bluuebunny/crossref_metadata_2025 huggingface dataset:
FK419, a Novel Nonpeptide GPIIb/IIIa Antagonist, Restores Microvascular Patency and Improves Outcome in the Guinea-Pig Middle Cerebral Artery Thrombotic Occlusion Model: Comparison with Tirofiban
Authors: Moriguchi Akira , Maeda Masashi , Mihara Kayoko , Aoki Toshiaki , Matsuoka Nobuya , Mutoh Seitaro
Published: January 2005

The antithrombotic efficacy of FK419, a novel nonpeptide platelet glycoprotein IIb/IIIa antagonist, was compared with tirofiban in guinea-pigs. FK419 and tirofiban similarly inhibited platelet aggregation in vitro (IC50 values: 0.43 ± 0.076 and 0.41 ± 0.053 μmol/L) and dispersed aggregated platelets (EC50 values: 2.3 ± 0.88 and 2.0 ± 0.81 μmol/L). FK419 inhibited retention of platelets and neutrophils in a collagen-coated bead column with greater potency than tirofiban (IC50 values of 0.90 ± 0.133 and 2.4 ± 0.21 μmol/L for platelet retention and 0.32 ± 0.078 and 0.57 ± 0.180 μmol/L for neutrophil retention). When FK419 or tirofiban were administered after photochemically induced middle cerebral artery (MCA) occlusion in guinea-pigs, they dose-dependently improved MCA patency. FK419 reduced neurological deficits and ischemic brain damage in a dose-dependent fashion, whereas tirofiban did not. Reduced regional cerebral blood flow in the striatum gradually returned to the preoccluded level with FK419 treatment; however, no restoration was observed with tirofiban even though the MCA was recanalized. These results indicate that FK419 ameliorates ischemic brain damage by not only lysing the obstructive thrombus in MCA but also preventing or restoring microcirculation deficits after occlusion/reperfusion, suggesting that FK419 would be an attractive intervention for the treatment of ischemic stroke patients.
Analysis of Combined Treatment of Embolic Stroke in Rat with r-tPA and a GPIIb/IIIa Inhibitor
Authors: Ding Guangliang , Jiang Quan , Zhang Li , Zhang Zheng Gang , Li Lian , Knight Robert A , Ewing James R , Wang Ying , Chopp Michael
Published: January 2005

Suppression of platelet activation improves the efficacy of thrombolytic therapy for stroke. Thus, combination treatment with recombinant tissue plasminogen activator (r-tPA) and 7E3 F(ab′)2, a GPIIb/IIIa inhibitor that binds the platelet to fibrin, may improve the efficacy of thrombolytic therapy in embolic stroke. Magnetic resonance imaging (MRI) was used to monitor treatment response in rats subjected to embolic middle cerebral artery (MCA) occlusion (MCAo). Animals were randomized into treated ( n = 12) and control ( n = 10) groups and received intravenous combination therapy or saline, respectively, 4 hours after MCAo. Magnetic resonance imaging (MRI) measurements performed 1 hour after MCAo showed no difference between groups. However, an increased incidence (50%) of MCA recanalization was found in the treated group at 24 hours compared with 20% in the control group. The area of low cerebral blood flow at 24 and 48 hours was significantly smaller in the combination treatment group, and the lesion size, as indicated from the T2 and T1 maps, differed significantly between groups. Fluorescence microscopy measurements of cerebral microvessels perfused with fluorescein isothiocyanate-dextran and measurements of infarct volume revealed that the combination treatment significantly increased microvascular patency and reduced infarct volume, respectively, compared with the control rats. The efficacy of combination treatment 4 hours after ischemia is reflected by MRI indices of tissue perfusion, MCA recanalization, and reduction of lesion volume. The treatment also reduced secondary microvascular perfusion deficits.
Identification of Neuroprotective Properties of Anti-MAG Antibody: A Novel Approach for the Treatment of Stroke?
Authors: Irving Elaine A , Vinson Mary , Rosin Claudia , Roberts Jenny C , Chapman Debra M , Facci Laura , Virley David J , Skaper Stephen D , Burbidge Stephen A , Walsh Frank S , Hunter A Jacqueline , Parsons Andrew A
Published: January 2005

The inhibitory activity of myelin-associated glycoprotein (MAG) on neurons is thought to contribute to the lack of regenerative capacity of the CNS after injury. The interaction of MAG and its neuronal receptors mediates bidirectional signaling between neurons and oligodendrocytes. The novel finding that an anti-MAG monoclonal antibody not only possesses the ability to neutralise the inhibitory effect of MAG on neurons but also directly protects oligodendrocytes from glutamate-mediated oxidative stress-induced cell death is reported here. Furthermore, administration of anti-MAG antibody (centrally and systemically) starting 1 hour after middle cerebral artery occlusion in the rat significantly reduced lesion volume at 7 days. This neuroprotection was associated with a robust improvement in motor function compared with animals receiving control IgG1. Together, these data highlight the potential for the use of anti-MAG antibodies as therapeutic agents for the treatment of stroke.
FK419, a Nonpeptide Platelet Glycoprotein IIb/IIIa Antagonist, Ameliorates Brain Infarction Associated with Thrombotic Focal Cerebral Ischemia in Monkeys: Comparison with Tissue Plasminogen Activator
Authors: Maeda Masashi , Moriguchi Akira , Mihara Kayoko , Aoki Toshiaki , Takamatsu Hiroyuki , Matsuoka Nobuya , Mutoh Seitaro , Goto Toshio
Published: January 2005

The binding of platelet glycoprotein (GP) IIb/IIIa to fibrinogen is the final common pathway in platelet aggregation, a process known to play a key role in the pathogenesis of ischemic brain damage. We compared the effects of FK419, a novel nonpeptide GPIIb/IIIa antagonist, with recombinant tissue plasminogen activator (rt-PA) on middle cerebral artery (MCA) patency and ischemic brain damage in a thrombotic stroke model in squirrel monkeys. FK419 not only inhibited in vitro platelet aggregation (IC50: 88 nmol/L), but also showed disaggregatory activity to aggregated platelet (EC50: 286 nmol/L). FK419 dose-dependently reduced the time to first reperfusion and total occlusion time of MCA blood flow when administered immediately after the termination of photoirradiation. FK419 reduced cerebral infarction and ameliorated neurologic deficits with similar dose-dependency. Although rt-PA reduced the time to first reperfusion, total occlusion time, and cerebral infarction, it did not significantly ameliorate neurologic deficits and induced petechial intracerebral hemorrhages. These results indicate: (1) FK419 restored cerebral blood flow after thrombotic occlusion of MCA, (2) FK419 reduced ischemic brain injury by its thrombolytic actions in a non-human primate stroke model, and (3) FK419 has superior antithrombotic efficacy and is safer than rt-PA.
High-Resolution Blood–Brain Barrier Permeability and Blood Volume Imaging Using Quantitative Synchrotron Radiation Computed Tomography: Study on an F98 Rat Brain Glioma
Authors: Adam Jean-François , Nemoz Christian , Bravin Alberto , Fiedler Stefan , Bayat Sam , Monfraix Sylvie , Berruyer Gilles , Charvet Anne Marie , Bas Jean-François Le , Elleaume Hélène , Estève François
Published: January 2005

The authors previously provided evidence of synchrotron radiation computed tomography (SRCT) efficacy for quantitative in vivo brain perfusion measurements using monochromatic X-ray beams. However, this technique was limited for small-animal studies by partial volume effects. In this paper, high-resolution absolute cerebral blood volume and blood–brain barrier permeability coefficient measurements were obtained on a rat glioma model using SRCT and a CCD camera (47 × 47 μm2 pixel size). This is the first report of in vivo high-resolution brain vasculature parameter assessment. The work gives interesting perspectives to quantify brain hemodynamic changes accurately in healthy and pathological small animals.
MRI Reveals That Early Changes in Cerebral Blood Volume Precede Blood–Brain Barrier Breakdown and Overt Pathology in MS-like Lesions in Rat Brain
Authors: Broom Kerry A , Anthony Daniel C , Blamire Andrew M , Waters Sara , Styles Peter , Perry Victor Hugh , Sibson Nicola R
Published: January 2005

Magnetic resonance imaging (MRI) is an established clinical tool for diagnosing multiple sclerosis (MS), the archetypal central nervous system neuroinflammatory disease. In this study, we have used a model of delayed-type hypersensitivity in the rat brain, which bears many of the hallmarks of an MS lesion, to investigate the development of MRI-detectable changes before the appearance of conventional indices of lesion development. In addition, we have correlated the MRI-detectable changes with the developing histopathology. Significant increases in regional cerebral blood volume (rCBV) preceded overt changes in blood–brain barrier (BBB) permeability, T2 relaxation and the diffusion properties of tissue water. Thus, changes in rCBV might be a more sensitive indicator of lesion onset than the conventional indices used clinically in MS patients, such as contrast enhancement. In addition, we show that BBB breakdown, and consequent edema formation, are more closely correlated with astrogliosis than any other histopathologic changes, while regions of T1 and T2 hypointensity appear to reflect hypercellularity.
Neuroprotection by Osteopontin in Stroke
Authors: Meller Robert , Stevens Susan L , Minami Manabu , Cameron Jennifer A , King Sonya , Rosenzweig Holly , Doyle Kristian , Lessov Nikola S , Simon Roger P , Stenzel-Poore Mary P
Published: January 2005

Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg–Gly–Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.
Near-Infrared Fluorescent Imaging of Cerebral Thrombi and Blood–Brain Barrier Disruption in a Mouse Model of Cerebral Venous Sinus Thrombosis
Authors: Kim Dong-Eog , Jaffer Farouc A , Weissleder Ralph , Tung Ching-Hsuan , Schellingerhout Dawid
Published: January 2005

An intravital microscopy imaging method was developed to visualize active cerebral thrombus and blood–brain barrier (BBB) disruption using Near Infrared Fluorescent (NIRF) probes. A circular craniotomy was made in CD-1 mice. Thrombi were formed by applying 10%-FeCl3 to the entire exposed superior sagittal sinus (SSS, 5 mm), or to the posterior 2.5 mm of the SSS for 5 mins. Control animals were pretreated with heparin (50 U/kg) before thrombus induction. Three hours after thrombus formation, a FXIIIa-targeted NIRF imaging probe (A15) was intravenously injected, and the SSS was imaged by intravital microscopy. This was followed by injection of indocyanine green (ICG) to assess BBB permeability. The A15 optical probe bound to thrombus, and the fluorescent signal emitted by the bound agent corresponded well with histologically confirmed thrombus. A15 initially remained intravascular, followed by excretion and subsequent decrease in all tissues except for thrombus, where it was retained. The subsequent ICG was also intravascular immediately after injection, but then began to leak into the cerebral parenchyma at 3 to 5 mins. The sites of leakage were adjacent to thrombosed areas. Heparin pretreatment prevented thrombus formation and reduced ICG leakage significantly. This demonstrates the feasibility of simultaneous in vivo monitoring of thrombus and BBB permeability in an animal model of cerebral venous thrombosis.
17β-Estradiol Pretreatment Reduces Bleeding and Brain Injury after Intracerebral Hemorrhagic Stroke in Male Rats
Authors: Auriat Angela , Plahta Wayne C , McGie Steven C , Yan Reginia , Colbourne Frederick
Published: January 2005

17 β-estradiol reduces cell death after global and focal ischemia and subarachnoid hemorrhage in rodents. Presently, we tested whether estrogen improves outcome after intracerebral hemorrhage (ICH) in male rats. Rats were implanted subcutaneously with 0.05, 0.25, or 0.50 mg pellets of estrogen (21-day release) or subjected to a sham procedure. Two weeks after implantation, they were given a striatal ICH via an infusion of collagenase. The three estrogen groups had significantly smaller lesions at a 7-day survival. Some rats had core temperature measured with an implanted telemetry probe, which also measured whole-body movements. Estrogen did not affect temperature nor activity levels after ICH. A second study with 0.25 mg pellets, administered once or twice, showed persistent histologic protection (30 days) and some functional benefit (e.g., elevated beam). A spectrophotometric hemoglobin assay showed that the 0.25 mg dose significantly reduced hemorrhagic blood volume at 12 hours after ICH. Regardless, estrogen did not lessen cerebral edema at 2 days after ICH and functional benefits were not consistently found on all tests (e.g., cylinder task). In summary, estrogen pretreatment reduces injury after ICH, in part by reducing bleeding. Estrogen may thus lessen injury and improve outcome after ICH in humans.
Focal Cerebral Ischemia Preferentially Affects Neurons Distant from Their Neighboring Microvessels
Authors: Mabuchi Takuma , Lucero Jacinta , Feng Anne , Koziol James A , del Zoppo Gregory J
Published: January 2005

Developing cerebral infarction obscures the relationship of neurons to their local supply microvessels. We tested the notion that in the basal ganglia (i) an ordered relationship between neurons and their nearest neighboring microvessel exists, and (ii) focal ischemia predictably affects neuron integrity based on microvessel–neuron proximity. Distances between individual microvessels and their nearest neurons ([m–n distance]s) were measured in normal primates and ischemic subjects undergoing middle cerebral artery occlusion for 2 hours. An ordered microvessel–neuron relationship exists in the normal nonischemic basal ganglia within the early hours of focal ischemia. During ischemia normal (n) and sensitive (n∗) neurons are interspersed. On average, neurons more distant from their nearest microvessel are most sensitive ([m–n distance]=16.2±11.2 μm versus [m–n∗ distance]=22.2±13.0 μm, 2 P≪0.00000001). Neurons not expressing glutamic acid decarboxylase were more likely to be sensitive than those with a normal microvessel–neuron relationship. In contrast, the [m–n distance] distribution of injured tyrosine hydroxylase-containing neurons was similar to those without tyrosine hydroxylase. Hence, the [m–n distance] relationship in the normal and ischemic basal ganglia is highly ordered, and distant neurons are consistently perturbed by ischemia, although this is not uniformly dependent on neurotransmitter type.
Modulation of p53 Degradation via MDM2-Mediated Ubiquitylation and the Ubiquitin–Proteasome System during Reperfusion after Stroke: Role of Oxidative Stress
Authors: Saito Atsushi , Hayashi Takeshi , Okuno Shuzo , Nishi Tatsuro , Chan Pak H
Published: January 2005

The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin–proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown. We examined the MDM2 pathway and the ubiquitin–proteasome system using a transient focal cerebral ischemia (tFCI) model and analyzed the interaction between p53 regulation and superoxide using copper/zinc superoxide dismutase (SOD1) transgenic mice after tFCI. p53 degradation and ubiquitylation were detected after tFCI. The accumulation of ubiquitylated p53 was inhibited and p53 degradation was facilitated by SOD1. Nuclear translocation and MDM2/Akt interaction were detected after tFCI and were inhibited by phosphatidylinositol 3-kinase inhibition and promoted by SOD1. Cytosolic translocation of the p53/MDM2 complex was detected after tFCI and was promoted by SOD1. Moreover, accumulation of multiubiquitin chains and direct oxidative injury to a proteasome were detected and inhibited by SOD1 after tFCI. These results suggest that SOD1 promotes the MDM2 pathway and the ubiquitin–proteasome system after tFCI and that production of reactive oxygen species after tFCI prevents p53 degradation by inhibiting both systems.
Hierarchy of bifurcations in the truncated and forced nonlinear Schrödinger model
Authors: Shlizerman Eli , Rom-Kedar Vered
Published: January 2005

The truncated forced nonlinear Schrödinger (NLS) model is known to mimic well the forced NLS solutions in the regime at which only one linearly unstable mode exists. Using a novel framework in which a hierarchy of bifurcations is constructed, we analyze this truncated model and provide insights regarding its global structure and the type of instabilities which appear in it. In particular, the significant role of the forcing frequency is revealed and it is shown that a parabolic resonance mechanism of instability arises in the relevant parameter regime of this model. Numerical experiments demonstrating the different types of chaotic motion which appear in the model are provided.
Modeling return to isotropy using kinetic equations
Authors: Perot Blair , Chartrand Chris
Published: January 2005

Kinetic equations modeling the behavior of the velocity probability density function (PDF) in homogeneous anisotropic decaying turbulence are hypothesized and their implications for return-to-isotropy are investigated. Anisotropic turbulent decay is a parametrically simple but theoretically complex turbulent flow that is dominated by nonlinear interactions. The physical implications of the Bhatnagar–Gross–Krook model, a relaxation model, and the Fokker–Planck model for the “collision” term in the PDF evolution equation are analyzed in detail. Using fairly general assumptions about the physics, three different parameter-free return-to-isotropy models are proposed. These models are compared with experimental data, classical models, and analytical limits. The final model expression is particularly interesting, and can easily be implemented in classic Reynolds stress transport models.
Experimental support for Townsend’s Reynolds number similarity hypothesis on rough walls
Authors: Flack Karen A. , Schultz Michael P. , Shapiro Thomas A.
Published: January 2005

The Reynolds number similarity hypothesis of Townsend [The Structure of Turbulent Shear Flow (Cambridge University Press, Cambridge, UK, 1976)] states that the turbulence beyond a few roughness heights from the wall is independent of the surface condition. The underlying assumption is that the boundary layer thickness δ is large compared to the roughness height k. This hypothesis was tested experimentally on two types of three-dimensional rough surfaces. Boundary layer measurements were made on flat plates covered with sand grain and woven mesh roughness in a closed return water tunnel at a momentum thickness Reynolds number Reθ of ∼14000. The boundary layers on the rough walls were in the fully rough flow regime (ks+⩾100) with the ratio of the boundary layer thickness to the equivalent sand roughness height δ∕ks greater than 40. The results show that the mean velocity profiles for rough and smooth walls collapse well in velocity defect form in the overlap and outer regions of the boundary layer. The Reynolds stresses for the two rough surfaces agree well throughout most of the boundary layer and collapse with smooth wall results outside of 3ks. Higher moment turbulence statistics and quadrant analysis also indicate the differences in the rough wall boundary layers are confined to y<5ks. The present results provide support for Townsend’s Reynolds number similarity hypothesis for uniform three-dimensional roughness in flows where δ∕ks⩾40.
Richtmyer–Meshkov instability of arbitrary shapes
Authors: Mikaelian Karnig O.
Published: January 2005

We consider the effect of a shock passing through an arbitrarily shaped interface y(x,0) between two fluids. The evolution of the interface into a new shape, written formally as y(x,t)=y(x,0)+tF(x), is found by applying the linear, classical Richtmyer–Meshkov instability result to each mode in the Fourier expansion of the original interface. We provide several examples where the new shape F(x) can be found analytically. For any interface y(x,0) we define an associated dual interface ydual(x,0) and show that F(x)=dydual(x,0)∕dx. Representing a shock by a new mathematical operator we find how y(x,0),ydual(x,0), and F(x) transform under the effect of a shock. Kink-singularities are found in F(x) when and where y(x,0) has a discontinuous change in its first derivative. These are the locations where jetting occurs. We briefly discuss the effects of nonlinearity, compressibility, viscosity, etc., all of which suppress kink-singularities, and present hydrocode simulations of shock tube and high-explosive-driven experiments to highlight the influence of compressibility, nonlinearity, and material strength.
Asymptotic analysis of liquid films dip-coated onto chemically micropatterned surfaces
Authors: Davis Jeffrey M.
Published: January 2005

The dip coating of chemically heterogeneous surfaces is a useful technique for attaining selective material deposition. For the case of vertical, wetting stripes surrounded by nonwetting regions, experiments have demonstrated that the thickness of the entrained film on the stripes is significantly different than on homogeneous surfaces because of the lateral confinement of the liquid. In the present work, the asymptotic matching of equations based on lubrication theory is used to determine the film thickness, and necessary restrictions on the capillary and Bond numbers are provided. The predictions are in excellent agreement with the existing experimental data, and the classical Landau–Levich formula for homogeneous surfaces is recovered from the analysis in the limit of very wide stripes.
One-particle two-time diffusion in three-dimensional homogeneous isotropic turbulence
Authors: Osborne D. R. , Vassilicos J. C. , Haigh J. D.
Published: January 2005

A model of turbulence based on a summation of Fourier modes with an imposed turbulent energy spectrum, E(k)∼k−p, is used to investigate the characteristics of one-particle diffusion in turbulent flow. The model is described and the general Eulerian field is investigated. Using a number of Lagrangian statistical measures the results from the model are compared with laboratory experiments [N. Mordant, P. Metz, O. Michel, and J.-F. Pinton, “Measurement of Lagrangian velocity in fully developed turbulence,” Phys. Rev. Lett. 87, 214501 (2001)]. The correlation structure and spectral properties of the real and modeled fields agree well under certain time dependency conditions. The correlation signature of Lagrangian accelerations is shown to reflect the persistence of the underlying streamline structure. Intermittency may influence these correlations but is not their primary cause.
Functional compartmentation of C4 photosynthesis in the triple layered chlorenchyma of Aristida (Poaceae)
Authors: Voznesenskaya Elena V. , Chuong Simon D. X. , Koteyeva Nuria K. , Edwards Gerald E. , Franceschi Vincent R.
Published: January 2005

The genus Aristida (Poaceae), is composed of species that have Kranz anatomy and C4 photosynthesis. Kranz anatomy typically consists of two photosynthetic cell types: a layer of mesophyll cells where atmospheric CO2 is fixed into C4 acids, and an internal, chlorenchymatous vascular bundle sheath to which C4 acids are transferred and then decarboxylated to donate CO2 to the C3 cycle. The anatomy of Aristida species is unusual as it has three distinct layers of chlorenchyma cells surrounding the vascular tissue: an inner bundle sheath, an outer bundle sheath and the mesophyll cells. In this study of Aristida purpurea Nutt. var. longiseta, the functions of the three layers of chlorenchyma cells relative to the C4 photosynthetic mechanism were determined using ultrastructural analysis, western blots, immunolocalisation of photosynthetic enzymes and starch histochemistry. The results indicate that mesophyll cells contain high levels of phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK), and function to capture CO2 in the C4 cycle. The inner bundle sheath, which is high in Rubisco and contains NADP-malic enzyme and glycine decarboxylase, functions to transfer CO2 to the C3 cycle through decarboxylation of C4 acids and by decarboxylation of glycine in the glycolate pathway. The outer chlorenchymatous sheath is where ADPG pyrophosphorylase is mainly located, and this cell layer functions as the primary site of starch storage. The outer sheath, which has low levels of Rubisco and PEPC, may also have a role in refixation of any CO2 that leaks from the inner bundle sheath cells.
Cross-modal integration in a dart-poison frog
Authors: Narins Peter M. , Grabul Daniela S. , Soma Kiran K. , Gaucher Philippe , Hödl Walter
Published: January 2005

The mechanisms by which the brain binds together inputs from separate sensory modalities to effect a unified percept of events are poorly understood. This phenomenon was studied in males of the dart-poison frogEpipedobates femoralis. These animals physically and vigorously defend their territories against conspecific calling intruders. In prior field studies with an electromechanical model frog, we were able to experimentally evoke this aggressive behavior only when an auditory cue (advertisement call) was presented simultaneously with a visual cue (vocal-sac pulsations). In the present field experiments, we used a modified version of the electromechanical model frog to present territorial males with visual and auditory cues separated by experimentally introduced temporal delays or spatial disparities to probe temporal and spatial integration in this animal. In temporal integration experiments, bimodal stimuli with temporal overlap during calling bouts consistently evoked aggressive behavior; stimuli lacking bimodal temporal overlap were relatively ineffective at the same task. In spatial integration studies, despite presenting the components of the bimodal stimulus with an initial spatial disparity of up to 12 cm, fighting behavior persisted. These results demonstrate that temporal and spatial integration may be reliably estimated in a freely behaving animal in its natural habitat and that we can use aggressive behavior in this species as an index of cross-modal integration in the field.
Assignment of an essential role for the
Neurospora frequency
gene in circadian entrainment to temperature cycles
Authors: Pregueiro Antonio M. , Price-Lloyd Nathan , Bell-Pedersen Deborah , Heintzen Christian , Loros Jennifer J. , Dunlap Jay C.
Published: January 2005

Circadian systems include slave oscillators and central pacemakers, and the cores of eukaryotic circadian clocks described to date are composed of transcription and translation feedback loops (TTFLs). In the model system
Neurospora
, normal circadian rhythmicity requires a TTFL in which a White Collar complex (WCC) activates expression of the
frequency
(
frq
) gene, and the FRQ protein feeds back to attenuate that activation. To further test the centrality of this TTFL to the circadian mechanism in
Neurospora
, we used low-amplitude temperature cycles to compare WT and
frq
-null strains under conditions in which a banding rhythm was elicited. WT cultures were entrained to these temperature cycles. Unlike those normal strains, however,
frq
-null mutants did not truly entrain to the same cycles. Their peaks and troughs always occurred in the cold and warm periods, respectively, strongly suggesting that the rhythm in
Neurospora
lacking
frq
function simply is driven by the temperature cycles. Previous reports suggested that a FRQ-less oscillator (FLO) could be entrained to temperature cycles, rather than being driven, and speculated that the FLO was the underlying circadian-rhythm generator. These inferences appear to derive from the use of a phase reference point affected by both the changing waveform and the phase of the oscillation. Examination of several other phase markers as well as results of additional experimental tests indicate that the FLO is, at best, a slave oscillator to the TTFL, which underlies circadian rhythm generation in
Neurospora
.
Ab initio
simulations of protein-folding pathways by molecular dynamics with the united-residue model of polypeptide chains
Authors: Liwo Adam , Khalili Mey , Scheraga Harold A.
Published: January 2005

We report the application of Langevin dynamics to the physics-based united-residue (UNRES) force field developed in our laboratory. Ten trajectories were run on seven proteins [PDB ID codes 1BDD (α; 46 residues), 1GAB (α; 47 residues), 1LQ7 (α; 67 residues), 1CLB (α; 75 residues), 1E0L (β; 28 residues), and 1E0G (α+β; 48 residues), and 1IGD (α+β; 61 residues)] with the UNRES force field parameterized by using our recently developed method for obtaining a hierarchical structure of the energy landscape. All α-helical proteins and 1E0G folded to the native-like structures, whereas 1IGD and 1E0L yielded mostly nonnative α-helical folds although the native-like structures are lowest in energy for these two proteins, which can be attributed to neglecting the entropy factor in the current parameterization of UNRES. Average folding times for successful folding simulations were of the order of nanoseconds, whereas even the ultrafast-folding proteins fold only in microseconds, which implies that the UNRES time scale is approximately three orders of magnitude larger than the experimental time scale because the fast motions of the secondary degrees of freedom are averaged out. Folding with Langevin dynamics required 2–10 h of CPU time on average with a single AMD Athlon MP 2800+ processor depending on the size of the protein. With the advantage of parallel processing, this process leads to the possibility to explore thousands of folding pathways and to predict not only the native structure but also the folding scenario of a protein together with its quantitative kinetic and thermodynamic characteristics.
Targeted mutagenesis using zinc-finger nucleases in
Arabidopsis
Authors: Lloyd Alan , Plaisier Christopher L. , Carroll Dana , Drews Gary N.
Published: January 2005

Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the
Arabidopsis
genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1–52 bp (median of 4 bp), 14 (13%) were simple insertions of 1–4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.
Chemical peristalsis
Authors: Astumian R. Dean
Published: January 2005

Molecules that emulate in part the remarkable capabilities of protein motors were recently chemically synthesized. A promising approach is based on physically interlocked macromolecular complexes such as rotaxanes and catenanes. Using the latter, Leigh
et al
. [Leigh, D. A., Wong, J. K. Y., Dehez, F. & Zerbetto, F. (2003)
Nature
424, 174–179] constructed a molecular rotor in which two small rings are induced by pulses of light to move unidirectionally around a third, larger ring. The mechanism is similar to that by which a peristaltic pump operates. Unlike macroscopic peristalsis, however, in which a traveling wave forces material through a series of one-way valves, the chemical peristaltic mechanism does not directly cause the small rings to move but only alters the energetics, with the motion itself arising by thermal activation over energy barriers. Engines operating by this mechanism are “Brownian” motors. Here we describe a minimal two-state mechanism for a catenane-based molecular motor. Although fluctuations caused by equilibrium processes cannot drive directed motion, nonequilibrium fluctuations, whether generated externally or by a far-from-equilibrium chemical reaction, can drive rotation even against an external torque. We discuss a possible architecture for input and output of information and energy between the motor and its environment and give a simple expression for the maximum thermodynamic efficiency. The proposed Brownian motor mechanism is consistent with the high efficiency observed by Yasuda
et al
. [Yasuda, Y., Noji, H., Kinoshita, K. & Yoshida, M. (1998)
Cell
93, 1117–1124] for the F
1
-ATP synthase operating as an ATP-powered molecular rotor.
Supernumerary neuromasts in the posterior lateral line of zebrafish lacking peripheral glia
Authors: López-Schier Hernán , Hudspeth A. J.
Published: January 2005

The lateral line and its associated sensory nerves develop from cephalic epithelial thickenings called neurogenic placodes. In the zebrafish, the transcription factor neurogenin 1 is essential for the generation of the sensory ganglion from the placode, but is dispensable for the migration of the primordium and the initial development of neuromasts. We find that inactivation of the gene encoding neurogenin 1 leads to the development of over twice the normal number of neuromasts along the posterior lateral line of zebrafish larvae. Mutation of the gene encoding another transcription factor, sox10, has a similar effect. After a normal number of proneuromasts is initially deposited by the migrating primordia, interneuromast cells divide and differentiate to form the extra neuromasts. Our results indicate that the development of these intercalary neuromasts occurs principally because of the absence of neural crest-derived peripheral glia, which evidently inhibit the assembly of interneuromast cells into neuromasts.
Glucose and fat metabolism in adipose tissue of acetyl-CoA carboxylase 2 knockout mice
Authors: Oh WonKeun , Abu-Elheiga Lutfi , Kordari Parichher , Gu Zeiwei , Shaikenov Tattym , Chirala Subrahmanyam S. , Wakil Salih J.
Published: January 2005

Acc2
-/-
mutant mice, when fed a high-fat/high-carbohydrate (HF/HC) diet, were protected against diet-induced obesity and diabetes. To investigate the role of acetyl-CoA carboxylase 2 (ACC2) in the regulation of energy metabolism in adipose tissues, we studied fatty acid and glucose oxidation in primary cultures of adipocytes isolated from wild-type and
Acc2
-/-
mutant mice fed either normal chow or a HF/HC diet. When fed normal chow, oxidation of [
14
C]palmitate in adipocytes of
Acc2
-/-
mutant mice was ≈80% higher than in adipocytes of WT mice, and it remained significantly higher in the presence of insulin. Interestingly, in addition to increased fatty acid oxidation, we also observed increased glucose oxidation in adipocytes of
Acc2
-/-
mutant mice compared with that of WT mice. When fed a HF/HC diet for 4-5 months, adipocytes of
Acc2
-/-
mutant mice maintained a 25% higher palmitate oxidation and a 2-fold higher glucose oxidation than WT mice. The mRNA level of glucose transporter 4 (GLUT4) decreased several fold in the adipose tissue of WT mice fed a HF/HC diet; however, in the adipose tissue of
Acc2
-/-
mutant mice, it was 7-fold higher. Moreover, lipolysis activity was higher in adipocytes of
Acc2
-/-
mutant mice compared with that in WT mice. These findings suggest that continuous fatty acid oxidation in the adipocytes of
Acc2
-/-
mutant mice, combined with a higher level of glucose oxidation and a higher rate of lipolysis, are major factors leading to efficient maintenance of insulin sensitivity and leaner
Acc2
-/-
mutant mice.
Potentiation of the ligand-binding activity of integrin α3β1 via association with tetraspanin CD151
Authors: Nishiuchi Ryoko , Sanzen Noriko , Nada Shigeyuki , Sumida Yasuhiro , Wada Yoshinao , Okada Masato , Takagi Junichi , Hasegawa Hitoshi , Sekiguchi Kiyotoshi
Published: January 2005

CD151, one of the tetraspanins, forms a stable complex with integrin α3β1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin α3β1–CD151 complexes, was capable of dissociating CD151 from integrin α3β1, thereby allowing us to deplete CD151 from purified integrin α3β1–CD151 complexes. The CD151-free integrin α3β1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin α3β1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated β1-containing integrins. These results raised the possibility that the association of integrin α3β1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin α3β1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin α3β1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin α3β1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.
The position of Hippopotamidae within Cetartiodactyla
Authors: Boisserie Jean-Renaud , Lihoreau Fabrice , Brunet Michel
Published: January 2005

The origin of late Neogene Hippopotamidae (Artiodactyla) involves one of the most serious conflicts between comparative anatomy and molecular biology: is Artiodactyla paraphyletic? Molecular comparisons indicate that Cetacea should be the modern sister group of hippos. This finding implies the existence of a fossil lineage linking cetaceans (first known in the early Eocene) to hippos (first known in the middle Miocene). The relationships of hippos within Artiodactyla are challenging, and the immediate affinities of Hippopotamidae have been studied by biologists for almost two centuries without resolution. Here, we compare opposing hypotheses implicating several “suiform” families. This morphological analysis of a comprehensive set of taxa and characters offers a robust solution to the origins of Hippopotamidae. This family appears to be deeply nested within the otherwise extinct artiodactyl family Anthracotheriidae, most precisely within the most advanced selenodont forms. The proposed sister group of hippos is the middle to late Miocene African semiaquaticLibycosaurus. Any close relationships of hippos with suoids, particularly with Tayassuidae, are rejected. Furthermore, the clade (Hippopotamidae, Anthracotheriidae) is proposed as the sister group of the Cetacea, offering broad morphological support for a molecular phylogeny, such support being also consistent with the fossil record. Corroboration of this relationship requires an exploration of anthracothere affinities with other Paleogene artiodactyls. Among those, the position of Ruminantia is a central question, still to be solved. Further progress in this debate is likely to come from morphological studies of paleontological data, whether known or still to be discovered.
A targeted mutation in Cacng4 exacerbates spike-wave seizures in stargazer (Cacng2) mice
Authors: Letts Verity A. , Mahaffey Connie L. , Beyer Barbara , Frankel Wayne N.
Published: January 2005

The voltage-dependent calcium channel γ4 subunit protein, CACNG4, is closely related to the γ2 subunit, CACNG2. Both are expressed primarily in the brain and share 53% amino acid identity. The
Cacng2
gene is disrupted in the stargazer mouse, with its distinctive phenotype including ataxia, frequent absence seizure episodes, and head elevation. A disruption within
Cacng4
was engineered to assess its particular function. The homozygous
Cacng4
-targeted mutant mouse appeared normal with no ataxic gait or absence seizures, suggesting that other members of the γ subunit family might functionally compensate for the absence of CACNG4. To test this hypothesis, the targeted
Cacng4
mutation was combined with alleles of
Cacng2
. Absence seizures were observed in combination with the stargazer 3J mutation, which itself does not have seizures, and increased seizure activity was observed in combination with the waggler allele. Furthermore, within the corticothalamic loop, where absence seizures arise, CACNG4 expression is restricted to the thalamus. Our studies show that the CACNG4 protein has seizure suppressing activity, but this effect is revealed only when CACNG2 expression is also compromised, suggesting that CACNG subunits have
in vivo
overlapping functions.
Inositol 1,4,5-trisphosphate receptor/GAPDH complex augments Ca
2+
release via locally derived NADH
Authors: Patterson Randen L. , van Rossum Damian B. , Kaplin Adam I. , Barrow Roxanne K. , Snyder Solomon H.
Published: January 2005

NADH regulates the release of calcium from the endoplasmic reticulum by modulation of inositol 1,4,5-trisphosphate receptors (IP
3
R), accounting for the augmented calcium release of hypoxic cells. We report selective binding of IP
3
R to GAPDH, whose activity leads to the local generation of NADH to regulate intracellular calcium signaling. This interaction requires cysteines 992 and 995 of IP
3
R and C150 of GAPDH. Addition of native GAPDH and NAD
+
to WT IP
3
R stimulates calcium release, whereas no stimulation occurs with C992S/995S IP
3
R that cannot bind GAPDH. Thus, the IP
3
R/GAPDH interaction likely enables cellular energy dynamics to impact calcium signaling.
Progress in Separating Plastic Materials for Recycling
Authors: Dodbiba G. , Fujita T.
Published: January 2005

In order to deal with the problems facing the plastics processing industry research, work is focused primarily on designing, developing and testing a variety of separation and sorting techniques able to recover plastics from wastes, which can be re‐used or re‐processed to form new products. In this regard, technologies developed in mineral processing can be of great help. Various techniques for separating plastics materials have been recently developed. These techniques can be divided in two main categories, i.e. wet separating techniques and dry separating techniques. This paper presents the recent progress in separating plastics reviewing the potential of the available techniques.
Densimetric Separation of Coal Using Magnetic Fluids
Authors: Svoboda J.
Published: January 2005

Ferrohydrostatic separation of particles in magnetic fluids is a sink‐float technique based on generalized Archimedes law whereby, in addition to the conventional force of gravity, a magnetically induced force also acts on the fluid. This additional magnetic pull creates a magnetically induced buoyancy force on a particle immersed in the fluid. This buoyancy force can be accurately controlled over a wide range and particles as dense as 20 000 kg/m3 can be made to float. The selectivity of separation is very high and mixtures of particles with a density differential as small as 30 kg/m3 can be separated. As a result of the ability of FHS to separate material into narrow, well‐defined density fractions, the technique is particularly suitable for densimetric analysis. Sink–float analysis of coal has been one of the areas of potential application of FHS. The cost of heavy liquids used for coal fractionation, and of their subsequent recovery and disposal are considerable and motivation to use FHS has therefore been strong. Numerous laboratory and pilot‐plant scale tests have been conducted in this direction, particularly in Eastern Europe and Japan. Investigation of ash and sulphur content, and the mass yield into density fractions, confirmed that heavy liquid and FHS techniques are very similar in their performance. Very selective FHS separators with automated density control have recently been designed and built. A technique for the recovery of ferrofluids has been developed and low‐cost ferrofluids are being manufactured on a production scale. Technology for accurate, environmentally friendly hands‐off densimetric analysis of coal by is thus available
Switching the Sorting Mode of Membrane Proteins from Cotranslational Endoplasmic Reticulum Targeting to Posttranslational Mitochondrial Import
Authors: Miyazaki Emi , Kida Yuichiro , Mihara Katsuyoshi , Sakaguchi Masao
Published: January 2005

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.