bluuebunny/crossref_metadata_2025 huggingface dataset:
The Missing Link in the Mystery of Normal Automaticity of Cardiac Pacemaker Cells
Authors: Lakatta Edward G. , Vinogradova Tatiana M. , Maltsev Victor A.
Published: March 2008

Earlier studies of the initiating event of normal automaticity of the heart's pacemaker cells, inspired by classical quantitative membrane theory, focused upon ion currents (IK, If) that determine the maximum diastolic potential and the early phase of the spontaneous diastolic depolarization (DD). These early DD events are caused by the prior action potential (AP) and essentially reflect a membrane recovery process. Events following the recovery process that ignite APs have not been recognized and remained a mystery until recently. These critical events are linked to rhythmic intracellular signals initiated by Ca2+ clock (i.e., sarcoplasmic reticulum [SR] cycling Ca2+). Sinoatrial cells, regardless of size, exhibit intense ryanodine receptor (RyR), Na+/Ca2+ exchange (NCX)‐1, and SR Ca2+ ATPase‐2 immunolabeling and dense submembrane NCX/RyR colocalization; Ca2+ clocks generate spontaneous stochastic but roughly periodic local subsarcolemmal Ca2+ releases (LCR). LCRs generate inward currents via NCX that exponentially accelerate the late DD. The timing and amplitude of LCR/INCX‐coupled events control the timing and amplitude of the nonlinear terminal DD and therefore ultimately control the chronotropic state by determining the timing of the ICaL activation that initiates the next AP. LCR period is precisely controlled by the kinetics of SR Ca2+ cycling, which, in turn, are regulated by 1) the status of protein kinase A‐dependent phosphorylation of SR Ca2+ cycling proteins; and 2) membrane ion channels ensuring the Ca2+ homeostasis and therefore the Ca2+ available to Ca2+ clock. Thus, the link between early DD and next AP, missed in earlier studies, is ensured by a precisely physiologically regulated Ca2+ clock within pacemaker cells that integrates multiple Ca2+‐dependent functions and rhythmically ignites APs during late DD via LCRs‐INCX coupling.
Cardiac Cell HypertrophyIn Vitro
Authors: Colella Matilde , Pozzan Tullio
Published: March 2008

Various conditions were used to investigate the importance of Ca2+ signaling in triggering hypertrophy in neonatal rat cardiomyocytes in vitro. An increase in cell size and sarcomere reorganization were induced not only by treatment with receptor agonists, such as angiotensin II, aldosterone, and norepinephrine, but also by a small depolarization caused by an increase in the KCl concentration of the medium. This latter treatment has no direct effects on receptor signaling. All these hypertrophic treatments caused a long‐lasting increase in the frequency of spontaneous [Ca2+] oscillations, causing nuclear translocation of transfected NFAT (GFP). Cyclosporine A inhibited hypertrophy and NFAT translocation, but not the increased oscillation frequency. We propose here that calcineurin–NFAT can act as integrators of the Ca2+ signal and can decode alterations in the frequency even of very rapid Ca2+ oscillations.
Sarcomere Mechanics in Uniform and Nonuniform Cardiac Muscle
Authors: Ter Keurs Henk E.D.J. , Shinozaki Tsuyoshi , Zhang Ying Ming , Wakayama Yuji , Sugai Yoshinao , Kagaya Yutaka , Miura Masahito , Boyden Penelope A. , Stuyvers Bruno D.M. , Landesberg Amir
Published: March 2008

Starling's law and the end‐systolic pressure–volume relationship (ESPVR) reflect the effect of sarcomere length (SL) on the development of stress (σ) and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a σ–SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to that of skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The σ−SL −[Ca2+]free relationships (σ–SL–Ca relationships) at submaximal [Ca2+] in intact and skinned trabeculae were similar, although the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the σ–SL–Ca relationship by using a kinetic model assuming that the rates of Tn‐C Ca2+ binding and/or cross‐bridge (XB) cycling are determined by either the SL, [Ca2+], or σ. We analyzed the correlation between the model results and steady‐state σ measurements at varied SL at [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that the dominant feedback mechanism is SL‐, σ‐, or [Ca2+]‐dependent, and that the feedback mechanism regulates Tn‐C Ca2+ affinity, XB kinetics, or the unitary XB force. The analysis strongly suggests that the feedback of the number of strong XBs to cardiac Tn‐C Ca2+ affinity is the dominant mechanism regulating XB recruitment. Using this concept in a model of twitch‐σ accurately reproduced the σ–SL–Ca relationship and the time courses of twitch σ and the intracellular [Ca2+]i. The foregoing concept has equally important repercussions for the nonuniformly contracting heart, in which arrhythmogenic Ca2+ waves arise from weakened areas in the cardiac muscle. These Ca2+ waves can reversibly be induced with nonuniform excitation–contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by the sarcomeres in the border zone during relaxation causes Ca2+ release from Tn‐C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). Modeling of the response of the cardiac twitch to rapid force changes using the feedback concept uniquely predicts the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn‐C. These results are consistent with the hypothesis that a force feedback to Ca2+ binding by Tn‐C is responsible for Starling's law and the ESPVR in the uniform myocardium and leads to a surge of Ca2+ released by the myofilaments during relaxation in the nonuniform myocardium, which initiates arrhythmogenic propagating Ca2+ release by the SR.
Modulation of Cardiac Performance by Motor Protein Gene Transfer
Authors: Herron Todd J. , Devaney Eric J. , Metzger Joseph M.
Published: March 2008

Cardiac muscle performance can be determined by factors intrinsic to each cardiac muscle cell, such as protein isoform expression. One protein whose expression plays a major role in determining cardiac performance is myosin. Myosin is the heart's molecular motor which transduces the chemical energy from ATP hydrolysis into the mechanical energy of each heartbeat. Alterations of myosin isoform expression are routinely associated with acquired and inherited cases of cardiomyopathy. For example, human heart failure is consistently associated with increased expression of a slow myosin motor isoform and a concomitant decreased expression of the heart's fast myosin motor isoform. Further, mutations of the cardiac myosin gene are the most common cause of inherited hypertrophic cardiomyopathy. Transgenic animal studies have provided insight into cardiac functional effects caused by myosin isoform gene switching (fast‐to‐slow myosin or slow‐to‐fast myosin) or by expression of a disease‐related mutant motor. More direct structure–function analysis using acute gene transfer of myosin motors provides evidence that the inotropic state of cardiac muscle can be affected by motor protein isoform shifting independent of intracellular calcium handling. Because most therapies for the diseased heart target intracellular calcium handling, acute gene transfer of cardiac molecular motors to modulate heart performance offers a novel therapeutic strategy for the compromised heart. Although the development of safe vectors for therapeutic myosin gene delivery are in their infancy, studies focused on acute genetic engineering of the heart's molecular motor will provide a foundation for therapeutic vector development and insight into mechanisms that contribute to cardiomyopathy.
Atrial‐Selective Sodium Channel Block as a Strategy for Suppression of Atrial Fibrillation
Authors: Burashnikov Alexander , Di Diego José M. , Zygmunt Andrew C. , Belardinelli Luiz , Antzelevitch Charles
Published: March 2008

Antiarrhythmic drug therapy remains the principal approach for suppression of atrial fibrillation (AF) and flutter (AFl) and prevention of their recurrence. Among the current strategies for suppression of AF/AFl is the development of antiarrhythmic agents that preferentially affect atrial, rather than ventricular electrical parameters. Inhibition of the ultrarapid delayed rectifier potassium current (IKur), present in the atria, but not in the ventricles, is an example of an atrial‐selective approach. Our recent study examined the hypothesis that sodium channel characteristics differ between atrial and ventricular cells and that atrial‐selective sodium channel block is another effective strategy for the management of AF. We have demonstrated very significant differences in the inactivation characteristics of atrial versus ventricular sodium channels and a striking atrial selectivity for the action of ranolazine, an inactivated‐state sodium channel blocker, to produce use‐dependent block of the sodium channels, leading to depression of excitability, development of post‐repolarization refractoriness (PRR), and suppression of AF. Lidocaine and chronic amiodarone, both predominantly inactivated‐state sodium channel blockers, also produced a preferential depression of sodium channel–dependent parameters (VMax conduction velocity, diastolic threshold of excitation, and PRR) in the atria. Propafenone, a predominantly open‐state sodium channel blocker, produced similar changes of electrophysiological parameters, which were was not atrial‐selective. The ability of ranolazine, chronic amiodarone, and propafenone to prolong the atrial action potential potentiated their ability to suppress AF in coronary‐perfused canine atrial preparations. In conclusion: Our data demonstrate important differences in the inactivation characteristics of atrial versus ventricular sodium channels and a striking atrial selectivity for the action of agents like ranolazine to produce use‐dependent block of sodium channels leading to suppression of AF. Our findings suggest that atrial‐selective sodium channel block may be a valuable strategy to combat AF.
Molecular Basis of Cardiac Action Potential Repolarization
Authors: Rudy Yoram
Published: March 2008

The action potential (AP) is generated by transport of ions through transmembrane ion channels. Rate dependence of AP repolarization is a fundamental property of cardiac cells, and its modification by disease or drugs can lead to fatal arrhythmias. Using a computational biology approach, we investigated the gating kinetics of the rapid (IKr) and slow (IKs) K+ currents during the AP in order to provide insight into the molecular basis of their role in AP repolarization. Results show that IKr intensifies during the late AP plateau by progressively recovering from inactivation and generating a pronounced late peak of open‐state occupancy. The delayed peak makes IKr an effective determinant of AP repolarization. IKs builds an available reserve of channels in closed states near the open state that can open rapidly to generate current during the AP repolarization phase. By doing so, IKs can provide repolarizing current when other currents (e.g., IKr) are compromised by disease or drugs, thus preventing excessive AP prolongation and arrhythmic activity.
Regulation of Endothelial Junctional Permeability
Authors: Vandenbroucke Emily , Mehta Dolly , Minshall Richard , Malik Asrar B.
Published: March 2008

The endothelium is a semi‐permeable barrier that regulates the flux of liquid and solutes, including plasma proteins, between the blood and surrounding tissue. The permeability of the vascular barrier can be modified in response to specific stimuli acting on endothelial cells. Transport across the endothelium can occur via two different pathways: through the endothelial cell (transcellular) or between adjacent cells, through interendothelial junctions (paracellular). This review focuses on the regulation of the paracellular pathway. The paracellular pathway is composed of adhesive junctions between endothelial cells, both tight junctions and adherens junctions. The actin cytoskeleton is bound to each junction and controls the integrity of each through actin remodeling. These interendothelial junctions can be disassembled or assembled to either increase or decrease paracellular permeability. Mediators, such as thrombin, TNF‐α, and LPS, stimulate their respective receptor on endothelial cells to initiate signaling that increases cytosolic Ca2+ and activates myosin light chain kinase (MLCK), as well as monomeric GTPases RhoA, Rac1, and Cdc42. Ca2+ activation of MLCK and RhoA disrupts junctions, whereas Rac1 and Cdc42 promote junctional assembly. Increased endothelial permeability can be reversed with “barrier stabilizing agents,” such as sphingosine‐1‐phosphate and cyclic adenosine monophosphate (cAMP). This review provides an overview of the mechanisms that regulate paracellular permeability.
Multi‐Scale Model of O2 Transport and Metabolism
Authors: Zhou Haiying , Lai Nicola , Saidel Gerald M. , Cabrera Marco E.
Published: March 2008

Regulation of pulmonary oxygen uptake (VO2p) during exercise depends on cellular energy demand, blood flow, ventilation, oxygen exchange across membranes, and oxygen utilization in the contracting skeletal muscle. In human and animal studies of metabolic processes that control cellular respiration in working skeletal muscle, pulmonary VO2 dynamics is measured at the mouth using indirect calorimetry. To provide information on the dynamic balance between oxygen delivery and oxygen consumption at the microvascular level, muscle oxygenation is measured using near‐infrared spectroscopy. A multi‐scale computational model that links O2 transport and cellular metabolism in the skeletal muscle was developed to relate the measurements and gain quantitative understanding of the regulation of VO2 at the cellular, tissue, and whole‐body level. The model incorporates mechanisms of oxygen transport from the airway openings to the cell, as well as the phosphagenic and oxidative pathways of ATP synthesis in the muscle cells.
Mapping Preconditioning's Signaling Pathways
Authors: Downey James M. , Krieg Thomas , Cohen Michael V.
Published: March 2008

Preconditioning the heart by exposure to brief cycles of ischemia–reperfusion causes it to become very resistant to ischemia‐induced infarction. This protection has been shown to depend on a large number of signal transduction components whose arrangements within the cardiomyocyte are unknown. To aid the translation of this phenomenon to the clinical setting, we have attempted to map the signal transduction pathways responsible for this protection. To resolve the signaling order we have injected a signal at an intermediate point in the system transduction pathway and monitored it at a downstream site. System analysis reveals both parallel and series signaling arrangements. Separate trigger and mediator phases could be identified. The trigger phase is now well mapped. During the preconditioning ischemia, autacoids—including adenosine, opioids, and bradykinin—are released from the heart. These substances occupy their respective Gi‐coupled receptors. Opioid and bradykinin receptors activate phosphatidylinositol 3‐kinase (PI3‐kinase) which, through phosphoinositide‐dependent protein kinase, causes activation of Akt. Opioid couples through transactivation of the epidermal growth factor receptor, while bradykinin's coupling to PI3‐kinase is unknown. PI3‐kinase causes extracellular signal regulated kinase (ERK)‐dependent activation of endothelial nitric oxide synthase. The resulting nitric oxide activates soluble guanylyl cyclase resulting in cyclic C‐GMP‐dependent protein kinase (PKG) activation through production of cyclic guanosine monophosphate. PKG initiates opening of ATP‐sensitive potassium channels on the inner membrane of the mitochondria. Potassium entry into mitochondria causes the generation of free radicals during reperfusion when oxygen is reintroduced. Through redox signaling, these radicals activate protein kinase C (PKC) and put the heart into the protected phenotype that persists for one to two hours. Although adenosine receptors activate PI3‐kinase, they also have a second direct coupling to PKC and thus bypass the mitochondrial pathway. The mediator phase occurs during the first minutes of reperfusion following the lethal ischemic insult and is still poorly defined. Briefly, PKC somehow potentiates adenosine's ability to activate signaling from low‐affinity A2b adenosine receptors. These receptors couple to the survival kinases, Akt and ERK, believed to inhibit the formation of deadly mitochondrial permeability transition pores through the phosphorylation of glycogen synthase kinase‐3β. The proposed signaling maps reveal many points at which drugs can trigger the protected phenotype.
The Identity and Regulation of the Mitochondrial Permeability Transition Pore
Authors: Juhaszova Magdalena , Wang Su , Zorov Dmitry B. , Bradley Nuss H. , Gleichmann Marc , Mattson Mark P. , Sollott Steven J.
Published: March 2008

The mitochondrial permeability transition (MPT) pore complex is a key participant in the machinery that controls mitochondrial fate and, consequently, cell fate. The quest for the pore identity has been ongoing for several decades and yet the main structure remains unknown. Established “dogma” proposes that the core of the MPT pore is composed of an association of voltage‐dependent anion channel (VDAC) and adenine nucleotide translocase (ANT). Recent genetic knockout experiments contradict this commonly accepted interpretation and provide a basis for substantial revision of the MPT pore identity. There is now sufficient evidence to exclude VDAC and ANT as the main pore structural components. Regarding MPT pore regulation, the role of cyclophilin D is confirmed and ANT may still serve some regulatory function, although the involvement of hexokinase II and creatine kinase remains unresolved. When cell protection signaling pathways are activated, we have found that the Bcl‐2 family members relay the signal from glycogen synthase kinase‐3 beta onto a target at or in close proximity to the pore. Our experimental findings in intact cardiac myocytes and neurons indicate that the current “dogma” related to the role of Ca2+ in MPT induction requires reevaluation. Emerging evidence suggests that after injury‐producing stresses, reactive oxygen species (but not Ca2+) are largely responsible for the pore induction. In this article we discuss the current state of knowledge and provide new data related to the MPT pore structure and regulation.
Experimental Molecular and Stem Cell Therapies in Cardiac Electrophysiology
Authors: Gepstein Lior
Published: March 2008

One of the most exciting fields in cardiovascular research today involves the possible use of stem cells, cell and gene therapies, and tissue engineering for the treatment of a variety of cardiovascular disorders. Here, we review on the possible applications of these emerging strategies in the field of cardiac electrophysiology. Initially, the elegant cell and gene therapy approaches proposed for the treatment of bradyarrhythmias are described. These gene therapy approaches are mainly focused on the generation of biological pacemakers either by altering the neurohumoral control of existing pacemaking cells (by overexpressing the β‐adrenergic receptor) or by converting quiescent cardiomyocytes into pacemaking cells by shifting the balance between diastolic repolarization and depolarization currents. An alternative approach explores the possibility of grafting pacemaking cells, which were either derived directly during the differentiation of human embryonic stem cells or engineered from mesenchymal stem cells, into the myocardium as a cell therapy strategy for biological pacemaking. We then describe the possible applications of similar strategies for the treatment of common tachyarrhythmias by overexpression of different ion channels, or their modifiers, either directly in host cardiomyocytes or ex vivo in cells that will be eventually transplanted into the heart. Next, we discuss the electrophysiological implications of cardiac stem cell therapy for heart failure. Finally, we address the obstacles, challenges, and avenues for further research required to make these novel strategies a clinical reality.
The Muscle Transcription Factor MyoD Promotes Osteoblast Differentiation by Stimulation of the Osterix Promoter
Authors: Hewitt Jocelyn , Lu Xiaghuai , Gilbert Linda , Nanes Mark S.
Published: March 2008

Transcription factors regulate tissue-specific differentiation of pluripotent mesenchyme to osteoblast (OB), myoblast (MB), and other lineages. Osterix (Osx) is an essential transcription factor for bone development because knockout results in lack of a mineralized skeleton. The proximal Osx promoter contains numerous binding sequences for MyoD and 14 repeats of a binding sequence for Myf5. These basic helix-loop-helix (bHLH) transcription factors have a critical role in MB differentiation and muscle development. We tested the hypothesis that bHLH transcription factors also support OB differentiation through regulation of Osx. Transfection of a MyoD expression vector into two primitive mesenchymal cell lines, C3H/10T1/2 and C2C12, stimulated a 1.2-kb Osx promoter-luciferase reporter 70-fold. Myf5 stimulated the Osx promoter 6-fold. Deletion analysis of the promoter revealed that one of three proximal bHLH sites is essential for MyoD activity. The Myf5 repeat conferred 60% of Myf5 activity with additional upstream sequence required for full activity. MyoD bound the active bHLH sequence and its 3′-flanking region, as shown by EMSA and chromatin immunoprecipitation assays. Real-time PCR revealed that primitive C2C12 and C3H/10T1/2 cells, pre-osteoblastic MC3T3 cells, and undifferentiated primary marrow stromal cells express the muscle transcription factors. C2C12 cells, which differentiate to MB spontaneously and form myotubules, were treated with bone morphogenetic protein 2 (BMP-2) to induce OB differentiation. BMP-2 stimulated expression of Osx and the differentiation marker alkaline phosphatase and blocked myotubule development. BMP-2 suppressed the muscle transcription factor myogenin, but expression of MyoD and Myf5 persisted. Silencing of MyoD inhibited BMP-2 stimulation of Osx and blocked the later appearance of bone alkaline phosphatase. MyoD support of Osx transcription contributes to early OB differentiation.
Small-Conductance Calcium-Activated Potassium Channels Control Excitability and Firing Dynamics in Gonadotropin-Releasing Hormone (GnRH) Neurons
Authors: Liu Xinhuai , Herbison Allan E.
Published: March 2008

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 ± 2.7 pA, mean decay time constant = 2167 msec, apamin IC50 = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 ± 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.
Prokineticin-1: A Novel Mediator of the Inflammatory Response in Third-Trimester Human Placenta
Authors: Denison Fiona C. , Battersby Sharon , King Anne E. , Szuber Michael , Jabbour Henry N.
Published: March 2008

Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.
Bioinformatics Analysis of Targeted Metabolomics—Uncovering Old and New Tales of Diabetic Mice under Medication
Authors: Altmaier Elisabeth , Ramsay Steven L. , Graber Armin , Mewes Hans-Werner , Weinberger Klaus M. , Suhre Karsten
Published: March 2008

Metabolomics is a powerful tool for identifying both known and new disease-related perturbations in metabolic pathways. In preclinical drug testing, it has a high potential for early identification of drug off-target effects. Recent advances in high-precision high-throughput mass spectrometry have brought the metabolomic field to a point where quantitative, targeted, metabolomic measurements with ready-to-use kits allow for the automated in-house screening for hundreds of different metabolites in large sets of biological samples. Today, the field of metabolomics is, arguably, at a point where transcriptomics was about 5 yr ago. This being so, the field has a strong need for adapted bioinformatics tools and methods. In this paper we describe a systematic analysis of a targeted quantitative characterization of more than 800 metabolites in blood plasma samples from healthy and diabetic mice under rosiglitazone treatment. We show that known and new metabolic phenotypes of diabetes and medication can be recovered in a statistically objective manner. We find that concentrations of methylglutaryl carnitine are oppositely impacted by rosiglitazone treatment of both healthy and diabetic mice. Analyzing ratios between metabolite concentrations dramatically reduces the noise in the data set, allowing for the discovery of new potential biomarkers of diabetes, such as the N-hydroxyacyloylsphingosyl-phosphocholines SM(OH)28:0 and SM(OH)26:0. Using a hierarchical clustering technique on partial η2 values, we identify functionally related groups of metabolites, indicating a diabetes-related shift from lysophosphatidylcholine to phosphatidylcholine levels. The bioinformatics data analysis approach introduced here can be readily generalized to other drug testing scenarios and other medical disorders.
Energetic Responses Are Triggered by Caudal Brainstem Melanocortin Receptor Stimulation and Mediated by Local Sympathetic Effector Circuits
Authors: Skibicka Karolina P. , Grill Harvey J.
Published: March 2008

The central melanocortin system is a critical contributor to energy balance control. Melanocortin receptors (MC-Rs) are widely distributed throughout forebrain and caudal brainstem nuclei. To assess the contribution of hindbrain MC-Rs to the control of energy expenditure, the MC3/4R agonist melanotan II (MTII) was delivered to either the fourth ventricle or medullary raphe of neurologically intact rats and chronic decerebrate (CD) rats, and interscapular brown adipose tissue (IBAT) temperature (TIBAT), core temperature (TC), heart rate (HR), and spontaneous activity were recorded. Fourth ventricular MTII (0.1, 1.0 nmol) significantly increased TIBAT, TC, and HR in intact rats (TC: +0.33 ± 0.08, +0.41 ± 0.09 C; HR: +40.84 ± 7.29, +69.04 ± 6.83 beats per minute) and in CDs (TC: +1.39 ± 0.67, +1.52 ± 0.37 C; HR: +83.21 ± 19.2, +107.38 ± 17.65 beats per minute). Response magnitude was greater in CD rats than in neurologically intact rats. TIBAT, TC, and HR were significantly increased after 10 pmol MTII delivery to the medullary raphe of intact rats, and here too, the response magnitude was greater in decerebrate rats. The hyperthermia, IBAT thermogenesis, and tachycardia observed in CD rats after fourth ventricular and hindbrain parenchymal MTII injections support the hypothesis that hindbrain MC-R stimulation engages endemic circuits that link sympathetic outflows to thermogenic and cardiac effectors, and that forebrain processing and forebrain-caudal brainstem communication are not required for response production.
Lower Thyroid Compensatory Reserve of Rat Pups after Maternal Hypothyroidism: Correlation of Thyroid, Hepatic, and Cerebrocortical Biomarkers with Hippocampal Neurophysiology
Authors: Taylor Matthew A. , Swant Jarod , Wagner John J. , Fisher Jeffrey W. , Ferguson Duncan C.
Published: March 2008

The developing central nervous system of the fetus and neonate is recognized as very sensitive to maternal or gestational hypothyroidism. Despite this recognition, there is still a lack of data concerning the relationship between thyroid-related biomarkers and neurological outcomes. We used propylthiouracil administered at 0, 3, or 10 ppm in drinking water from gestational d 2 until weaning to create hypothyroid conditions to study the relationship between hypothalamic-pituitary-thyroid axis compensation and impaired neurodevelopment. In addition to serum T3, T4, free T4, and TSH concentrations, cerebrocortical T3 concentration (cT3), hepatic type I and cerebrocortical type II (D2) 5′-deiodinase activity, and thyroidal mRNA for thyroglobulin and sodium iodide symporter were measured. Extracellular recordings from the CA1 region in hippocampal slices were obtained from both postnatal d 21–32 (pups) and postnatal d 90–110 (adults) rats to assess neurophysiological effects. Thyroidal mRNA for thyroglobulin and sodium iodide symporter were increased in pups but not in dams. Both propylthiouracil doses increased cerebrocortical D2 activity approximately 5-fold in pups but only 10 ppm increased D2 activity in dams. In dams, cT3 concentrations were maintained at 3 ppm but fell 75% at 10 ppm. cT3 concentration in pups fell 50% at 3 ppm and more than 90% at 10 ppm. In both 3 and 10 ppm pups, hippocampal baseline synaptic activity correlated negatively with cerebrocortical D2 activity. In 3 ppm adults, impaired long-term potentiation was evident. In summary, during depletion of serum T4, D2 activity served as a sensitive marker of tissue thyroid status, an indicator of the brain’s compensatory response to maintain cT3, and correlated with a neurophysiological outcome.
Vitamin D-Binding Protein Influences Total Circulating Levels of 1,25-Dihydroxyvitamin D3 but Does Not Directly Modulate the Bioactive Levels of the Hormone in Vivo
Authors: Zella Lee A. , Shevde Nirupama K. , Hollis Bruce W. , Cooke Nancy E. , Pike J. Wesley
Published: March 2008

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)2D3. Modest changes in the basal expression of genes involved in 1,25(OH)2D3 metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)2D3 in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)2D3 that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)2D3 is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)2D3 necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)2D3, produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)2D3 and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone’s biologically active pool.
Cyclic Adenosine 3′,5′-Monophosphate Responsive Element Binding Protein Phosphorylation Is Required But Not Sufficient for Activation of Corticotropin-Releasing Hormone Transcription
Authors: Liu Ying , Kamitakahara Anna , Kim Alice Joohee , Aguilera Greti
Published: March 2008

cAMP is a major regulator of CRH transcription. However, receptors activating CRH neurons (α-adrenergic and glutamatergic) do not signal through cAMP, suggesting that calcium phospholipid-dependent signaling synergizes with small elevations of intracellular cAMP. To test this hypothesis, we examined the relationship between activation of CRH transcription, cAMP production, and cAMP response element binding protein (CREB) phosphorylation in neuronal cultures treated with the adenylyl cyclase stimulator, forskolin, the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or their combination. Forskolin, at threshold concentrations for cAMP production and CREB phosphorylation, induced CRH promoter-driven luciferase activity in 4B cells (EC50 = 0.7 μm) and CRH primary transcript in hypothalamic neurons (EC50 = 0.6 μm). PMA alone failed to activate CRH transcription despite being as effective as forskolin in phosphorylating CREB (Ser133 and Ser121). Although PMA potentiated the effect of low forskolin concentrations on CRH transcription and CREB phosphorylation, there was no correlation between phosphorylated CREB levels and activation of CRH transcription. Similarly, the calcium/calmodulin-dependent kinase inhibitor, KN-93, enhanced PMA plus forskolin-stimulated CREB phosphorylation and inhibited CRH transcription. Suppression of CREB phosphorylation by the protein kinase A inhibitor, H89, or the CREB dominant negative, A-CREB, did not affect basal but blocked forskolin-stimulated transcription. This study shows that calcium phospholipid-dependent pathways potentiate the ability of small elevations of intracellular cAMP to activate CRH transcription, providing a mechanism by which non-cAMP-dependent regulators induce CRH gene expression. In addition, the data indicate that phosphorylated CREB is essential but not sufficient for activation of CRH transcription, suggesting that full promoter stimulation requires the interaction of phosphorylated CREB with a coactivator.
Elocalcitol Inhibits Inflammatory Responses in Human Thyroid Cells and T Cells
Authors: Borgogni E. , Sarchielli E. , Sottili M. , Santarlasci V. , Cosmi L. , Gelmini S. , Lombardi A. , Cantini G. , Perigli G. , Luconi M. , Vannelli G. B. , Annunziato F. , Adorini L. , Serio M. , Crescioli C.
Published: March 2008

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves’ disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)γ levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNγ and TNFα-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNγ pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.
Estrogen Inhibits Cardiac Hypertrophy: Role of Estrogen Receptor-β to Inhibit Calcineurin
Authors: Pedram Ali , Razandi Mahnaz , Lubahn Dennis , Liu Jinghua , Vannan Mani , Levin Ellis R.
Published: March 2008

Estrogen has been reported to prevent development of cardiac hypertrophy in female rodent models and in humans. However, the mechanisms of sex steroid action are incompletely understood. We determined the cellular effects by which 17β-estradiol (E2) inhibits angiotensin II (AngII)-induced cardiac hypertrophy in vivo. Two weeks of angiotensin infusion in female mice resulted in marked hypertrophy of the left ventricle, exacerbated by the loss of ovarian steroid hormones from oophorectomy. Hypertrophy was 51% reversed by the administration of E2 (insertion of 0.1 mg/21-d-release tablets). The effects of E2 were mainly mediated by the estrogen receptor (ER) β-isoform, because E2 had little effect in ERβ-null mice but comparably inhibited AngII-induced hypertrophy in wild-type or ERα-null mice. AngII induced a switch of myosin heavy chain production from α to β, but this was inhibited by E2 via ERβ. AngII-induced ERK activation was also inhibited by E2 through the β-receptor. E2 stimulated brain natriuretic peptide protein expression and substantially prevented ventricular interstitial cardiac fibrosis (collagen deposition) as induced by AngII. Importantly, E2 inhibited calcineurin activity that was stimulated by AngII, related to E2 stimulating the modulatory calcineurin-interacting protein (MCIP) 1 gene and protein expression. E2 acting mainly through ERβ mitigates the important signaling by AngII that produces cardiac hypertrophy and fibrosis in female mice.
Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation
Authors: Ajamian Lara , Abrahamyan Levon , Milev Miroslav , Ivanov Pavel V. , Kulozik Andreas E. , Gehring Niels H. , Mouland Andrew J.
Published: March 2008

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55Gag, viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55Gag. Pr55Gag synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.
Splicing regulation: From a parts list of regulatory elements to an integrated splicing code
Authors: Wang Zefeng , Burge Christopher B.
Published: March 2008

Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and introniccis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicingcis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions.
TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex
Authors: Hashimi Hassan , Zíková Alena , Panigrahi Aswini K. , Stuart Kenneth D. , Lukeš Julius
Published: March 2008

The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1).
Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster
Authors: Gonsalvez Graydon B. , Praveen Kavita , Hicks Amanda J. , Tian Liping , Matera A. Gregory
Published: March 2008

Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, we examined this topic in detail and found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, we found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, we constructed a fly strain that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. We therefore conclude that dart5 is required for viability when SMN is limiting.
Does Component Sharing Help or Hurt Reliability? An Empirical Study in the Automotive Industry
Authors: Ramdas Kamalini , Randall Taylor
Published: March 2008

Component sharing—the use of a component on multiple products within a firm's product line—is widely practiced as a means of offering high variety at low cost. Although many researchers have examined trade-offs involved in component sharing, little research has focused on the impact of component sharing on quality. In this paper, we examine how component sharing impacts one dimension of quality—reliability—defined as mean time to failure. Design considerations suggest that a component designed uniquely for a product will result in higher reliability due to the better fit of the component within the architecture of the product. On the other hand, the learning curve literature suggests that greater experience with a component can improve conformance quality, and can increase reliability via learning from end-user feedback. The engineering literature suggests that improved conformance in turn increases reliability. Sharing a component across multiple products increases experience, and hence, should increase reliability. Using data from the automotive industry, we find support for the hypothesis that higher component reliability is associated with higher cumulative experience with a component. Further, we find support for the hypothesis that higher component reliability is associated with a component that has been designed uniquely for a product. This finding suggests that the popular design strategy of component sharing can in some cases compromise product quality, via reduced reliability.
The Effect of Product Introduction Delays on Operating Performance
Authors: Hendricks Kevin B. , Singhal Vinod R.
Published: March 2008

This paper provides empirical evidence on the effect of product introduction delays on accounting-based measures of operating performance. Based on a diverse set of 450 publicly traded firms that experienced product introduction delays, we find that delays have a statistically significant negative effect on profitability. Depending on the method used to estimate abnormal performance, the median abnormal decline in return on assets (ROA) ranges from 2.70% to 3.44% over a three-year period around the year of the delay announcement. The median decline in sales over assets ranges from 5.92% to 10.99%, and the median decline in return on sales ranges from 1.48% to 3.06%. Cross-sectional regression analysis indicates that the impact of delays on abnormal ROA is more negative for smaller firms, and for firms that are more profitable before the delay. Furthermore, the impact is more negative for firms that operate in industries that are larger and more profitable. We also find a positive association between abnormal ROA and abnormal stock price performance around the product introduction delay announcements.