bluuebunny/crossref_metadata_2025 huggingface dataset:
Timing of the initial muscle biopsy does not affect the measured muscle protein fractional synthesis rate during basal, postabsorptive conditions
Authors: Smith Gordon I. , Villareal Dennis T. , Lambert Charles P. , Reeds Dominic N. , Mohammed B. Selma , Mittendorfer Bettina
Published: November 2009

The muscle protein fractional synthesis rate (FSR) is determined by monitoring the incorporation of an amino acid tracer into muscle protein during a constant-rate intravenous tracer infusion. Commonly two sequential muscle biopsies are obtained some time after starting the tracer infusion. However, other protocols, including those with an initial biopsy before starting the tracer infusion to measure the background enrichment and those with only a single biopsy after several hours of tracer infusion have been used. To assess the validity of these approaches, we compared the muscle protein FSR obtained by calculating the difference in [ ring-2H5]phenylalanine and [5,5,5-2H3]leucine incorporation into muscle protein at ∼3.5 h after starting the tracer infusion and 1) at 60 min; 2) before starting the tracer infusion (background enrichment); 3) a population average muscle protein background enrichment; and 4) by measuring the tracer incorporation into muscle protein at ∼3.5 h assuming essentially no background enrichment. Irrespective of the tracer used, the muscle protein FSR calculated from the difference in the muscle protein labeling several hours after starting the tracer infusion and either the labeling at 60 min or the background enrichment were not different (e.g., 0.049 ± 0.007%/h vs. 0.049 ± 0.007%/h, respectively, with [2H5]phenylalanine; P = 0.99). However, omitting the initial biopsy and assuming no background enrichment yielded average FSR values that were ∼15% (with [2H5]phenylalanine) to 80% (with [2H3]leucine) greater ( P ≤ 0.059); using a population average background enrichment reduced the difference to ∼3% ( P = 0.76) and 22% ( P = 0.52) with [2H5]phenylalanine and [2H3]leucine, respectively. We conclude that during basal, postabsorptive conditions, valid muscle protein FSR values can be obtained irrespective of the timing of the initial biopsy so long as the protein labeling in two sequential biopsies is measured whereas the single biopsy approach should be avoided.
Diagrammatic analysis of the respiratory action of the diaphragm
Authors: Wilson Theodore A. , De Troyer Andre
Published: November 2009

During isolated phrenic nerve stimulation, the muscles of the diaphragm shorten by 40–50% of their optimal length, and the force in the muscle and transdiaphragmatic pressure (Pdi) depend on the final muscle length. The muscle shortening depends on the load imposed on the diaphragm by pleural and abdominal pressures during a particular maneuver. The mechanics of the interaction between the diaphragm and the load is well understood, but the force-length properties of the diaphragm are nonlinear, and an algebraic analysis of the interaction is clumsy. Here we describe a graphical analysis of the interaction. The variable muscle length is transformed into an equivalent variable, i.e., volume displaced by the diaphragm (Vdi), to obtain the characteristic line for the diaphragm, a graph of Pdi vs. Vdi for a given level of activation. The load is described by the same variables. Therefore, load lines can be drawn on the same graph, and the equilibrium point for the diaphragm is given by the intersection of the load line with the characteristic line of the diaphragm. Graphical analyses of the volume dependence of the respiratory effects of diaphragm and intercostal muscle activation and for the interaction between them are shown.
Carbohydrate exerts a mild influence on fluid retention following exercise-induced dehydration
Authors: Osterberg Kristin L. , Pallardy Shannon E. , Johnson Richard J. , Horswill Craig A.
Published: November 2009

Rapid and complete rehydration, or restoration of fluid spaces, is important when acute illness or excessive sweating has compromised hydration status. Many studies have investigated the effects of graded concentrations of sodium and other electrolytes in rehydration solutions; however, no study to date has determined the effect of carbohydrate on fluid retention when electrolyte concentrations are held constant. The purpose of this study was to determine the effect of graded levels of carbohydrate on fluid retention following exercise-induced dehydration. Fifteen heat-acclimatized men exercised in the heat for 90 min with no fluid to induce 2–3% dehydration. After a 30-min equilibration period, they received, over the course of 60 min, one of five test beverages equal to 100% of the acute change in body mass. The experimental beverages consisted of a flavored placebo with no electrolytes (P), placebo with electrolytes (P + E), 3%, 6%, and 12% carbohydrate solutions with electrolytes. All beverages contained the same type and concentration of electrolytes (18 meq/l Na+, 3 meq/l K+, 11 meq/l Cl−). Subjects voided their bladders at 60, 90, 120, 180, and 240 min, and urine specific gravity and urine volume were measured. Blood samples were taken before exercise and 30, 90, 180, and 240 min following exercise and were analyzed for glucose, sodium, hemoglobin, hematocrit, renin, aldosterone, and osmolality. Body mass was measured before and after exercise and a final body mass was taken at 240 min. There were no differences in percent dehydration, sweat loss, or fluid intake between trials. Fluid retention was significantly greater for all carbohydrate beverages compared with P (66.3 ± 14.4%). P + E (71.8 ± 9.9%) was not different from water, 3% (75.4 ± 7.8%) or 6% (75.4 ± 16.4%) but was significantly less than 12% (82.4 ± 9.2%) retention of the ingested fluid. No difference was found between the carbohydrate beverages. Carbohydrate at the levels measured exerts a mild influence on fluid retention in postexercise recovery.
Role of Supplementary Eye Field in Saccade Initiation: Executive, Not Direct, Control
Authors: Stuphorn Veit , Brown Joshua W. , Schall Jeffrey D.
Published: November 2009

The goal of this study was to determine whether the activity of neurons in the supplementary eye field (SEF) is sufficient to control saccade initiation in macaque monkeys performing a saccade countermanding (stop signal) task. As previously observed, many neurons in the SEF increase the discharge rate before saccade initiation. However, when saccades are canceled in response to a stop signal, effectively no neurons with presaccadic activity display discharge rate modulation early enough to contribute to saccade cancellation. Moreover, SEF neurons do not exhibit a specific threshold discharge rate that could trigger saccade initiation. Yet, we observed more subtle relations between SEF activation and saccade production. The activity of numerous SEF neurons was correlated with response time and varied with sequential adjustments in response latency. Trials in which monkeys canceled or produced a saccade in a stop signal trial were distinguished by a modest difference in discharge rate of these SEF neurons before stop signal or target presentation. These findings indicate that neurons in the SEF, in contrast to counterparts in the frontal eye field and superior colliculus, do not contribute directly and immediately to the initiation of visually guided saccades. However the SEF may proactively regulate saccade production by biasing the balance between gaze-holding and gaze-shifting based on prior performance and anticipated task requirements.
Origins of Cortical Layer V Surround Receptive Fields in the Rat Barrel Cortex
Authors: Wright Nicholas , Fox Kevin
Published: November 2009

Layer IV of the barrel cortex contains an anatomical map of the contralateral whisker pad, which serves as a useful reference in relating receptive field properties of cells to the cortical columns in which they reside. Recent studies have shown that the degree to which the surround receptive fields of layer IV cells are generated intracortically or subcortically depends on whether they lie in barrel or septal columns. To investigate whether this is true for layer V cells, we blocked intracortical activity in the barrel cortex by infusing muscimol from the cortical surface and measuring spike responses to sensory stimulation in the presence of locally iontophoresed bicuculline. Layer V cells beneath barrels had small subcortically generated single- or double-whisker center receptive fields and larger intracortically generated six to seven whisker surround receptive fields. Conversely, septally located cells received multiwhisker input from both subcortical and cortical sources. Most properties of layer Va and layer Vb cells were very similar. However, layer Vb barrel neurons showed a relative lack of phasic inhibition evoked from sensory input compared with layer Va cells. The direct thalamic input to the layer V cells was not sufficient to evoke a sensory response in the absence of input from superficial layers. These findings suggest that despite the apparent overt similarity of layer V receptive fields, barrel and septal subdivisions process different sources of information within the barrel cortex.
Global Gene Expression Analysis of Rodent Motor Neurons Following Spinal Cord Injury Associates Molecular Mechanisms With Development of Postinjury Spasticity
Authors: Wienecke J. , Westerdahl A-C. , Hultborn H. , Kiehn O. , Ryge J.
Published: November 2009

Spinal cord injury leads to severe problems involving impaired motor, sensory, and autonomic functions. After spinal injury there is an initial phase of hyporeflexia followed by hyperreflexia, often referred to as spasticity. Previous studies have suggested a relationship between the reappearance of endogenous plateau potentials in motor neurons and the development of spasticity after spinalization. To unravel the molecular mechanisms underlying the increased excitability of motor neurons and the return of plateau potentials below a spinal cord injury we investigated changes in gene expression in this cell population. We adopted a rat tail-spasticity model with a caudal spinal transection that causes a progressive development of spasticity from its onset after 2 to 3 wk until 2 mo postinjury. Gene expression changes of fluorescently identified tail motor neurons were studied 21 and 60 days postinjury. The motor neurons undergo substantial transcriptional regulation in response to injury. The patterns of differential expression show similarities at both time points, although there are 20% more differentially expressed genes 60 days compared with 21 days postinjury. The study identifies targets of regulation relating to both ion channels and receptors implicated in the endogenous expression of plateaux. The regulation of excitatory and inhibitory signal transduction indicates a shift in the balance toward increased excitability, where the glutamatergic N-methyl-d-aspartate receptor complex together with cholinergic system is up-regulated and the γ-aminobutyric acid type A receptor system is down-regulated. The genes of the pore-forming proteins Cav1.3 and Nav1.6 were not up-regulated, whereas genes of proteins such as nonpore-forming subunits and intracellular pathways known to modulate receptor and channel trafficking, kinetics, and conductivity showed marked regulation. On the basis of the identified changes in global gene expression in motor neurons, the present investigation opens up for new potential targets for treatment of motor dysfunction following spinal cord injury.
Conditioned Eyelid Movement Is not a Blink
Authors: Schade Powers Alice , Coburn-Litvak Pamela , Evinger Craig
Published: November 2009

Based on kinematic properties and distinct substrates, there are different classes of eyelid movement described as eyeblinks. We investigate whether the eyelid movements made in response to a conditioned stimulus (CS) are a category of eyelid movements distinct from blinks. Human subjects received 60 trials of classical eyelid conditioning with a tone as the CS and electrical stimulation of the supraorbital branch of the trigeminal nerve as the unconditioned stimulus (UCS). Before and after training, reflex blinks were elicited with the UCS. The kinematics of conditioned responses (CRs) differed significantly from those of reflex blinks. The slope of the amplitude-maximum velocity function was steeper for reflex blinks than for CRs, and reflex blink duration was significantly shorter than CR duration. Unlike reflex blinks, for which maximum velocity was independent of blink duration, the maximum velocity of CRs depended on CR duration. These quantitative and qualitative differences indicated that CRs were a unique class of eyelid movements distinct from blinks and eyelid movements with vertical saccadic gaze shifts.
Circadian Regulation of A-Type Potassium Currents in the Suprachiasmatic Nucleus
Authors: Itri Jason N. , Vosko Andrew M. , Schroeder Analyne , Dragich Joanna M. , Michel Stephan , Colwell Christopher S.
Published: November 2009

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN) of the hypothalamus. Understanding the ionic mechanisms underlying these rhythms is an important goal of research in chronobiology. Previous work has shown that SCN neurons express A-type potassium currents (IAs), but little is known about the properties of this current in the SCN. We sought to characterize some of these properties, including the identities of IA channel subunits found in the SCN and the circadian regulation of IA itself. In this study, we were able to detect significant hybridization for Shal-related family members 1 and 2 (Kv4.1 and 4.2) within the SCN. In addition, we used Western blot to show that the Kv4.1 and 4.2 proteins are expressed in SCN tissue. We further show that the magnitude of the IA current exhibits a diurnal rhythm that peaks during the day in the dorsal region of the mouse SCN. This rhythm seems to be driven by a subset of SCN neurons with a larger peak current and a longer decay constant. Importantly, this rhythm in neurons in the dorsal SCN continues in constant darkness, providing an important demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. We conclude that the anatomical expression, biophysical properties, and pharmacological profiles measured are all consistent with the SCN IA current being generated by Kv4 channels. Additionally, these data suggest a role for IA in the regulation of spontaneous action potential firing during the transitions between day/night and in the integration of synaptic inputs to SCN neurons throughout the daily cycle.
Ca2+ Dependence of the Binomial Parameters p and n at the Mouse Neuromuscular Junction
Authors: Wang Xueyong , Pinter Martin J. , Rich Mark M.
Published: November 2009

The Ca2+ dependence of synaptic quantal release is generally thought to be restricted to probability of vesicular release. However, some studies have suggested that the number of release sites ( n) at the neuromuscular junction (NMJ) is also Ca2+ dependent. In this study, we recorded endplate currents over a wide range of extracellular Ca2+ concentrations and found the expected Ca2+ dependency of release. A graphical technique was used to estimate p (probability of release) and n using standard binomial assumptions. The results suggested n was Ca2+ dependent. The data were simulated using compound binomial statistics with variable n (Ca2+ dependent) or fixed n (Ca2+ independent). With fixed n, successful simulation of increasing Ca2+ required that p increase abruptly at some sites from very low to high values. Successful simulation with variable n required the introduction of previously silent release sites ( p = 0) with high values of p. Thus the success of both simulations required abrupt, large increases of p at a subset of release sites with initially low or zero p. Estimates of the time course of release obtained by deconvolving evoked endplate currents with average miniature endplate currents decreased slightly as Ca2+ increased, thus arguing against sequential release of multiple quanta at higher Ca2+ levels. Our results suggest that the apparent Ca2+ dependence of n at the NMJ can be explained by an underlying Ca2+ dependence of a spatially variable p such that p increases abruptly at a subset of sites as Ca2+ is increased.
Functional Interactions Within the Parahippocampal Region Revealed by Voltage-Sensitive Dye Imaging in the Isolated Guinea Pig Brain
Authors: Biella Gerardo , Spaiardi Paolo , Toselli Mauro , de Curtis Marco , Gnatkovsky Vadym
Published: November 2009

The massive transfer of information from the neocortex to the entorhinal cortex (and vice versa) is hindered by a powerful inhibitory control generated in the perirhinal cortex. In vivo and in vitro experiments performed in rodents and cats support this conclusion, further extended in the present study to the analysis of the interaction between the entorhinal cortex and other parahippocampal areas, such as the postrhinal and the retrosplenial cortices. The experiments were performed in the in vitro isolated guinea pig brain by a combined approach based on electrophysiological recordings and fast imaging of optical signals generated by voltage-sensitive dyes applied to the entire brain by arterial perfusion. Local stimuli delivered in different portions of the perirhinal, postrhinal, and retrosplenial cortex evoked local responses that did not propagate to the entorhinal cortex. Neither high- and low-frequency-patterned stimulation nor paired associative stimuli facilitated the propagation of activity to the entorhinal region. Similar stimulations performed during cholinergic neuromodulation with carbachol were also ineffective in overcoming the inhibitory network that controls propagation to the entorhinal cortex. The pharmacological inactivation of GABAergic transmission by local application of bicuculline (1 mM) in area 36 of the perirhinal cortex facilitated the longitudinal (rostrocaudal) propagation of activity into the perirhinal/postrhinal cortices but did not cause propagation into the entorhinal cortex. Bicuculline injection in both area 35 and medial entorhinal cortex released the inhibitory control and allowed the propagation of the neural activity to the entorhinal cortex. These results demonstrate that, as for the perirhinal-entorhinal reciprocal interactions, also the connections between the postrhinal/retrosplenial cortices and the entorhinal region are subject to a powerful inhibitory control.
Modulation of Spontaneous and GABA-Evoked Tonic α4β3δ and α4β3γ2L GABAAReceptor Currents by Protein Kinase A
Authors: Tang Xin , Hernandez Ciria C. , Macdonald Robert L.
Published: November 2009

Protein kinase A (PKA) has been reported to regulate synaptic αβγ γ-aminobutyric acid type A (GABAA) receptor currents, but whether PKA regulates GABAAreceptor peri- and extrasynaptic tonic currents is unknown. GABAAreceptors containing α4 subunits are important in mediating tonic inhibition and exist as both α4βδ and α4βγ receptors in the brain. To mimic GABA-independent and GABA-dependent tonic currents, we transfected HEK 293T cells with α4β3δ or α4β3γ2L subunits and recorded spontaneous currents in the absence of applied GABA and steady-state currents in the presence of 1 μM GABA. Both α4β3δ and α4β3γ2L receptors displayed spontaneous currents, but PKA activation increased spontaneous α4β3δ currents substantially more than spontaneous α4β3γ2L currents. The increase in spontaneous α4β3δ currents was due to an increase in single-channel open frequency. In contrast, PKA activation did not alter steady-state tonic currents recorded in the presence of 1 μM GABA. We concluded that PKA had a GABA concentration–dependent effect on α4β3δ and α4β3γ2L currents. In the absence of GABA, spontaneous α4β3δ and, to a lesser extent, α4β3γ2L currents could provide a basal, tonic current that could be regulated by PKA. With increasing concentrations of extracellular GABA, however, tonic α4β3δ and α4β3γ2L currents would become more GABA dependent and less PKA sensitive. Thus in brain regions with fluctuating extracellular GABA levels, the dynamic range of GABA-activated tonic currents would be set by PKA and the increase in tonic current produced by increasing GABA would be reduced by PKA-mediated phosphorylation. When ambient GABA reaches micromolar concentrations, PKA would have no effect on steady-state tonic currents.
Neuron-Specific Cholinergic Modulation of a Forebrain Song Control Nucleus
Authors: Shea Stephen D. , Koch Henner , Baleckaitis Daniel , Ramirez Jan-Marino , Margoliash Daniel
Published: November 2009

Cholinergic activation profoundly affects vertebrate forebrain networks, but pathway, cell type, and modality specificity remain poorly understood. Here we investigated cell-specific cholinergic modulation of neurons in the zebra finch forebrain song control nucleus HVC using in vitro whole cell recordings. The HVC contains projection neurons that exclusively project to either another song motor nucleus RA (robust nucleus of the arcopallium) (HVC-RAn) or the basal ganglia Area X (HVC-Xn) and these populations are synaptically coupled by a network of GABAergic interneurons. Among HVC-RAn, we observed two physiologically distinct classes that fire either phasically or tonically to injected current. Muscarine excited phasic HVC-RAn and most HVC-Xn. Effects were observed under conditions of blockade of fast synaptic transmission and were reversed by atropine. In contrast, unlike what is commonly observed in mammalian systems, HVC interneurons were inhibited by muscarine and these effects were reversed by atropine. Thus cholinergic modulation reconfigures the HVC network in a more complex fashion than that implied by monolithic “gating.” The two projection pathways are decoupled through suppression of the inhibitory network that links them, whereas each is simultaneously predominantly excited. We speculate that fluctuating cholinergic tone in HVC could modulate the interaction of song motor commands with basal ganglia circuitry associated with song perception and modification. Furthermore, if the in vitro distinction between RA-projecting neurons that we observed is also present in vivo, then the song system motor pathway exhibits greater physiological diversity than has been commonly assumed.
Presynaptic Modulation of Ia Afferents in Young and Old Adults When Performing Force and Position Control
Authors: Baudry Stéphane , Maerz Adam H. , Enoka Roger M.
Published: November 2009

The present work investigated presynaptic modulation of Ia afferents in the extensor carpi radialis (ECR) when young and old adults exerted a wrist extension force either to support an inertial load (position control) or to achieve an equivalent constant torque against a rigid restraint (force control) at 5, 10, and 15% of the maximal force. H reflexes were evoked in the ECR by stimulating the radial nerve above the elbow. A conditioning stimulus was applied to the median nerve above the elbow to assess presynaptic inhibition of homonymous Ia afferents (D1 inhibition) or at the wrist (palmar branch) to assess the ongoing presynaptic inhibition of heteronymous Ia afferents that converge onto the ECR motor neuron pool (heteronymous Ia facilitation). The young adults had less D1 inhibition and greater heteronymous Ia facilitation during the position task (79 and 132.1%, respectively) compared with the force task (69.1 and 115.1%, respectively, P < 0.05). In contrast, the old adults exhibited no difference between the two tasks for either D1 inhibition (∼72%) or heteronymous Ia facilitation (∼114%). Contraction intensity did not influence the amount of D1 inhibition or heteronymous Ia facilitation for either group of subjects. The amount of antagonist coactivation was similar between tasks for young adults, whereas it was greater in the position task for old adults ( P = 0.02). These data indicate that in contrast to young adults, old adults did not modulate presynaptic inhibition of Ia afferents when controlling the position of a compliant load but rather increased coactivation of the antagonist muscle.
Rapid Rise of Extracellular pH Evoked by Neural Activity Is Generated by the Plasma Membrane Calcium ATPase
Authors: Makani Sachin , Chesler Mitchell
Published: November 2009

In hippocampus, synchronous activation of CA1 pyramidal neurons causes a rapid, extracellular, population alkaline transient (PAT). It has been suggested that the plasma membrane Ca2+-ATPase (PMCA) is the source of this alkalinization, because it exchanges cytosolic Ca2+for external H+. Evidence supporting this hypothesis, however, has thus far been inconclusive. We addressed this long-standing problem by measuring surface alkaline transients (SATs) from voltage-clamped CA1 pyramidal neurons in juvenile mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes placed against the somata. In saline containing benzolamide (a poorly permeant carbonic anhydrase blocker), a 2-s step from −60 to 0 mV caused a mean SAT of 0.02 unit pH. Addition of 5 mM HEPES to the artificial cerebrospinal fluid diminished the SAT by 91%. Nifedipine reduced the SAT by 53%. Removal of Ca2+from the saline abolished the SAT, and addition of BAPTA to the patch pipette reduced it by 79%. The inclusion of carboxyeosin (a PMCA inhibitor) in the pipette abolished the SAT, whether it was induced by a depolarizing step, or by simulated, repetitive, antidromic firing. The peak amplitude of the “antidromic” SAT of a single cell averaged 11% of the PAT elicited by comparable real antidromic activation of the CA1 neuronal population. Caloxin 2A1, an extracellular PMCA peptide inhibitor, blocked both the SAT and PAT by 42%. These results provide the first direct evidence that the PMCA can explain the extracellular alkaline shift elicited by synchronous firing.
Human Skeletal Muscle Drug Transporters Determine Local Exposure and Toxicity of Statins
Authors: Knauer Michael J. , Urquhart Bradley L. , Meyer zu Schwabedissen Henriette E. , Schwarz Ute I. , Lemke Christopher J. , Leake Brenda F. , Kim Richard B. , Tirona Rommel G.
Published: November 2009

Rationale:The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity.

Objective:We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro.

Methods and Results:We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance–associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1.

Conclusions:These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.
Roles for Endoplasmic Reticulum–Associated Degradation and the Novel Endoplasmic Reticulum Stress Response Gene Derlin-3 in the Ischemic Heart
Authors: Belmont Peter J. , Chen Wenqiong J. , San Pedro Matthew N. , Thuerauf Donna J. , Gellings Lowe Nicole , Gude Natalie , Hilton Brett , Wolkowicz Roland , Sussman Mark A. , Glembotski Christopher C.
Published: November 2009

Rationale
:

Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery.

Objective
:

Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart.

Methods and Results
:

In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia–induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia–induced cardiomyocyte death.

Conclusions
:

This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.
Cyclooxygenase-2–Dependent Prostacyclin Formation and Blood Pressure Homeostasis
Authors: Yu Ying , Stubbe Jane , Ibrahim Salam , Song Wen-liang , Symth Emer M. , Funk Colin D. , FitzGerald Garret A.
Published: November 2009

Rationale:Cyclooxygenase (COX)-derived prostanoids (PGs) are involved in blood pressure homeostasis. Both traditional nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX-1 and COX-2 and NSAIDs designed to be selective for inhibition of COX-2 cause sodium retention and elevate blood pressure.

Objective:To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements.

Methods and Results:COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI2receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI2metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, inner renal medullary expression of the receptor for PGI2, but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI2observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI2receptor agonist increased urine volume and sodium excretion in mice.

Conclusions:These studies suggest that dysregulated expression of the COX-2 dependent, PGI2biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.
Inhibition of Semaphorin As a Novel Strategy for Therapeutic Angiogenesis
Authors: Moriya Junji , Minamino Tohru , Tateno Kaoru , Okada Sho , Uemura Akiyoshi , Shimizu Ippei , Yokoyama Masataka , Nojima Aika , Okada Mitsuhiro , Koga Hisashi , Komuro Issei
Published: November 2009

Rationale
:

The axon-guiding molecules known as semaphorins and their receptors (plexins) regulate the vascular pattern and play an important role in the development of vascular network during embryogenesis. Semaphorin (Sema)3E is one of the class 3 semaphorins, and plexinD1 is known to be its receptor. Although these molecules have a role in embryonic vascular development, it remains unclear whether the Sema3E/plexinD1 axis is involved in postnatal angiogenesis.

Objective
:

The objective of this study was to elucidate the role of Sema3E/plexinD1 in postnatal angiogenesis.

Methods and Results
:

Sema3E inhibited cell growth and tube formation by suppressing the vascular endothelial growth factor (VEGF) signaling pathway. Expression of Sema3E and plexinD1 was markedly upregulated in ischemic limbs of mice (2.5- and 4.5-fold increase for Sema3E and plexinD1, respectively), and inhibition of this pathway by introduction of the plexinD1-Fc gene or disruption of Sema3E led to a significant increase of blood flow recovery (1.6- and 1.5-fold increase for the plexinD1-Fc gene treatment and Sema3E disruption, respectively). Hypoxia activated the tumor suppressor protein p53, thereby upregulating Sema3E expression. Expression of p53 and Sema3E was enhanced in diabetic mice compared with normal mice (2- and 1.3-fold increase for p53 and Sema3E, respectively). Consequently, neovascularization after VEGF treatment was poor in the ischemic tissues of diabetic mice, whereas treatment with VEGF plus plexinD1-Fc markedly improved neovascularization.

Conclusions
:

These results indicate that inhibition of Sema3E may be a novel strategy for therapeutic angiogenesis, especially when VEGF is ineffective.
Adiponectin
Authors: Tao Ling , Wang Yajing , Gao Erhe , Zhang Hangxiang , Yuan Yuexing , Lau Wayne B. , Chan Lawrence , Koch Walter J. , Ma Xin L.
Published: November 2009

Rationale
:

Patients treated with peroxisome proliferator-activated receptor (PPAR)-γ agonist manifest favorable metabolic profiles associated with increased plasma adiponectin (APN). However, whether increased APN production as a result of PPAR-γ agonist treatment is an epiphenomenon or is causatively related to the cardioprotective actions of PPAR-γ remains unknown.

Objective
:

To determine the role of APN in rosiglitazone (RSG) cardioprotection against ischemic heart injury.

Methods and Results
:

Adult male wild-type (WT) and APN knockdown/knockout (APN
+/−
and APN
−/−
) mice were treated with vehicle or RSG (20 mg/kg per day), and subjected to coronary artery ligation 3 days after beginning treatment. In WT mice, RSG (7 days) significantly increased adipocyte APN expression, elevated plasma APN levels (2.6-fold), reduced infarct size (17% reduction), decreased apoptosis (0.23±0.02% versus 0.47±0.04% TUNEL-positive in remote nonischemic area), attenuated oxidative stress (48.5% reduction), and improved cardiac function (
P
<0.01). RSG-induced APN production and cardioprotection were significantly blunted (
P
<0.05 versus WT) in APN
+/−
, and completely lost in APN
−/−
(
P
>0.05 versus vehicle-treated APN
−/−
mice). Moreover, treatment with RSG for up to 14 days significantly improved the postischemic survival rate of WT mice (
P
<0.05 versus vehicle group) but not APN knockdown/knockout mice.

Conclusions
:

The cardioprotective effects of PPAR-γ agonists are critically dependent on its APN stimulatory action, suggesting that under pathological conditions where APN expression is impaired (such as advanced type 2 diabetes), the harmful cardiovascular effects of PPAR-γ agonists may outweigh its cardioprotective benefits.
Sex-Specific Role of Thioredoxin in Neuroprotection Against Iron-Induced Brain Injury Conferred by Estradiol
Authors: Chen Tzu-Yin , Tsai Ke-Li , Lee Tzu-Ying , Chiueh Chuang Chin , Lee Wen-Sen , Hsu Chin
Published: November 2009

Background and Purpose—

Accumulation of iron after intracerebral hemorrhage causes free radical formation and oxidative damage resulting in liquefaction. The aim of this study was the investigation of molecular mechanisms underlying estrogen-mediated neuroprotective effect against iron-induced brain injury in vivo.

Methods—

Age-matched male and female Sprague-Dawley rats were stereotaxically infused with either ferrous citrate (FC) or saline (10 μL) into the right caudate nucleus. Beta-estradiol 3-benzoate (E
2
) capsule was implanted subcutaneously at 24 hours before infusion of FC. The severity of brain injury and neurological deficits were measured by histological quantification and forelimb asymmetry test, respectively. The role of thioredoxin (Trx) in E
2
-mediated neuroprotective effect was examined by intrastriatal administration of a Trx reductase inhibitor, 5,5-dithiobis-(2-nitrobenzoic acid), and small interfering RNA.

Results—

FC induced greater brain injury in male rats than females. E
2
treatment reduced FC-induced brain injury in both sexes. E
2
significantly increased protein level and activity of Trx in the caudate nucleus of females but not males. Administration of female rats with 5,5-dithiobis-(2-nitrobenzoic acid) or Trx small interfering RNA to the caudate nucleus decreased the protective effect of E
2
against FC-induced injury. The protein and mRNA levels of estrogen receptorα, but not estrogen receptorβ, were more abundant in the caudate nucleus of female rats.

Conclusions—

Increase of brain Trx activity might play an important role in the E
2
-mediated neuroprotective effect against FC-induced brain injury in female rats. Understanding of the sex differences in the Trx-mediated neuroprotective effect by E
2
might help in improving treatment of brain dysfunction after hemorrhagic stroke and/or head trauma.
The Postischemic Environment Differentially Impacts Teratoma or Tumor Formation After Transplantation of Human Embryonic Stem Cell-Derived Neural Progenitors
Authors: Seminatore Christine , Polentes Jerome , Ellman Ditte , Kozubenko Nataliya , Itier Valerie , Tine Samir , Tritschler Laurent , Brenot Marion , Guidou Emmanuelle , Blondeau Johanna , Lhuillier Mickael , Bugi Aurore , Aubry Laetitia , Jendelova Pavla , Sykova Eva , Perrier Anselme L. , Finsen Bente , Onteniente Brigitte
Published: November 2009

Background and Purpose—

Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors.

Methods—

Four differentiation stages were identified on the basis of quantitative polymerase chain reaction expression of pluripotency, proliferation, and differentiation markers. Neural progenitors were transplanted at these 4 stages into rats with no, small, or large middle cerebral artery occlusion lesions. The fate of each transplant was compared with their pretransplantation status 1 to 4 months posttransplantation.

Results—

The influence of the postischemic environment was limited to graft survival and occurrence of nonneuroectodermal structures after transplantation of very immature neural progenitors. Both effects were lost with differentiation. We identified a particular stage of differentiation characterized in vitro by a rebound of proliferative activity that produced highly proliferative grafts susceptible to threaten surrounding host tissues.

Conclusion—

The effects of the ischemic environment on the formation of teratoma by transplanted human embryonic stem cell-derived neural progenitors are limited to early differentiation stages that will likely not be used for stem cell therapy. In contrast, hyperproliferation observed at later stages of differentiation corresponds to an intrinsic activity that should be monitored to avoid tumorigenesis.
Impact of Glucose Tolerance Status on Development of Ischemic Stroke and Coronary Heart Disease in a General Japanese Population
Authors: Doi Yasufumi , Ninomiya Toshiharu , Hata Jun , Fukuhara Masayo , Yonemoto Koji , Iwase Masanori , Iida Mitsuo , Kiyohara Yutaka
Published: November 2009

Background and Purpose—

Few studies have shown the association between glucose tolerance status defined by a 75-g oral glucose tolerance test and the development of different types of cardiovascular disease.

Methods—

A total of 2421 community-dwelling Japanese subjects aged 40 to 79 years who underwent the oral glucose tolerance test were followed up for 14 years.

Results—

In multivariable analysis, the risks of ischemic stroke in both sexes and coronary heart disease (CHD) in women were significantly higher in subjects with diabetes determined by the World Health Organization criteria than in those with normal glucose tolerance even after adjustment for other confounding factors, but such association was not seen for CHD in men (ischemic stroke: adjusted hazard ratio [HR]=2.54,
P
=0.002 in men; adjusted HR=2.02,
P
=0.03 in women; CHD: adjusted HR=1.26,
P
=0.47 in men; adjusted HR=3.46,
P
=0.002 in women). Similar associations were observed for fasting plasma glucose levels of ≥7.0 mmol/L (ischemic stroke: adjusted HR=2.15,
P
=0.03 in men; adjusted HR=2.10,
P
=0.045 in women; CHD: adjusted HR=1.29,
P
=0.47 in men; adjusted HR=3.83,
P
=0.003 in women) and for 2-hour postload glucose levels of ≥11.1 mmol/L (ischemic stroke: adjusted HR=2.71,
P
=0.003 in men; adjusted HR=2.19,
P
=0.03 in women; CHD: adjusted HR=1.58,
P
=0.16 in men; adjusted HR=4.44,
P
<0.001 in women). The age-adjusted incidences of ischemic stroke and CHD did not significantly increase in subjects with impaired fasting glycemia or impaired glucose tolerance in either sex.

Conclusions—

Our findings suggest that diabetes is an independent risk factor for ischemic stroke in both sexes and CHD in women in the Japanese population.
Strength Training Improves Upper-Limb Function in Individuals With Stroke
Authors: Harris Jocelyn E. , Eng Janice J.
Published: November 2009

Background and Purpose—

After stroke, maximal voluntary force is reduced in the arm and hand muscles, and upper-limb strength training is 1 intervention with the potential to improve function.

Methods—

We performed a meta-analysis of randomized controlled trials. Electronic databases were searched from 1950 through April 2009. Strength training articles were assessed according to outcomes: strength, upper-limb function, and activities of daily living. The standardized mean difference (SMD) was calculated to estimate the pooled effect size with random-effect models.

Results—

From the 650 trials identified, 13 were included in this review, totaling 517 individuals. A positive outcome for strength training was found for grip strength (SMD=0.95,
P
=0.04) and upper-limb function (SMD=0.21,
P
=0.03). No treatment effect was found for strength training on measures of activities of daily living. A significant effect for strength training on upper-limb function was found for studies including subjects with moderate (SMD=0.45,
P
=0.03) and mild (SMD=0.26,
P
=0.01) upper-limb motor impairment. No trials reported adverse effects.

Conclusions—

There is evidence that strength training can improve upper-limb strength and function without increasing tone or pain in individuals with stroke.
Genetically Reduced Soluble Epoxide Hydrolase Activity and Risk of Stroke and Other Cardiovascular Disease
Authors: Lee Julie , Dahl Morten , Grande Peer , Tybjærg-Hansen Anne , Nordestgaard Børge G.
Published: November 2009

Background and Purpose—

The development of stroke has been linked to lowered levels of epoxyeicosatrienoic acids in the cerebral microvasculature. These substances are metabolized by the enzyme-soluble epoxide hydrolase encoded by the
EPHX2
gene. We tested whether genetically reduced soluble epoxide hydrolase activity is associated with risk of ischemic stroke, myocardial infarction, and ischemic heart disease.

Methods—

We genotyped participants from the Copenhagen City Heart Study (n=10 352), the Copenhagen General Population Study (n=26 042), the Copenhagen Carotid Stroke Study (n=398 cases+796 control subjects), and the Copenhagen Ischemic Heart Disease Study (n=4901 cases+9798 control subjects) for the R103C, R287Q, and Arg
402-403ins
variants in the
EPHX2
gene and recorded hospital admissions due to ischemic stroke, myocardial infarction, and ischemic heart disease.

Results—

The hazard/odds ratio for ischemic stroke did not differ from 1.0 for any of the
EPHX2
genotypes or genotype combinations in the Copenhagen City Heart Study (
P
for trend=0.15 to 0.76), in the Copenhagen General Population Study (
P
for trend=0.75 to 0.95), and the Copenhagen Carotid Stroke Study (
P
for trend=0.08 to 1.00). Similar results were obtained for myocardial infarction and ischemic heart disease in the 3 studies.

Conclusions—

Our results show with significant power that genetically reduced soluble epoxide hydrolase activity is not a major risk factor for ischemic stroke, myocardial infarction, or ischemic heart disease in the Danish population. This suggests that the relationship between the
EPHX2
gene and risk of ischemic stroke and other cardiovascular disease does not exist or its effect size is likely to be quite small.
Activation of Sphingosine 1-Phosphate Receptor-1 by FTY720 Is Neuroprotective After Ischemic Stroke in Rats
Authors: Hasegawa Yu , Suzuki Hidenori , Sozen Takumi , Rolland William , Zhang John H.
Published: November 2009

Background and Purpose—

FTY720 is a known sphingosine 1–phosphate receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).

Methods—

One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and 1 mg/kg of FTY720, another selective sphingosine 1–phosphate receptor-1 agonist SEW2871 (5 mg/kg), or 0.25 mg/kg of FTY720 plus a sphingosine 1–phosphate antagonist, VPC23019 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling were conducted at 24 hours after MCAO.

Results—

FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW2871 showed similar neuroprotective effects to FTY720, whereas VPC 20319 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC23019 blocked the antiapoptotic effects of FTY720.

Conclusions—

These data suggest that activation of sphingosine 1–phosphate-1 by FTY720 reduces neuronal death after transient MCAO.