bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurone‐to‐astrocyte signalling in the brain represents a distinct multifunctional unit
Authors: Fellin Tommaso , Carmignoto Giorgio
Published: June 2004

Astrocytes can respond to neurotransmitters released at the synapse by generating elevations in intracellular Ca2+ concentration ([Ca2+]i) and releasing glutamate that signals back to neurones. This discovery opens new perspectives for the possible participation of these glial cells in actual information processing by the brain and raises the hypothesis that astrocyte activation by neuronal signals plays a key role in distinct, functional events. Depending on the level of neuronal activity, the [Ca2+]i response that is activated by neurotransmitters can either remain restricted to an astrocytic process or it can propagate as an intracellular [Ca2+]i wave to other astrocytic processes in contact with different neurones, astrocytes, microglia or endothelial cells of cerebral arterioles. Glutamate release triggered by the [Ca2+]i rise at the astrocytic process represents a feedback, short‐distance signal that affects synaptic transmission locally. The release of glutamate as well as of other compounds far away from the site of initial activation represents a feedforward, long‐distance signal that can be involved in the regulation of distinct processes. For instance, through the release of vasoactive molecules from the astrocytic processes in contact with cerebral arterioles, the neurone–astrocyte–endothelial cell signalling pathway plays a pivotal role in the neuronal control of vascular tone. In this article we will review recent results that should persuade us to reshape our current thinking on the roles of astroglial cells in the brain. We propose that neurones and astrocytes represent an integral unit that has a distinctive role in different fundamental events in brain function. Furthermore, while recent findings provide important evidences for the vesicular hypothesis of glutamate release, we discuss also the proposals for a possible physiological role of hemichannels and purinergic P2X7 receptors in glutamate release from astrocytes. A full clarification of the functional significance of the bidirectional communication that astrocytes establish with neurones as well as with other brain cells represents one of the most intriguing challenges in neurobiological research at the moment and should fuel stimulating debates in years to come.
Sustained and generalized extracellular fluid expansion following heat acclimation
Authors: Patterson Mark J. , Stocks Jodie M. , Taylor Nigel A. S.
Published: June 2004

We measured intra‐ and extravascular body‐fluid compartments in 12 resting males before (day 1; control), during (day 8) and after (day 22) a 3‐week, exercise–heat acclimation protocol to investigate plasma volume (PV) changes. Our specific focus was upon the selective nature of the acclimation‐induced PV expansion, and the possibility that this expansion could be sustained during prolonged acclimation. Acclimation was induced by cycling in the heat, and involved 16 treatment days (controlled hyperthermia (90 min); core temperature = 38.5°C) and three experimental exposures (40 min rest, 96.9 min (s.d. 9.5 min) cycling), each preceded by a rest day. The environmental conditions were a temperature of 39.8°C (s.d. 0.5°C) and relative humidity of 59.2% (s.d. 0.8%). On days 8 and 22, PV was expanded and maintained relative to control values (day 1: 44.0 ± 1.8; day 8: 48.8 ± 1.7; day 22: 48.8 ± 2.0 ml kg−1; P < 0.05). The extracellular fluid compartment (ECF) was equivalently expanded from control values on days 8 (279.6 ± 14.2versus 318.6 ± 14.3 ml kg−1; n= 8; P < 0.05) and 22 (287.5 ± 10.6 versus 308.4 ± 14.8 ml kg−1; n= 12; P < 0.05). Plasma electrolyte, total protein and albumin concentrations were unaltered following heat acclimation (P > 0.05), although the total plasma content of these constituents was elevated (P < 0.05). The PV and interstitial fluid (ISF) compartments exhibited similar relative expansions on days 8 (15.0 ± 2.2%versus 14.7 ± 4.1%; P > 0.05) and 22 (14.4 ± 3.6%versus 6.4 ± 2.2%; P= 0.10). It is concluded that the acclimation‐induced PV expansion can be maintained following prolonged heat acclimation. In addition, this PV expansion was not selective, but represented a ubiquitous expansion of the extracellular compartment.
The influence of gender and upper airway resistance on the ventilatory response to arousal in obstructive sleep apnoea in humans
Authors: Jordan Amy S. , McEvoy R. Doug , Edwards Jill K. , Schory Karen , Yang Chang‐Kook , Catcheside Peter G. , Fogel Robert B. , Malhotra Atul , White David P.
Published: June 2004

The termination of obstructive respiratory events is typically associated with arousal from sleep. The ventilatory response to arousal may be an important determinant of subsequent respiratory stability/instability and therefore may be involved in perpetuating obstructive respiratory events. In healthy subjects arousal is associated with brief hyperventilation followed by more prolonged hypoventilation on return to sleep. This study was designed to assess whether elevated sleeping upper airway resistance (RUA) alters the ventilatory response to arousal and subsequent breathing on return to sleep in patients with obstructive sleep apnoea (OSA). Inspired minute ventilation (VI), RUA and end‐tidal CO2 pressure (PET,CO2) were measured in 22 patients (11 men, 11 women) with OSA (mean ±s.e.m., apnoea–hypopnoea index (AHI) 48.9 ± 5.9 events h−1) during non‐rapid eye movement (NREM) sleep with low RUA (2.8 ± 0.3 cmH2O l−1 s; optimal continuous positive airway pressure (CPAP) = 11.3 ± 0.7 cmH2O) and with elevated RUA (17.6 ± 2.8 cmH2O l−1 s; sub‐optimal CPAP = 8.4 ± 0.8 cmH2O). A single observer, unaware of respiratory data, identified spontaneous and tone‐induced arousals of 3–15 s duration preceded and followed by stable NREM sleep. VI was compared between CPAP levels before and after spontaneous arousal in 16 subjects with tone‐induced arousals in both conditions. During stable NREM sleep at sub‐optimal CPAP, PET,CO2 was mildly elevated (43.5 ± 0.8 versus 42.5 ± 0.8 Torr). However, baseline VI (7.8 ± 0.3 versus 8.0 ± 0.3 l min−1) was unchanged between CPAP conditions. For the first three breaths following arousal, VI was higher for sub‐optimal than optimal CPAP (first breath: 11.2 ± 0.9 versus 9.3 ± 0.6 l min−1). The magnitude of hypoventilation on return to sleep was not affected by the level of CPAP and both obstructive and central respiratory events were rare following arousal. Similar results occurred after tone‐induced arousals which led to larger responses than spontaneous arousals. VI for the first breath following arousal under optimal CPAP was greater in men than women (11.0 ± 0.4 versus 7.6 ± 0.6 l min−1). These results demonstrate that the ventilatory response to arousal is influenced by pre‐arousal airway resistance and gender. Whether this contributes to the perpetuation of respiratory events and the pathogenesis of OSA is unclear.
Mechanisms of deep brain stimulation: an intracellular study in rat thalamus
Authors: Anderson Trent , Hu Bin , Pittman Quentin , Kiss Zelma H. T.
Published: June 2004

High‐frequency deep brain stimulation (DBS) in the thalamus alleviates most kinds of tremor, yet its mechanism of action is unknown. Studies in subthalamic nucleus and other brain sites have emphasized non‐synaptic factors. To explore the mechanism underlying thalamic DBS, we simulated DBS in vitro by applying high‐frequency (125 Hz) electrical stimulation directly into the sensorimotor thalamus of adult rat brain slices. Intracellular recordings revealed two distinct types of membrane responses, both of which were initiated with a depolarization and rapid spike firing. However, type 1 responses repolarized quickly and returned to quiescent baseline during simulated DBS whereas type 2 responses maintained the level of membrane depolarization, with or without spike firing. Individual thalamic neurones exhibited either type 1 or type 2 response but not both. In all neurones tested, simulated DBS‐evoked membrane depolarization was reversibly eliminated by tetrodotoxin, glutamate receptor antagonists, and the Ca2+ channel antagonist Cd2+. Simulated DBS also increased the excitability of thalamic cells in the presence of glutamate receptor blockade, although this non‐synaptic effect induced no spontaneous firing such as that found in subthalamic nucleus neurones. Our data suggest that high‐frequency stimulation when applied in the ventral thalamus can rapidly disrupt local synaptic function and neuronal firing thereby leading to a ‘functional deafferentation’ and/or ‘functional inactivation’. These mechanisms, driven primarily by synaptic activation, help to explain the paradox that lesions, muscimol and DBS in thalamus all effectively stop tremor.
Zinc is both an intracellular and extracellular regulator of KATP channel function
Authors: Prost Anne‐Lise , Bloc Alain , Hussy Nicolas , Derand Renaud , Vivaudou Michel
Published: June 2004

Extracellular Zn2+ has been identified as an activator of pancreatic KATP channels. We further examined the action of Zn2+ on recombinant KATP channels formed with the inward rectifier K+ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn2+, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of KATP channels. External Zn2+, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn2+ dose‐dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half‐maximal effects at 1.8 and 60 μm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn2+ on the SUR subunit. Effects of internal Zn2+ were not due to Zn2+ leaking out, since they were unaffected by the presence of a Zn2+ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn2+. Therefore, Zn2+ is an endogenous KATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting KATP channels, and suggest a new role for Zn2+ as an intracellular signalling molecule.
Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells
Authors: Pushkin Alexander , Abuladze Natalia , Gross Eitan , Newman Debra , Tatishchev Sergei , Lee Ivan , Fedotoff Olga , Bondar Galyna , Azimov Rustam , Ngyuen Matt , Kurtz Ira
Published: June 2004

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C‐terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1‐ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C‐terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L958DDV and D986NDD in the wild‐type kNBC1‐ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1‐mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.
Enhanced effect of gap junction uncouplers on macroscopic electrical properties of reperfused myocardium
Authors: Rodriguez‐Sinovas Antonio , García‐Dorado David , Ruiz‐Meana Marisol , Soler‐Soler Jordi
Published: June 2004

Transient inhibition of gap junction (GJ)‐mediated communication with heptanol during myocardial reperfusion limits infarct size. However, inhibition of cell coupling in normal myocardium may be arrhythmogenic. The purpose of this study was to test the hypothesis that the consequences of GJ inhibition may be magnified in reperfused myocardium compared with normal tissue, thus allowing the inhibition of GJs in reperfused tissue while only minimally modifying overall macroscopic cell coupling in normal myocardium. Concentration–response curves were defined for the effects of heptanol, 18α‐glycyrrhetinic acid, halothane, and palmitoleic acid on conduction velocity, tissue electrical impedance, developed tension and lactate dehydrogenase (LDH) release in normoxically perfused rat hearts (n= 17). Concentrations lacking significant effects on tissue impedance were added during the initial 15 min of reperfusion in hearts submitted to 60 min (n= 43) or 30 min (n= 35) of ischaemia. These concentrations markedly increased myocardial electrical impedance (resistivity and phase angle) in myocardium reperfused after either 30 or 60 min of ischaemia, and reduced reperfusion‐induced LDH release after 1 h of ischaemia by 83.6, 57.9, 51.7 and 52.5% for heptanol, 18α‐glycyrrhetinic acid, halothane and palmitoleic acid, respectively. LDH release was minimal in hearts submitted to 30 min of ischaemia, independently of group allocation. In conclusion, the present results strongly support the hypothesis that intercellular communication in postischaemic myocardium may be effectively reduced by concentrations of GJ inhibitors affecting only minimally overall electrical impedance in normal myocardium. Reduction of cell coupling during initial reperfusion was consistently associated with attenuated lethal reperfusion injury.
Ca2+ channel regulation by transforming growth factor‐β1 and bone morphogenetic protein‐2 in developing mice myotubes
Authors: Mejia‐Luna Lizbeth , Avila Guillermo
Published: June 2004

In skeletal muscle myogenesis, precursor cells or myoblasts fuse to form multinucleated cells (myotubes), which then further develop into functional muscle. We investigated if the inhibition of myogenesis by transforming growth factor‐β1 (TGF‐β1) and bone morphogenetic protein‐2 (BMP‐2) involve regulation of voltage‐dependent Ca2+ channels. Primary cultured myoblasts were kept in fusion medium (0–6 days) in either the absence (control conditions) or the presence of 40 pm TGF‐β1 or 5 nm BMP‐2. Subsequently, the developing myotubes were transferred to a growth factor‐free recording solution, and subjected to whole cell patch‐clamp experiments. At day 0, 14% of non‐fusing myoblasts exhibited T‐current, whereas the L‐current was practically absent. Under control conditions, however, the percentage of T‐ and L‐channel‐expressing myotubes increased sharply, from 25% at day 1 to ∼100% at days 2–6. In addition, parallel increases were determined for Ca2+‐currents density and cell membrane capacitance (Cm), which is proportional to the size of myotubes. Interestingly, at days 1–2 TGF‐β1 and BMP‐2 eliminated the T‐current on initial 14% of T‐channel‐expressing myoblasts. Moreover, at day 6 the growth factors significantly reduced the maximal values of both T‐current density (80%) and Cm (60%). The effect of BMP‐2 was selective on T‐channels, whereas TGF‐β1 decreased also the L‐current density (90%). A similar reduction in maximal conductance of the Ca2+ channels was determined, in the absence of significant alterations in other essential properties of the channels, including the time course and voltage dependence of activation and inactivation. The results suggest these growth factors markedly reduce the number of functional T‐ (both TGF‐β1 and BMP‐2) and L‐channels (only TGF‐β1) in the surface of the plasma membrane, and contribute to explaining the associated effects on myogenesis.
Atypical Ca2+‐induced Ca2+ release from a sarco‐endoplasmic reticulum Ca2+‐ATPase 3‐dependent Ca2+ pool in mouse pancreatic β‐cells
Authors: Beauvois Melanie C. , Arredouani Abdelilah , Jonas Jean‐Christophe , Rolland Jean‐François , Schuit Frans , Henquin Jean‐Claude , Gilon Patrick
Published: June 2004

The contribution of Ca2+ release from intracellular stores to the rise in the free cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic β‐cells. Depolarization of β‐cells by 45 mm K+ (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca2+]c responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca2+]c peak (TCP) (40–120 s after the onset of depolarization). Simultaneous measurements of [Ca2+]c and voltage‐dependent Ca2+ current established that the TCP did not result from a larger Ca2+ current. Abolition of the TCP by thapsigargin and its absence in sarco‐endoplasmic reticulum Ca2+‐ATPase 3 (SERCA3) knockout mice show that it is caused by Ca2+ mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of β‐cells but required a rise in [Ca2+]c pointing to a Ca2+‐induced Ca2+ release (CICR). This CICR did not involve inositol 1,4,5‐trisphosphate (IP3) receptors (IP3Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified β‐cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP3R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K+, but plays little role in response to glucose in non‐obese mouse pancreatic β‐cells.
Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition
Authors: Ruiz‐Meana Marisol , Pina Pilar , Garcia‐Dorado David , Rodríguez‐Sinovas Antonio , Barba Ignasi , Miró‐Casas Elisabet , Mirabet Maribel , Soler‐Soler Jordi
Published: June 2004

Post‐ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re‐energization following 1 h of simulated ischaemia (NaCN/2‐deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin‐sensitive swelling and calcein release associated with re‐energization in rat heart mitochondria submitted to simulated ischaemia or to Ca2+ stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re‐energized cardiomyocytes that was reversed by exposure to 3 mm glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.
Ca2+ dependency of ‘Ca2+‐independent’ exocytosis in SPOC1 airway goblet cells
Authors: Rossi Andrea H. , Sears Patrick R. , Davis C. William
Published: June 2004

SPOC1 airway goblet cells secrete mucin in response to P2Y2 receptor agonists and to secretagogues, phorbol 12‐myristate 13‐acetate (PMA) and ionomycin, which mobilize elements of the phospholipase C pathway, PKC and Ca2+, respectively. Previous studies demonstrated that mucin secretion from SLO‐permeabilized, EGTA‐buffered SPOC1 cells was stimulated by PMA at low Ca2+ levels (< 0.1 μm), consistent with the notion that regulated exocytosis may occur by Ca2+‐independent pathways. We tested the alternative hypothesis that PMA‐induced mucin secretion is, in fact, a Ca2+‐dependent process under the conditions of low bulk Ca2+, one that is permitted in the typical SLO‐permeabilized cell model by the slow binding kinetics of EGTA. Both IP3 and elevated bulk Ca2+ activated mucin secretion in SPOC1 cells buffered by EGTA, suggesting that IP3 generates a local Ca2+ gradient in the vicinity of the secretory granules to the degree necessary to trigger exocytosis. BAPTA, which binds Ca2+ approximately 100‐fold faster than EGTA, diminished IP3‐induced mucin release over a range of concentrations by ≥ 69%, yet maintained an essentially normal mucin secretory response to elevated bulk Ca2+ in permeabilized SPOC1 cells. BAPTA also diminished the mucin secretory response of permeabilized cells to PMA, relative to the EGTA‐buffered control: at PMA below 30 nm, BAPTA abolished the secretory response, and at higher concentrations it was reduced significantly relative to the EGTA‐buffered controls. PMA‐induced secretion in EGTA was insensitive to heparin. These results suggest that Ca2+ is released locally during PMA‐induced exocytosis, by an IP3‐independent mechanism.
Creep Does Not Contribute to Fatigue in Bovine Trabecular Bone
Authors: Moore T. L. A. , O’Brien F. J. , Gibson L. J.
Published: June 2004

In both cortical and trabecular bone loaded in fatigue, the stress-strain loops translate along the strain axis. Previous studies have suggested that this translation is the result of creep associated with the mean stress applied in the fatigue test. In this study, we measured the residual strain (corresponding to the translation of the stress-strain loops) in fatigue tests on bovine trabecular bone and compared it to an upper bound estimate of the creep strain in each test. Our results indicate that the contribution of creep to the translation of the stress-strain loops is negligible in bovine trabecular bone. These results, combined with models for fatigue in lower density bone, suggest that that creep does not contribute to the fatigue of normal human bone. Creep may make a significant contribution to fatigue in low-density osteoporotic bone in which trabeculae have resorbed, reducing the connectivity of the trabecular structure.
A Molecular Rotor as Viscosity Sensor in Aqueous Colloid Solutions
Authors: Akers W. , Haidekker M. A.
Published: June 2004

Background: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. Method of approach: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. Results: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of −5.5% to 5.1%). Conclusions: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.
Flow-Induced Endothelial Surface Reorganization and Minimization of Entropy Generation Rate
Authors: Liu Haidong , Narusawa Uichiro
Published: June 2004

Effects of hydrodynamic shear on the shape of the endothelial surface are examined based on evaluations of the rate of entropy generation at the cell surface. A linear solution of the flow over a sinusoidally varying endothelial surface is used to evaluate the entropy generation rate on the cell surface for which measured cell dimensions are available. Both the local rate of entropy generation (equivalent to the rate of energy dissipation by viscous shear) at the peak of a cell and the total entropy generation rate over the cell surface are minimized under conditions of a constant cell surface area and a constant cell peak height; which yields horizontal cell dimensions that are close to those obtained experimentally.
Simultaneous In Vitro Measurement of Patellofemoral Kinematics and Forces
Authors: Zavatsky Amy B. , Oppold Paul T. , Price Andrew J.
Published: June 2004

This study involved the development and testing of a system for the simultaneous in vitro measurement of tibiofemoral kinematics and patellofemoral kinematics and forces. Knee motion was tracked using a Vicon 370, and patellofemoral force was measured using a six degree-of-freedom transducer based on the design of Singerman et al. Using this system, twelve knee specimens were tested in supine leg extension under a simulated quadriceps force. The comprehensive set of results corresponds well to the individual results of previous studies. The measurement system will be of value in assessing the effects of total knee arthroplasty on patellar function.
Measuring Alignment of the Hindfoot
Authors: Tuijthof Gabrielle J. M. , Herder Just L. , Scholten Peter E. , Niek van Dijk C. , Pistecky Peter V.
Published: June 2004

In subtalar arthrodesis operations, correction of the hindfoot alignment is performed in about half of the cases. To improve the quality of the operation, a measurement system was developed which reliably measures the hindfoot angle pre-, per-, and postoperatively. This device was evaluated by measuring subjects in standing weightbearing position and in prone nonweightbearing position. The results were compared with hindfoot angles constructed on posterior photographic images. The results are similar to other studies (all maximum values): intratester accuracy 1.4°, intertester accuracy 2.2°, intratester reliability 0.9, and intertester reliability 0.74. The proposed device will improve the quality of correction, because it enables peroperative measurement of hindfoot alignment.
A 2-D Model of Flow-Induced Alterations in the Geometry, Structure, and Properties of Carotid Arteries
Authors: Gleason R. L. , Taber L. A. , Humphrey J. D.
Published: June 2004

Evidence from diverse investigations suggests that arterial growth and remodeling correlates well with changes in mechanical stresses from their homeostatic values. Ultimately, therefore, there is a need for a comprehensive theory that accounts for changes in the 3-D distribution of stress within the arterial wall, including residual stress, and its relation to the mechanisms of mechanotransduction. Here, however, we consider a simpler theory that allows competing hypotheses to be tested easily, that can provide guidance in the development of a 3-D theory, and that may be useful in modeling solid-fluid interactions and interpreting clinical data. Specifically, we present a 2-D constrained mixture model for the adaptation of a cylindrical artery in response to a sustained alteration in flow. Using a rule-of-mixtures model for the stress response and first order kinetics for the production and removal of the three primary load-bearing constituents within the wall, we illustrate capabilities of the model by comparing responses given complete versus negligible turnover of elastin. Findings suggest that biological constraints may result in sub-optimal adaptations, consistent with reported observations. To build upon this finding, however, there is a need for significantly more data to guide the hypothesis testing as well as the formulation of specific constitutive relations within the model.
Finite-Element Modeling of the Hemodynamics of Stented Aneurysms
Authors: Stuhne and Gordan R. , Steinman David A.
Published: June 2004

Background. Computational fluid dynamics (CFD) simulations are used to analyze the wall shear stress distribution and flow streamlines near the throat of a stented basilar side-wall aneurysm. Previous studies of stented aneurysm flows used low mesh resolution, did not include mesh convergence analyses, and depended upon conformal meshing techniques that apply only to very artificial stent geometries. Method of Approach. We utilize general-purpose computer assisted design and unstructured mesh generation tools that apply in principle to stents and vasculature of arbitrary complexity. A mesh convergence analysis for stented steady flow is performed, varying node spacing near the stent. Physiologically realistic pulsatile simulations are then performed using the converged mesh. Results. Artifact-free resolution of the wall shear stress field on stent wires requires a node spacing of approximately 1/3 wire radius. Large-scale flow features tied to the velocity field are, however, captured at coarser resolution (nodes spaced by about one wire radius or more). Conclusions. Results are consistent with previous work, but our methods yield more detailed insights into the complex flow dynamics. However, routine applications of CFD to anatomically realistic cases still depend upon further development of dedicated algorithms, most crucially to handle geometry definition and mesh generation for complicated stent deployments.
Honey Bee Nest Thermoregulation: Diversity Promotes Stability
Authors: Jones Julia C. , Myerscough Mary R. , Graham Sonia , Oldroyd Benjamin P.
Published: June 2004

A honey bee colony is characterized by high genetic diversity among its workers, generated by high levels of multiple mating by its queen. Few clear benefits of this genetic diversity are known. Here we show that brood nest temperatures in genetically diverse colonies (i.e., those sired by several males) tend to be more stable than in genetically uniform ones (i.e., those sired by one male). One reason this increased stability arises is because genetically determined diversity in workers' temperature response thresholds modulates the hive-ventilating behavior of individual workers, preventing excessive colony-level responses to temperature fluctuations.
Mapping the Antigenic and Genetic Evolution of Influenza Virus
Authors: Smith Derek J. , Lapedes Alan S. , de Jong Jan C. , Bestebroer Theo M. , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Fouchier Ron A. M.
Published: June 2004

The antigenic evolution of influenza A (H3N2) virus was quantified and visualized from its introduction into humans in 1968 to 2003. Although there was remarkable correspondence between antigenic and genetic evolution, significant differences were observed: Antigenic evolution was more punctuated than genetic evolution, and genetic change sometimes had a disproportionately large antigenic effect. The method readily allows monitoring of antigenic differences among vaccine and circulating strains and thus estimation of the effects of vaccination. Further, this approach offers a route to predicting the relative success of emerging strains, which could be achieved by quantifying the combined effects of population level immune escape and viral fitness on strain evolution.
Human Immunodeficiency Virus Type 1 Infection of Human Brain-Derived Progenitor Cells
Authors: Lawrence Diane M. P. , Durham Linda C. , Schwartz Lynnae , Seth Pankaj , Maric Dragan , Major Eugene O.
Published: June 2004

Although cells of monocytic lineage are the primary source of human immunodeficiency virus type 1 (HIV-1) in the brain, other cell types in the central nervous system, including astrocytes, can harbor a latent or persistent HIV-1 infection. In the present study, we examined whether immature, multipotential human brain-derived progenitor cells (nestin positive) are also permissive for infection. When exposed to IIIB and NL4-3 strains of HIV-1, progenitor cells and progenitor-derived astrocytes became infected, with peak p24 levels of 100 to 500 pg/ml at 3 to 6 days postinfection. After 10 days, virus production was undetectable but could be stimulated by the addition of tumor necrosis factor alpha (TNF-α). To bypass limitations to receptor entry, we compared the fate of infection in these cell populations by transfection with the infectious HIV-1 clone, pNL4-3. Again, transfected progenitors and astrocytes produced virus for 7 days but diminished to low levels beyond 8 days posttransfection. During the nonproductive phase, TNF-α stimulated virus production from progenitors as late as 5 weeks posttransfection. Astrocytes produced 5- to 20-fold more infectious virus (27 ng of p24/106cells) than progenitors at the peak of 3 days posttransfection. Differentiation of infected progenitors toward an astrocyte phenotype increased virus production to levels consistent with infected astrocytes, suggesting a phenotypic difference in viral replication. Using this cell culture system of multipotential human brain-derived progenitor cells, we provide evidence that progenitor cells may be a reservoir for HIV-1 in the brains of AIDS patients.
Sensitivity of NS3 Serine Proteases from Hepatitis C Virus Genotypes 2 and 3 to the Inhibitor BILN 2061
Authors: Thibeault Diane , Bousquet Christiane , Gingras Rock , Lagacé Lisette , Maurice Roger , White Peter W. , Lamarre Daniel
Published: June 2004

Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (Ki, 80 to 90 nM) compared to genotype 1 enzymes (Ki, 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, withKivalues below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.
Elevated Expression of GM3 in Receptor-Bearing Targets Confers Resistance to Human Immunodeficiency Virus Type 1 Fusion
Authors: Rawat Satinder S. , Gallo Stephen A. , Eaton Julie , Martin Thomas D. , Ablan Sherimay , KewalRamani Vineet N. , Wang Ji Ming , Blumenthal Robert , Puri Anu
Published: June 2004

GM3, a major ganglioside of T lymphocytes, promotes human immunodeficiency virus type 1 (HIV-1) entry via interactions with HIV-1 receptors and the viral envelope glycoprotein (Env). Increased GM3 levels in T lymphocytes and the appearance of anti-GM3 antibodies in AIDS patients have been reported earlier. In this study, we investigated the effect of GM3 regulation on HIV-1 entry by utilizing a mouse cell line (B16F10), which expresses exceptionally high levels of GM3. Strikingly, B16 cells bearing CD4, CXCR4, and/or CCR5 were highly resistant to CD4-dependent HIV-1 Env-mediated membrane fusion. In contrast, these targets supported membrane fusion mediated by CD4-requiring HIV-2, SIV, and CD4-independent HIV-1 Envs. Coreceptor function was not impaired by GM3 overexpression as indicated by Ca
2+
fluxes mediated by the CXCR4 ligand SDF-1α and the CCR5 ligand MIP-1β. Reduction in GM3 levels of B16 target cells resulted in a significant recovery of CD4-dependent HIV-1 Env-mediated fusion. We propose that GM3 in the plasma membrane blocks HIV-1 Env-mediated fusion by interfering with the lateral association of HIV-1 receptors. Our findings offer a novel mechanism of interplay between membrane lipids and receptors by which host cells may escape viral infections.
The N-Terminal 85 Amino Acids of the
Barley Stripe Mosaic Virus
γb Pathogenesis Protein Contain Three Zinc-Binding Motifs
Authors: Bragg Jennifer N. , Lawrence Diane M. , Jackson Andrew O.
Published: June 2004

Barley stripe mosaic virus
RNAγ encodes γb, a cysteine-rich protein that affects pathogenesis. Nine of the eleven cysteines are concentrated in two clusters, designated C1 (residues 1 to 23) and C2 (residues 60 to 85), that are arranged in zinc finger-like motifs. A basic motif (BM) rich in lysine and arginine (residues 19 to 47) resides between the C1 and C2 clusters. We have demonstrated that γb binds zinc and that the C1, BM, and C2 motifs have independent zinc-binding activities. To evaluate the requirements for binding, mutations were introduced into each region. Cysteine residues at positions 7, 9, 10, 19, and 23 in the C1 motif were replaced with serines. In the BM, asparagines were substituted for lysines at positions 26 and 35, glutamine for arginine at position 25, and glycines for arginines at positions 33 and 36. The C2 mutations included cysteine replacements with serines at positions 60, 64, 71, and 81, and a histidine-to-leucine change at position 85. These mutations destroyed zinc-binding activity in each of the isolated motifs. γb derivatives containing mutations in only two of the motifs retained the ability to bind zinc, whereas a γb derivative containing mutations inactivating all three motifs destroyed the ability to bind zinc. Plants inoculated with transcripts containing combinations of the C1, BM, and C2 mutations elicited a “null” phenotype in barley characteristic of γb deletion mutants and also delayed the appearance and reduced the size of local lesions in
Chenopodium amaranticolor
. These results show that zinc binding of each of the motifs is critical for the biological activity of γb.
Human Immunodeficiency Virus Type 1-Specific Immune Responses in Primates upon Sequential Immunization with Adenoviral Vaccine Carriers of Human and Simian Serotypes
Authors: Reyes-Sandoval A. , Fitzgerald J. C. , Grant R. , Roy S. , Xiang Z. Q. , Li Y. , Gao G. P. , Wilson J. M. , Ertl H. C. J.
Published: June 2004

Two triple immunization vaccine regimens with adenoviral vectors with E1 deleted expressing Gag of human immunodeficiency virus type 1 were tested for induction of T- and B-cell-mediated-immune responses in mice and in nonhuman primates. The vaccine carriers were derived from distinct serotypes of human and simian adenoviruses that fail to elicit cross-neutralizing antibodies expected to dampen the effect of booster immunizations. Both triple immunization regimens induced unprecedented frequencies of gamma interferon-producing CD8
+
T cells to Gag in mice and monkeys that remained remarkably stable over time. In addition, monkeys developed Gag-specific interleukin-2-secreting T cells, presumably belonging to the CD4
+
T-cell subset, and antibodies to both Gag and the adenoviral vaccine carriers.
Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes
Authors: Moradpour Darius , Evans Matthew J. , Gosert Rainer , Yuan Zhenghong , Blum Hubert E. , Goff Stephen P. , Lindenbach Brett D. , Rice Charles M.
Published: June 2004

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn
7
-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides
Authors: Real Eléonore , Rain Jean-Christophe , Battaglia Véronique , Jallet Corinne , Perrin Pierre , Tordo Noël , Chrisment Peggy , D'Alayer Jacques , Legrain Pierre , Jacob Yves
Published: June 2004

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.
Exchanging the Yellow Fever Virus Envelope Proteins with Modoc Virus prM and E Proteins Results in a Chimeric Virus That Is Neuroinvasive in SCID Mice
Authors: Charlier Nathalie , Molenkamp Richard , Leyssen Pieter , Paeshuyse Jan , Drosten Christian , Panning Marcus , De Clercq Erik , Bredenbeek Peter J. , Neyts Johan
Published: June 2004

A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the “not-known vector” flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [3H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.
Structure of D-63 from Sulfolobus Spindle-Shaped Virus 1: Surface Properties of the Dimeric Four-Helix Bundle Suggest an Adaptor Protein Function
Authors: Kraft Paul , Kümmel Daniel , Oeckinghaus Andrea , Gauss George H. , Wiedenheft Blake , Young Mark , Lawrence C. Martin
Published: June 2004

Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH ≤ 4.0) hot springs (≥70°C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.