil in amplifying the ground motion and resulting damage is illustrated. Although no major structural failures occurred, there were many examples of poor performance, and in some cases failures, of unreinforced masonry walls. Concerns are expressed over the abundance of unreinforced masonry, particularly in schools and buildings such as hospitals, for which postdisaster performance must be ensured. Key words: earthquake, Saguenay, soils, structures, codes, masonry.
Les dommages dus au tremblement de terre du Saguenay du 25 novembre 1988
Authors: Tinawi René , Mitchell Denis , Law Tim
Published: July 2008

The November 25, 1988, Saguenay earthquake prompted a site visit by a team representing the Canadian National Committee on Earthquake Engineering. This paper contains selected ground motion records in the form of acceleration-time histories, obtained from the Geological Survey of Canada, and corresponding response spectra. The horizontal acceleration spectrum obtained for Chicoutimi is compared with the design base shear coefficients from the 1980, 1985, and 1990 National Building Codes of Canada. Failures of natural slopes and embankments as well as both architectural and structural damage are discussed. The significant role played by the presence of soft subsoil in amplifying the ground motion and resulting damage is illustrated. Although no major structural failures occurred, there were many examples of poor performance, and in some cases failures, of unreinforced masonry walls. Concerns are expressed over the abundance of unreinforced masonry, particularly in schools and buildings such as hospitals, for which postdisaster performance must be ensured. Key words: earthquake, Saguenay, soils, structures, codes, masonry.
Seismic design of base-isolated highway bridges utilizing lead–rubber bearings
Authors: Ghobarah A. , Ali H. M.
Published: July 2008

A study is made of the seismic behaviour of base-isolated highway bridges with the objective of developing design procedures in the form of code-type approach. The recommendations of current codes concerning the use of energy dissipation mechanisms for the seismic design of bridges are reviewed. A model representing the bridge deck, piers, and the base-isolation system is used to evaluate the response of the bridge to a selected earthquake time history record and to evaluate the effects of various design parameters on the dynamic response. The results of this analysis are used in the development of design guidelines for the isolated bridge system. It was found that base isolation affects the design forces on piers and abutments as well as the deck displacements. An optimum design should provide a reasonable balance between the shear forces on supports and tolerable displacements. Two design procedures are proposed based on the time history and inelastic response spectra approaches. Simplified charts are presented which aid in the seismic design of new bridges as well as in the upgrading of existing ones. Key words: dynamic, seismic, design, highway, bridges, earthquake, base isolation.
River discharge and tidal controls on salt-wedge position and implications for channel shoaling: Fraser River, British Columbia
Authors: Kostaschuk R. A. , Atwood L. A.
Published: July 2008

Salinity and current surveys in the Main Channel of the Fraser River estuary, British Columbia, show that a well-defined salt-wedge intrusion migrates along the channel. Bivariate statistical analyses reveal that when discharge is nearly constant, the position of the salt-wedge is controlled by tidal height. Multivariate analyses of data with large ranges in discharge and tidal height indicate that intrusion location is a function of both discharge and tides. Multiple regression models provide predictive relations for salt-wedge position. Application of a regression model indicates that channel shoaling at the mouth of the river is related to the low-tide position of the salt-wedge. Key words: Fraser River estuary, salt-wedge position, channel shoaling.
Dynamic and fatigue responses of prestressed concrete girders with openings
Authors: Kennedy John B. , Chami Saddallah , Grace Nabil F.
Published: July 2008

The influences of opening size, eccentricity, and location on the natural frequencies, mode shapes, damping, and deformation response of prestressed beams and girders with openings under both dynamic and cyclic loadings are presented. A theoretical study was conducted by the finite element method to determine the above structural responses for prestressed rectangular, T- and I-beams. The results were verified and substantiated by tests on six post-tensioned concrete beams. Good agreement was shown between the theoretical and experimental results. Furthermore, it was shown that cyclic loading can have a significant influence on the amplitudes of vibration and the structural response of the top and bottom chords of the opening, and a very significant effect on the tensile strains on the side of the opening resulting from the initial prestressing. Design suggestions are made. Key words: design, dynamic, fatigue, frequencies, girders, openings, prestressed concrete, stresses, structures, tests.
BENEFITS OF AMBIGUITY IN NEW PRODUCT DEVELOPMENT
Authors: BRUN ERIC , SÆTRE ALF STEINAR , GJELSVIK MARTIN
Published: July 2008

In this paper we challenge the traditional notion of ambiguity as an undesirable element in New Product Development (NPD) and explore how companies sometimes sustain or even increase ambiguity during their NPD projects. Based on qualitative analysis of case data from four NPD projects in the medical-device industry, we present a model by which this process can be better understood. We identify four ways that NPD projects can benefit from temporarily sustaining ambiguity: retaining fallback options, saving costs, saving time, and retaining ideas.
THE STRATEGIC IMPACT OF THE INTERNET ON MAGAZINE PUBLISHING
Authors: ELLONEN HANNA-KAISA , KUIVALAINEN OLLI , JANTUNEN ARI
Published: July 2008

This paper discusses the impact of the Internet on the magazine publisher's competitive advantage on three analytic levels: the products, the company and the industry. The analysis is based on interview data from the Finnish magazine publishing industry. On the product level, the Internet is a means of differentiation, while on the company level firm-specific resources and capabilities determine how companies can react to the strategic changes on the product level. However, it seems that the Internet has not significantly changed the five forces that shape the nature and state of competition in the magazine publishing industry.
RAPID COMMERCIALIZATION OF ACQUIRED INNOVATIONS — A COLLABORATIVE MODEL BASED ON CASE STUDIES IN CHINESE COMPANIES
Authors: SUN HONGYI , CHOW AUGUSTINE , LO CHOITUNG
Published: July 2008

Many Chinese companies rely on externally acquired innovations rather than developing their own. The best way to rapidly commercialize externally acquired innovations in a Chinese company, however, is still a rather new research topic. This paper reports research towards development of an integrated model for commercialization of acquired innovations. Development of the preliminary model was based upon a review of the literature. The preliminary model covers four main factors, namely, technological collaboration, organizational collaboration, customer collaboration and resource collaboration. Managers from four entrepreneurial Chinese companies evaluated the model. Each of the four case companies had acquired innovations from external sources. Our empirical results indicate that the factors of partner matches/relationships, product champions, pioneer users and financial alliances tend to be more important than other factors. The managers also added two more factors, namely, government regulations and university support. This paper provides initial empirical evidence that demonstrates the multifaceted determinants of rapid commercialization of acquired innovations in the context of Chinese manufacturing. Our findings suggest that the innovative firms operating in China integrate technological collaboration, government policies and university research activities in order to facilitate commercialization of their acquired innovations. In summary, collaborations both inside and outside of companies are critical for successful commercialization.
PATENT CITATIONS INDICATING PRESENT VALUE OF THE BIOTECHNOLOGY BUSINESS
Authors: NIKULAINEN TUOMO , HERMANS RAINE , KULVIK MARTTI
Published: July 2008

In this paper our aim is to assess the potential of using patent statistics in predicting the future sales anticipations and present value of a company active in the science-based industry. Instead of using conventional patent and patent application counts as indicators, we constructed patent citation weighted portfolios for each company. This way the heterogeneity of patents can be taken into account. Our data covers biotechnology patents held by Finnish biotechnology companies. Empirical results imply that particularly backward citations are related to present value estimations of the companies. In contrast to some previous studies, forward citations do not seem to predict the present value estimations. The findings provide some important implications on interpreting the significance of patent citations regarding valuation of science-based companies, and planning technology and innovation policies.
Functional characterization of NIPA2, a selective Mg2+ transporter
Authors: Goytain Angela , Hines Rochelle M. , Quamme Gary A.
Published: July 2008

We used microarray analysis to identify renal cell transcripts that were upregulated with low magnesium. One transcript, identified as NIPA2 (nonimprinted in Prader-Willi/Angelman syndrome) subtype 2, was increased over twofold relative to cells cultured in normal magnesium. The deduced sequence comprises 129 amino acids with 8 predicted transmembrane regions. As the secondary structure of NIPA2 conformed to a membrane transport protein, we expressed it in Xenopus oocytes and determined that it mediated Mg2+ uptake with two-electrode voltage-clamp and fluorescence studies. Mg2+ transport was electrogenic, voltage dependent, and saturable, demonstrating a Michaelis affinity constant of 0.31 mM. Unlike other reported Mg2+ transporters, NIPA2 was very selective for the Mg2+ cation. NIPA2 mRNA is found in many tissues but particularly abundant in renal cells. With the use of immunofluorescence, it was shown that NIPA2 protein was normally localized to the early endosomes and plasma membrane and was recruited to the plasma membrane in response to low extracellular magnesium. We conclude that NIPA2 plays a role in magnesium metabolism and regulation of renal magnesium conservation.
V-ATPase expression in the mouse olfactory epithelium
Authors: Păunescu Teodor G. , Jones Abigail C. , Tyszkowski Robert , Brown Dennis
Published: July 2008

The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa “A” subunit) and ATP6V1E1 (the ubiquitous 31-kDa “E” subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 (“B1,” typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 (“B2”) in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection.
Renal expression and localization of SLC9A3 sodium/hydrogen exchanger and its possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss)
Authors: Ivanis Goran , Braun Marvin , Perry Steve F.
Published: July 2008

Experiments were performed to assess the possible involvement of the Na+/H+exchanger isoform 3 (NHE3; SLC9A3) in renal acid-base regulation in adult rainbow trout ( Oncorhynchus mykiss). NHE3 mRNA was expressed at high levels in the kidney relative to its paralog, NHE2. The results of in situ hybridization demonstrated an abundance of NHE3 mRNA in renal tubules. The combination of immunocytochemistry and histological staining revealed that NHE3 was confined to the apical membrane of proximal tubules, where it was colocalized with the vacuolar-type H+-ATPase. Levels of NHE3 protein (assessed by Western blotting) were increased during hypercapnia, likely as a result of increased transcription, as indicated by increasing levels of NHE3 mRNA (as determined by real-time PCR). Plasma cortisol concentration was increased during hypercapnia, and administration of exogenous cortisol caused a marked increase in NHE3 mRNA and protein. Thus we speculate that the elevation of plasma cortisol during hypercapnia contributes to transcriptional activation of NHE3 that ultimately promotes acid-base regulation by stimulating H+secretion and HCO3−reabsorption.
Distinct patterns of fat metabolism in skeletal muscle of normal-weight, overweight, and obese humans
Authors: Velan S. Sendhil , Said Nicholas , Durst Christopher , Frisbee Stephanie , Frisbee Jefferson , Raylman Raymond R. , Thomas M. Albert , Rajendran Vazhaikkurichi M. , Spencer Richard G. , Alway Stephen E.
Published: July 2008

The link between body weight, lipid metabolism, and health risks is poorly understood and difficult to study. Magnetic resonance spectroscopy (MRS) permits noninvasive investigation of lipid metabolism. We extended existing two-dimensional MRS techniques to permit quantification of intra- and extramyocellular lipid (IMCL and EMCL, respectively) compartments and their degree of unsaturation in human subjects and correlated these results with body mass index (BMI). Using muscle creatine for normalization, we observed a statistically significant ( P < 0.01) increase in the IMCL-to-creatine ratio with BMI ( n = 8 subjects per group): 5.9 ± 1.7 at BMI < 25, 10.9 ± 1.82 at 25 < BMI < 30, and 13.1 ± 0.87 at BMI > 30. Similarly, the degree of IMCL unsaturation decreased significantly ( P < 0.01) with BMI: 1.51 ± 0.08 at BMI < 25, 1.30 ± 0.11 at 25 < BMI < 30, and 0.90 ± 0.14 at BMI > 30. We conclude that important aspects of lipid metabolism can be evaluated by two-dimensional MRS and propose that degree of unsaturation measured noninvasively may serve as a biomarker for lipid metabolic defects associated with obesity.
Intracellular pH regulates superoxide production by the macula densa
Authors: Liu Ruisheng , Carretero Oscar A. , Ren Yilin , Wang Hong , Garvin Jeffrey L.
Published: July 2008

We hypothesized that elevated macula densa intracellular pH (pHi) during tubuloglomerular feedback enhances O2−production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O2−production increased from 0.53 ± 0.09 to 2.62 ± 0.54 U/min ( P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O2−production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O2−production induced by NaCl was blunted by 40%. To study the effect of pHion O2−in intact macula densa cells, we measured O2−while pHiwas changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O2−production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10−5M), O2−production induced by elevating pHiwas blocked. Finally, we measured the optimum pH for O2−production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pHiis ∼8 for O2−production. We found that elevated pHienhances O2−production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O2−production is pH sensitive, with an optimum pHiof 8.
Stable expression of HIF-1α in tubular epithelial cells promotes interstitial fibrosis
Authors: Kimura Kuniko , Iwano Masayuki , Higgins Debra F. , Yamaguchi Yukinari , Nakatani Kimihiko , Harada Koji , Kubo Atsushi , Akai Yasuhiro , Rankin Erinn B. , Neilson Eric G. , Haase Volker H. , Saito Yoshihiko
Published: July 2008

Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1α. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1α, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1α) histologically and used the anti-HIF-1α agent [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1α could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL−/−mice and in all VHL−/−mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1α appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment.
Short-duration galvanic vestibular stimulation evokes prolonged balance responses
Authors: Son Gregory Martin Lee , Blouin Jean-Sébastien , Inglis John Timothy
Published: July 2008

The application of galvanic vestibular stimulation (GVS) evokes distinct responses in lower limb muscles involved in the control of balance. The purpose of this study was to investigate the balance and lower limb muscle responses to short-duration GVS and to determine whether these responses are modulated by small changes in center of gravity (CoG) and baseline muscle activity occurring during quiet standing. Twelve subjects stood quietly on a force plate with their feet together and were instructed to look straight ahead. One thousand twenty-four GVS stimuli (4 mA, 20-ms pulses) were delivered bilaterally to the mastoid processes in a bipolar, binaural configuration. Bilateral surface electromyography (EMG) from soleus (Sol) and tibialis anterior (TA) and ground reaction forces were recorded. EMG and force responses were trigger averaged at the onset of the GVS pulse. Short-duration GVS applied during quiet standing with the head facing forward evoked characteristic balance responses and biphasic modulation of all muscles with the same polarity for ipsilateral Sol and TA. The amplitude of the GVS-evoked muscle responses was modulated by both the estimated position of the subject's CoG and the background activation of the recorded muscle. Muscle-dependent modulations of the GVS-evoked muscle responses were observed: the Sol responses decreased, while the TA responses increased when the CoG position shifted toward the heels. The well-defined balance responses evoked by short-duration GVS are important to acknowledge when studying the vestibulo-motor responses in healthy subjects and patient populations.
Functional deficits and insulin-like growth factor-I gene expression following tourniquet-induced injury of skeletal muscle in young and old rats
Authors: Hammers David W. , Merritt Edward K. , Matheny Wayne , Adamo Martin L. , Walters Thomas J. , Estep J. Scot , Farrar Roger P.
Published: July 2008

This study investigated the effect of age on recovery of skeletal muscle from an ischemia-reperfusion (I/R)-induced injury. Young (6 mo old) and old (24–27 mo old) Sprague-Dawley rats underwent a 2-h bout of hindlimb ischemia induced by a pneumatic tourniquet (TK). The TK was released to allow reperfusion of the affected limb, and animals were divided into 7- and 14-day recovery groups. Maximum plantar flexor force production was assessed in both 7- and 14-day recovery groups of both ages, followed by histological evaluation. Subsequent analysis of IGF-I gene expression and intracellular signaling in 7-day recovery muscles was performed by RT-PCR and Western blotting, respectively. Old rats had significantly greater deficits in force production and exhibited more evidence of histological pathology than young at both 7 and 14 days postinjury. In addition, old rats demonstrated an attenuated upregulation of IGF-I mRNA and induction of proanabolic signaling compared with young in response to injury. We conclude that aged skeletal muscle exhibits more damage and/or defective regeneration following I/R and identify an age-associated decrease in local IGF-I responsiveness as a potential mechanism for this phenomenon.
NMDA Receptor Antagonists Reveal Age-Dependent Differences in the Properties of Visual Cortical Plasticity
Authors: de Marchena Jacqueline , Roberts Adam C. , Middlebrooks Paul G. , Valakh Vera , Yashiro Koji , Wilfley Lindsey R. , Philpot Benjamin D.
Published: July 2008

The suggestion that NMDA receptor (NMDAR)-dependent plasticity is subunit specific, with NR2B-types required for long-term depression (LTD) and NR2A-types critical for the induction of long-term potentiation (LTP), has generated much attention and considerable debate. By investigating the suggested subunit-specific roles of NMDARs in the mouse primary visual cortex over development, we report several important findings that clarify the roles of NMDAR subtypes in synaptic plasticity. We observed that LTD was not attenuated by application of ifenprodil, an NR2B-type antagonist, or NVP-AAM007, a less selective NR2A-type antagonist. However, we were surprised that NVP-AAM007 completely blocked adult LTP (postnatal day (P) 45–90), while only modestly affecting juvenile LTP (P21-28). To assess whether this developmental transition reflected an increasing role for NR2A-type receptors with maturity, we characterized the specificity of NVP-AAM007. We found not only that NVP-AAM007 lacks discernable subunit specificity but also that the effects of NVP-AAM077 on LTP could be mimicked using subsaturating concentrations of APV, a global NMDAR antagonist. These results indicate that the effects of NVP-AAM077 on synaptic plasticity are largely explained by nonspecific blockade of NMDARs. Moreover our findings are the first to reveal a developmental increase in the sensitivity of LTP to NMDAR antagonism. We suggest that discrepant reports describing the effect of NVP-AAM077 on LTP may be partially explained by this developmental shift in the properties of LTP. These results indicate that the degree of NMDAR activation required for LTP increases with development, providing insight into a novel underlying mechanism governing the properties of synaptic plasticity.
Amacrine-to-Amacrine Cell Inhibition in the Rabbit Retina
Authors: Hsueh Hain-Ann , Molnar Alyosha , Werblin Frank S.
Published: July 2008

We studied the interactions between excitation and inhibition in morphologically identified amacrine cells in the light-adapted rabbit retinal slice under patch clamp. The majority of on amacrine cells received glycinergic off inhibition. About half of the off amacrine cells received glycinergic on inhibition. Neither class received any GABAergic inhibition. A minority of on, off, and on–off amacrine cells received both glycinergic on and GABAergic off inhibition. These interactions were found in cells with diverse morphologies having both wide and narrow processes that stratify in single or multiple layers of the inner plexiform layer (IPL). Most on–off amacrine cells received no inhibition and have monostratified processes confined to the middle of the IPL. The most common interaction between amacrine cells that we measured was “crossover inhibition,” where off inhibits on and on inhibits off. Although the morphology of amacrine cells is diverse, the interactions between excitation and inhibition appear to be relatively limited and specific.
Neural Substrate of Modified and Unmodified Pathways for Learning in Monkey Vestibuloocular Reflex
Authors: Ramachandran Ramnarayan , Lisberger Stephen G.
Published: July 2008

To understand how the brain learns, we need to identify the full neural circuit for a behavior; characterize how neural responses in the circuit change during behavioral learning; and understand the nature, location, and control of the cellular changes that are responsible for learning. This goal seems attainable for the vestibuloocular reflex (VOR), where the neural circuit basis for learning is already partially understood. The current hypothesis for VOR learning postulates cellular changes in the cerebellar cortex and the vestibular nucleus. It suggests that the brain stem contains two parallel pathways that have been modeled on the basis of extensive biological data as unmodified and modified VOR pathways with frequency-dependent internal gains and different time delays. We now show a correspondence between the responses of different groups of neurons in the vestibular nucleus and the signals emanating from the two pathways in the model. Floccular target neurons (FTNs) and position-vestibular-pause neurons (PVPs) were identified by their discharge during eye movements and by the presence or absence of inhibition by floccular stimulation. FTNs had response gains and phases that coincided with predictions for pathways that are modified in association with learning, whereas PVPs had responses in agreement with predictions for the unmodified pathways. The quantitative agreement of prior model predictions and new data supports the identity of FTNs and PVPs as brain stem interneurons in the modified and unmodified VOR pathways. Other aspects of the data make predictions about how vestibular inputs are transformed as they pass through the two pathways.
Dysregulated Information Processing by Medium Spiny Neurons in Striatum of Freely Behaving Mouse Models of Huntington's Disease
Authors: Miller Benjamin R. , Walker Adam G. , Shah Anand S. , Barton Scott J. , Rebec George V.
Published: July 2008

Huntington's disease (HD) is an autosomal dominant condition that compromises behavioral output. Dysfunction of medium spiny neurons (MSNs), which are the sole output system of the striatum, is thought to underlie HD pathophysiology. What is not known is how HD alters MSN information processing during behavior, which likely drives the HD behavioral phenotype. We recorded from populations of MSNs in two freely behaving and symptomatic HD mouse models: R6/2 transgenics are based on a C57BL/6J*CBA/J background and show robust behavioral symptoms, whereas knock-in (KI) mice have a 129sv background and express relatively mild behavioral signs. At the single-unit level, we found that the MSN firing rate was elevated in R6/2 but not in KI mice compared with their respective wild-type (WT) controls. In contrast, burst activity, which corresponds to periods of high-frequency firing, was altered in both HD models compared with WT. At the population level, we found that correlated firing between pairs of MSNs was a prominent feature in WT that was reduced in both HD models. Similarly, coincident bursts, which are bursts between pairs of neurons that overlap in time and occur more often in pairs of MSNs that exhibit correlated firing, were decreased in HD mice. Our results indicate an important role in both bursting and correlated burst firing for information processing in MSNs. Dysregulation of this processing scheme, moreover, is a key component of HD pathophysiology regardless of the severity of HD symptoms, genetic construct, and background strain of the mouse models.