T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

PubMed

Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

2015-02-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase.

PubMed

Yamamoto, Sachiko; Takehara, Munenori; Ushimaru, Makoto

2017-01-01

SERCA maintains intracellular Ca 2+ homeostasis by sequestering cytosolic Ca 2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:1 9-cis ) and linoleamide (18:2 9,12-cis ), induce an increase in intracellular Ca 2+ levels, which might be caused by their inhibition of SERCA. Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca 2+ -ATPase activity of SERCA was examined. The Ca 2+ -ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC 50 15-53μM) and partially by oleamide (IC 50 8.3-34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:1 9-trans ), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca 2+ and the apparent maximum velocity of Ca 2+ -ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca 2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca 2+ affinity of the enzyme. Our findings imply a novel role of these PFAAs as modulators of intracellular Ca 2+ homeostasis via regulation of SERCA activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

PubMed

Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Phospholamban mutants compete with wild type for SERCA binding in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruber, Simon J.; Haydon, Suzanne; Thomas, David D., E-mail: ddt@umn.edu

2012-04-06

Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCAmore » activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.« less

Characterization of sarcoplasmic reticulum Ca(2+) ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy.

PubMed

Guglielmi, V; Oosterhof, A; Voermans, N C; Cardani, R; Molenaar, J P; van Kuppevelt, T H; Meola, G; van Engelen, B G; Tomelleri, G; Vattemi, G

2016-06-01

Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes.

PubMed

Corvazier, Elisabeth; Bredoux, Raymonde; Kovács, Tünde; Enouf, Jocelyne

2009-03-01

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

PubMed

Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

2018-05-01

The cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

PubMed Central

Nassal, Michelle M. J.; Wan, Xiaoping; Laurita, Kenneth R.; Cutler, Michael J.

2015-01-01

Background Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias. Methods Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol. Results As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca2+ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20). Conclusions Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic. PMID:26352986

Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.

PubMed

Asahi, M; Kimura, Y; Kurzydlowski, K; Tada, M; MacLennan, D H

1999-11-12

In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

PubMed

Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

2017-06-02

Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

PubMed

Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

2001-07-20

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Luteolin Modulates SERCA2a Leading to Attenuation of Myocardial Ischemia/ Reperfusion Injury via Sumoylation at Lysine 585 in Mice.

PubMed

Du, Yinping; Liu, Ping; Xu, Tongda; Pan, Defeng; Zhu, Hong; Zhai, Nana; Zhang, Yanbin; Li, Dongye

2018-01-01

The myocardial sarcoplasmic reticulum calcium ATPase (SERCA2a) is a pivotal pump responsible for calcium cycling in cardiomyocytes. The present study investigated the effect of luteolin (Lut) on restoring SERCA2a protein level and stability reduced by myocardial ischemia/reperfusion (I/R) injury. We verified a hypothesis that Lut protected against myocardial I/R injury by regulating SERCA2a SUMOylation. The hemodynamic data, myocardial infarct size of intact hearts, apoptotic analysis, mitochondrial membrane potential (ΔΨm), the level of SERCA2a SUMOylation, and the activity and expression of SERCA2a were examined in vivo and in vitro to clarify the cardioprotective effects of Lut after SUMO1 was knocked down or over-expressed. The putative SUMO conjugation sites in mouse SERCA2a were investigated as the possible regulatory mechanism of Lut. Initially, we found that Lut reversed the SUMOylation and stability of SERCA2a as well as the expression of SUMO1, which were reduced by I/R injury in vitro. Furthermore, Lut increased the expression and activity of SERCA2a partly through SUMO1, thus improving ΔΨm and reducing apoptotic cells in vitro and promoting the recovery of heart function and reducing infarct size in vivo. We also demonstrated that SUMO acceptor sites in mouse SERCA2a involving lysine 585, 480 and 571. Among the three acceptor sites, Lut enhanced SERCA2a stability via lysine 585. Our results suggest that Lut regulates SERCA2a through SUMOylation at lysine 585 to attenuate myocardial I/R injury. © 2018 The Author(s). Published by S. Karger AG, Basel.

Artemisinin dimer anti-cancer activity correlates with heme-catalyzed ROS generation and ER stress induction

PubMed Central

Stockwin, Luke H.; Han, Bingnan; Yu, Sherry X.; Hollingshead, Melinda G.; ElSohly, Mahmoud A.; Gul, Waseem; Slade, Desmond; Galal, Ahmed M.; Newton, Dianne L.

2009-01-01

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anti-cancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the anti-oxidant L-NAC, the iron chelator desferroxamine, and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity while inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage; iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian ER-resident SERCA Ca2+-ATPases. A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity. PMID:19533749

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban.

PubMed

Martin, Peter D; James, Zachary M; Thomas, David D

2018-06-05

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial function in an experimental model of heart failure in large animals.

PubMed

Byrne, M J; Power, J M; Preovolos, A; Mariani, J A; Hajjar, R J; Kaye, D M

2008-12-01

Abnormal excitation-contraction coupling is a key pathophysiologic component of heart failure (HF), and at a molecular level reduced expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) is a major contributor. Previous studies in small animals have suggested that restoration of SERCA function is beneficial in HF. Despite this promise, the means by which this information might be translated into potential clinical application remains uncertain. Using a recently established cardiac-directed recirculating method of gene delivery, we administered adeno-associated virus 2 (AAV2)/1SERCA2a to sheep with pacing-induced HF. We explored the effects of differing doses of AAV2/1SERCA2a (low 1 x 10(10) d.r.p.; medium 1 x 10(12) d.r.p. and high 1 x 10(13) d.r.p.) in conjunction with an intra-coronary delivery group (2.5 x 10(13) d.r.p.). At the end of the study, haemodynamic, echocardiographic, histopathologic and molecular biologic assessments were performed. Cardiac recirculation delivery of AAV2/1SERCA2a elicited a dose-dependent improvement in cardiac performance determined by left ventricular pressure analysis, (+d P/d t(max); low dose -220+/-70, P>0.05; medium dose 125+/-53, P<0.05; high dose 287+/-104, P<0.05) and echocardiographically (fractional shortening: low dose -3+/-2, P>0.05; medium dose 1+/-2, P>0.05; high dose 6.5+/-3.9, P<0.05). In addition to favourable haemodynamic effects, brain natriuretic peptide expression was reduced consistent with reversal of the HF molecular phenotype. In contrast, direct intra-coronary infusion did not elicit any effect on ventricular function. As such, AAV2/1SERCA2a elicits favourable functional and molecular actions when delivered in a mechanically targeted manner in an experimental model of HF. These observations lay a platform for potential clinical translation.

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

PubMed

Little, Alexander G; Seebacher, Frank

2013-09-15

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the role of TH in modulating SERCA1 expression during cold exposure may have predisposed it to regulate endothermic thermogenesis.

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy.

PubMed

Ceholski, Delaine K; Trieber, Catharine A; Young, Howard S

2012-05-11

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

Carbonylation Contributes to SERCA2a Activity Loss and Diastolic Dysfunction in a Rat Model of Type 1 Diabetes

PubMed Central

Shao, Chun Hong; Capek, Haley L.; Patel, Kaushik P.; Wang, Mu; Tang, Kang; DeSouza, Cyrus; Nagai, Ryoji; Mayhan, William; Periasamy, Muthu; Bidasee, Keshore R.

2011-01-01

OBJECTIVE Approximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ATP-driven pump that translocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized. RESEARCH DESIGN AND METHODS The streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca2+ uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss. RESULTS After 6–7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca2+ was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO's effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction. CONCLUSIONS These data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes. PMID:21300842

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.« less

Intramyocardial injection of SERCA2a-expressing lentivirus improves myocardial function in doxorubicin-induced heart failure.

PubMed

Mattila, Minttu; Koskenvuo, Juha; Söderström, Mirva; Eerola, Kim; Savontaus, Mikko

2016-07-01

Doxorubicin is an effective anticancer drug. The major limitation to its use is the induction of dose-dependent cardiomyopathy. No specific treatment exists for doxorubicin-induced cardiomyopathy and treatments used for other forms of heart failure have only limited beneficial effects. The contraction-relaxation cycle of the heart is controlled by cytosolic calcium concentrations, which, in turn, are critically regulated by the activity of the sarcoplasmic reticulum Ca(2) (+) ATPase (SERCA2a) pump. We hypothesized that SERCA2a gene transfer would ameliorate doxorubicin-induced cardiomyopathy. Lentiviral vectors LV-SERCA2a-GFP and LV-GFP were constructed and in vitro gene transfer of LV-SERCA2a-GFP confirmed SERCA2a expression by western blot analysis. Heart failure was induced by giving a single intraperitoneal injection of doxorubicin. LV-SERCA2a-GFP, LV-GFP vectors and phosphate-buffered saline (PBS) were injected under echocardiographic control to the anterior wall of the left ventricle. Echocardiography analyses were performed on the injection day and 28 days postinjection. On the injection day, there were no significant differences in the average ejection fractions (EFs) among SERCA2a (40.0%), GFP (41.1%) and PBS (39.4%) injected animals. On day 28, EF in the SERCA2a group had increased by 16.6 ± 6.7% to 46.4 ± 2.1%. By contrast, EFs in the GFP (40.2 ± 1.3%) and PBS (40.6 ± 1.4%) groups remained at pre-injection levels. In addition, end systolic and end diastolic left ventricle volumes were significantly smaller in the SERCA2a group compared to controls. SERCA2a gene transfer significantly improves left ventricle function and dimensions in doxorubicin-induced cardiomyopathy, thus making LV-SERCA2a gene transfer an attractive treatment modality for doxorubicin-induced heart failure. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Prevention of Atrial Fibrillation by Using Sarcoplasmic Reticulum Calcium ATPase Pump Overexpression in a Rabbit Model of Rapid Atrial Pacing.

PubMed

Wang, Hong Li; Zhou, Xian Hui; Li, Zhi Qiang; Fan, Ping; Zhou, Qi Na; Li, Yao Dong; Hou, Yue Mei; Tang, Bao Peng

2017-08-16

BACKGROUND Recent research suggests that abnormal Ca2+ handling plays a role in the occurrence and maintenance of atrial fibrillation (AF). Therefore, Ca2+ release and ingestion depend on properties of the ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+ATPase2a (SERCA2a). This study aimed to detect whether SERCA2a gene overexpression has a preventive effect on atrial fibrillation caused by rapid pacing right atrium. MATERIAL AND METHODS Forty-eight New Zealand white rabbits were randomly divided into a control group, AF group, AAV9/GFP group, and AAV9/SERCA2a group. The right atrium was rapidly paced at 600 beats/min for 30 days after an intraperitoneal injection of an adeno-associated virus expressing the SERCA2a gene and GFP. The AF induction rate and the effective refraction period (ERP) were measured after 0, 4, 8, 12, and 24 h of pacing. Western blot analysis was used to test for the expression of SERCA2a. Changes in atrial tissue structure were observed by H&E staining and electron microscopy. RESULTS The AF induction rate was higher in the AF groups than in the AAV9/SERCA2a group at different time points of pacing. After 12 h of pacing, ERP was significantly prolonged in the AAV9/SERCA2a group compared to the AF and AAV9/GFP groups (p<0.05). SERCA2a protein expression was significantly lower in the AF and AAV9/GFP groups compared to the control group (p<0.05), while expression was significantly higher in the AAV9/SERCA2a group than in the AF and AAV9/GFP groups (p<0.05). The myocardial structure of the AAV9/SERCA2a group was significantly improved compared with the AF group, indicating that SERCA2a overexpression relieved the structural remodeling of atrial fibrillation. CONCLUSIONS SERCA2a overexpression is capable of suppressing ERP shortening and AF induced by rapid pacing atrium. SERCA2a gene therapy is expected to be a new anti-atrial fibrillation strategy.

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat.

PubMed

Murphy, Robyn M; Larkins, Noni T; Mollica, Janelle P; Beard, Nicole A; Lamb, Graham D

2009-01-15

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 +/- 2 and 10 +/- 1 micromol (l fibre volume)(-1), quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (approximately 20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca2+-ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (approximately 20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca2+ in the SR whilst maintaining free [Ca2+] in the SR at sufficiently low levels that Ca2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

Novel role of Sarco/endoplasmic reticulum calcium ATPase 2 in development of colorectal cancer and its regulation by F36, a curcumin analog.

PubMed

Fan, Lu; Li, Ang; Li, Wanshuai; Cai, Peifen; Yang, Baofang; Zhang, Minxia; Gu, Yanhong; Shu, Yongqian; Sun, Yang; Shen, Yan; Wu, Xuefeng; Hu, Gang; Wu, Xudong; Xu, Qiang

2014-10-01

Sarco/endoplasmic reticulum calcium ATPase (SERCA) enzymes play important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. Here, we reported that SERCA2 expression was positively correlated with tumor node metastasis (TNM) stages (n=75, P=0.0251) and grades (n=63, P=0.0146) of patients with colorectal cancer. The animal experiments demonstrated that SERCA2 expression was consistent with PCNA staining of intestinal tissues of male C57BL/6J-Apc(Min/)JNju mice. Besides, SERCA2 expression was also increased in undifferentiated HT-29 cells as compared with that in differentiated HT-29gal cells. Moreover, SERCA2 overexpression promoted proliferation and migration of SW480 cells via activating MAPK and AKT signaling pathways, while silence of SERCA2 inhibited the proliferation and migration of SW480 cells. In addition, we identified that a curcumin analog, F36, exhibited more potent inhibitory effect in colorectal cancer cells than curcumin through inhibiting SERCA2 expression. Taken together, our findings indicate that SERCA2 is involved in the malignant progress of colorectal cancer and maybe a therapeutic target for colorectal cancer treatment. Curcumin analog F36 shows enhanced anti-cancer activity in colorectal cancer cells by targeting SERCA2. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats.

PubMed

Vetter, Roland; Rehfeld, Uwe; Reissfelder, Christoph; Fechner, Henry; Seppet, Enn; Kreutz, Reinhold

2011-03-01

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

Effects of moderate heart failure and functional overload on rat plantaris muscle

NASA Technical Reports Server (NTRS)

Spangenburg, Espen E.; Lees, Simon J.; Otis, Jeff S.; Musch, Timothy I.; Talmadge, Robert J.; Williams, Jay H.

2002-01-01

It is thought that changes in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) of skeletal muscle contribute to alterations in skeletal muscle function during congestive heart failure (CHF). It is well established that exercise training can improve muscle function. However, it is unclear whether similar adaptations will result from exercise training in a CHF patient. Therefore, the purpose of this study was to determine whether skeletal muscle during moderate CHF adapts to increased activity, utilizing the functional overload (FO) model. Significant increases in plantaris mass of the CHF-FO and sham-FO groups compared with the CHF and control (sham) groups were observed. Ca(2+) uptake rates were significantly elevated in the CHF group compared with all other groups. No differences were detected in Ca(2+) uptake rates between the CHF-FO, sham, and sham-FO groups. Increases in Ca(2+) uptake rates in moderate-CHF rats were not due to changes in SERCA isoform proportions; however, FO may have attenuated the CHF-induced increases through alterations in SERCA isoform expression. Therefore, changes in skeletal muscle Ca(2+) handling during moderate CHF may be due to alterations in regulatory mechanisms, which exercise may override, by possibly altering SERCA isoform expression.

SERCA directs cell migration and branching across species and germ layers

PubMed Central

Lansdale, Nick; Navarro, Sonia; Truong, Thai V.; Bower, Dan J.; Featherstone, Neil C.; Connell, Marilyn G.; Al Alam, Denise; Frey, Mark R.; Trinh, Le A.; Fernandez, G. Esteban; Warburton, David; Fraser, Scott E.; Bennett, Daimark; Jesudason, Edwin C.

2017-01-01

ABSTRACT Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding. PMID:28821490

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

PubMed

Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

PubMed

Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-11-27

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

PubMed Central

Meraviglia, Viviana; Sacchetto, Roberta; Motta, Benedetta M.; Corti, Corrado; D’Elia, Yuri; Rosato-Siri, Marcelo D.; Suffredini, Silvia; Pompilio, Giulio; Pramstaller, Peter P.; Stilli, Donatella

2018-01-01

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. PMID:29385061

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle.

PubMed

Lamboley, C R; Murphy, R M; McKenna, M J; Lamb, G D

2014-03-15

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

PubMed

Betzer, Cristine; Lassen, Louise Berkhoudt; Olsen, Anders; Kofoed, Rikke Hahn; Reimer, Lasse; Gregersen, Emil; Zheng, Jin; Calì, Tito; Gai, Wei-Ping; Chen, Tong; Moeller, Arne; Brini, Marisa; Fu, Yuhong; Halliday, Glenda; Brudek, Tomasz; Aznar, Susana; Pakkenberg, Bente; Andersen, Jens Peter; Jensen, Poul Henning

2018-05-01

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca 2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca 2+ levels followed by a later Ca 2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca 2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome.

PubMed

Kranias, Evangelia G; Hajjar, Roger J

2012-06-08

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

Improved exercise capacity and reduced systemic inflammation after adenoviral-mediated SERCA-2a gene transfer.

PubMed

Gupta, Dipin; Palma, Jon; Molina, Ezequiel; Gaughan, John P; Long, Walter; Houser, Steven; Macha, Mahender

2008-04-01

We hypothesized that sarcoplasmic reticulum Ca2+ ATPase pump (SERCA-2a) gene delivery would have beneficial effects upon exercise capacity and markers of inflammation in the setting of heart failure. A pressure-overload model of experimental heart failure was used in rats. Following a decrease in fractional shortening of >or=25%, animals underwent intracoronary adenoviral-mediated gene transfection using SERCA-2a. Heart failure animals were randomized to receive the SERCA-2a gene, the beta galactosidase (control) gene, or followed without any further intervention. Exercise and hemodynamic testing were performed, and myocardial and systemic markers of inflammation were assayed after 7 and 21 d. Animals receiving Ad.SERCA-2a showed an increase in exercise tolerance (499.0 +/- 14.9 versus 312.8 +/- 10.5 s, P < 0.0001) relative to Ad.Gal group. Groups treated with Ad.SERCA-2a had significantly decreased serum levels of the inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor-alpha compared with Ad.Gal-treated animals. Serum levels of atrial natriuretic peptide were decreased in animals receiving Ad.SERCA-2a compared with animals receiving Ad.Gal at day 7 (0.35 +/- 0.03 versus 0.52 +/- 0.11 pg/mL, P = 0.001). Myocardial levels of the proapoptotic protein bax were reduced in Ad.SERCA-2a -treated animals compared with those receiving Ad.Gal at day 7 (protein level/actin: 0.24 +/- 0.05 versus 0.33 +/- 0.04, P = 0.04) and day 21 (protein level/actin: 0.61 +/- 0.04 versus 0.69 +/- 0.01, P = 0.001). Genetic modulation of heart failure using the SERCA-2a gene was associated with improvement in cardiac function and exercise capacity as well as improvements in heart-failure associated inflammatory markers.

Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells

PubMed Central

Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie

2005-01-01

Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1–3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration. PMID:16250893

Hyperthyroidism causes mechanical insufficiency of myocardium with possibly increased SR Ca2+-ATPase activity.

PubMed

Takeuchi, Koh; Minakawa, M; Otaki, M; Odagiri, S; Itoh, K; Murakami, A; Yaku, H; Kitamura, N

2003-12-01

Hyperthyroidism is known to affect multiple organ functions, and thyroid hormone has been known to improve myocardial function in a failing heart. The purpose of this study is to elucidate the functional and metabolic effects of thyroid hormone on myocardium in a rat model exposed to long-term excess thyroid hormone, particularly focusing on the SR Ca(2+)-ATPase (SERCA2) function. 3,5,3'-Triiodo-L-thyronine (T3), or the vehicle, was subcutaneously given for 4 weeks (T3 and control [C] group). Bolus I.V. Thapsigargin (TG) was used to test the SERCA2 function (C-TG and T3-TG) in Langendorff perfused heart. Myocardial functions such as LV-developed pressure (LVDP; mmHg), +/- dP/dt (mmHg/s), tau (ms), and oxygen consumption (MVO(2); ml/min/g wt) were measured. SERCA2 and GLUT4 protein level were also evaluated by Western immunoblotting. Left ventricle to body weight (LV/BW) ratio was significantly higher in the T3 group. Both negative dP/dt and tau were significantly decreased by TG. It is interesting that the decrement of negative dP/dt and tau attained by TG was significantly larger in the hyperthyroid group (T3-TG) than in a normal heart (C-TG). SERCA2 and GLUT4 protein levels were not significantly different between control and the T3 group. We conclude that prolonged exposure to thyroid hormone causes hypertrophy of the myocardium and an augmentation of the SR Ca(2+) ATPase activity. Care must be taken in hyperthyroid heart during the ischemia-reperfusion process where the SRECA2 function is inhibited.

Concomitant Intravenous Nitroglycerin With Intracoronary Delivery of AAV1.SERCA2a Enhances Gene Transfer in Porcine Hearts

PubMed Central

Karakikes, Ioannis; Hadri, Lahouaria; Rapti, Kleopatra; Ladage, Dennis; Ishikawa, Kiyotake; Tilemann, Lisa; Yi, Geng-Hua; Morel, Charlotte; Gwathmey, Judith K; Zsebo, Krisztina; Weber, Thomas; Kawase, Yoshiaki; Hajjar, Roger J

2012-01-01

SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts. PMID:22215018

MALONDIALDEHYDE AND 4-HYDROXYNONENAL ADDUCTS ARE NOT FORMED ON CARDIAC RYANODINE RECEPTOR (RyR2) AND SARCO(ENDO)PLASMIC RETICULUM Ca2+ ATPase (SERCA2) IN DIABETES

PubMed Central

Moore, Caronda J.; Shao, Chun Hong; Nagai, Ryoji; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

2013-01-01

Recently, we reported an elevated level of glucose-derived carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca2+ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes. This study used Western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, Nε-carboxy(methyl)lysine (CML), pentosidine and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca2+ kinetics in myocytes from control, diabetic and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from control or 8 weeks diabetic rats with altered evoked Ca2+ transients. However, CML, pentosidine, and pyrraline adducts were elevated 3–5 fold (p<0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-reactive carbonyl species scavenger) reduced CML, pentosidine and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca2+ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca2+ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca2+ dysregulation. PMID:23354458

Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

PubMed

Ahmad, Shama; Ahmad, Aftab; Hendry-Hofer, Tara B; Loader, Joan E; Claycomb, William C; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L; Chandler, Joshua D; Day, Brian J; Veress, Livia A; White, Carl W

2015-04-01

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

PubMed Central

Ahmad, Aftab; Hendry-Hofer, Tara B.; Loader, Joan E.; Claycomb, William C.; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L.; Chandler, Joshua D.; Day, Brian J.; Veress, Livia A.; White, Carl W.

2015-01-01

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881

Fiber-specific regulation of Ca(2+)-ATPase isoform expression by thyroid hormone in rat skeletal muscle.

PubMed

van der Linden, C G; Simonides, W S; Muller, A; van der Laarse, W J; Vermeulen, J L; Zuidwijk, M J; Moorman, A F; van Hardeveld, C

1996-12-01

We studied the effect of thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the expression of sarcoplasmic reticulum (SR) fast- and slow-type Ca(2+)-ATPase isoforms, SERCA1 and SERCA2a, respectively, and total SR Ca(2+)-ATPase activity in rat skeletal muscle. Cross sections and homogenates of soleus and extensor digitorum longus muscles from hypo-, eu-, and hyperthyroid rats were examined, and expression of Ca(2+)-ATPase isoforms in individual fibers was compared with expression of fast (MHC II) and slow (MHC I) myosin heavy chain isoforms. In both muscles, T3 induced a coordinated and full conversion to a fast-twitch phenotype in one-half of the fibers that were slow twitch in the absence of T3. The conversion was partial in the other one-half of the fibers, giving rise to a mixed phenotype. The stimulation by T3 of total SERCA expression in all fibers was reflected by increased SR Ca(2+)-ATPase activity. The time course of the T3-induced changes of SERCA isoform expression was examined 1-14 days after the start of daily T3 treatment of euthyroid rats. SERCA1 expression was stimulated by T3 at a pretranslational level in all fibers. SERCA2a mRNA expression was transiently stimulated and disappeared in a subset of fibers. In these fibers SR Ca(2+)-ATPase activity was high because of high SERCA1 protein levels. These data suggest that the ultimate downregulation of SERCA2a expression, which is always associated with high SR Ca(2+)-ATPase activities, occurs at a pretranslational level.

Sodium Accumulation in SERCA Knockout-Induced Heart Failure

PubMed Central

Li, Liren; Louch, William E.; Niederer, Steven A.; Aronsen, Jan M.; Christensen, Geir; Sejersted, Ole M.; Smith, Nicolas P.

2012-01-01

In cardiomyocytes, a major decrease in the level of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) can severely impair systolic and diastolic functions. In mice with cardiomyocyte-specific conditional excision of the Serca2 gene (SERCA2 KO), end-stage heart failure developed between four and seven weeks after gene deletion combined with [Na+]i elevation and intracellular acidosis. In this study, to investigate the underpinning changes in Ca2+ dynamics and metabolic homeostasis, we developed data-driven mathematical models of Ca2+ dynamics in the ventricular myocytes of the control, four-week, and seven-week SERCA2 knockout (KO) mice. The seven-week KO model showed that elevated [Na+]i was due to increased Na+ influxes through the Na+/Ca2+ exchanger (NCX) and the Na+/H+ exchanger, with the latter exacerbated by intracellular acidosis. Furthermore, NCX upregulation in the seven-week KO model resulted in increased ATP consumption for ion transport. Na+ accumulation in the SERCA KO due to NCX upregulation and intracellular acidosis potentially play a role in the development of heart failure, by initiating a reinforcing cycle involving: a mismatch between ATP demand and supply; an increasingly compromised metabolism; a decreased pHi; and, finally, an even greater [Na+]i elevation. PMID:22824267

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

PubMed Central

Gravina, Fernanda S.; van Helden, Dirk F.; Kerr, Karen P.; de Oliveira, Ramatis B.; Jobling, Phillip

2014-01-01

Background/Aims The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca2+ release from sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) -dependent intracellular Ca2+ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking. Methodology/Principal Findings Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K+ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca2+ stores, but were inhibited in low Ca2+ solution or in the presence of nifedipine, an inhibitor of L-type Ca2+channels. ICs were found in small numbers in the mouse cervix but not in the vagina. Conclusions/Significance Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca2+ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges. PMID:25337931

Phasic contractions of the mouse vagina and cervix at different phases of the estrus cycle and during late pregnancy.

PubMed

Gravina, Fernanda S; van Helden, Dirk F; Kerr, Karen P; de Oliveira, Ramatis B; Jobling, Phillip

2014-01-01

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca2+ release from sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) -dependent intracellular Ca2+ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking. Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K+ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca2+ stores, but were inhibited in low Ca2+ solution or in the presence of nifedipine, an inhibitor of L-type Ca2+channels. ICs were found in small numbers in the mouse cervix but not in the vagina. Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca2+ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.

A natural compound jaceosidin ameliorates endoplasmic reticulum stress and insulin resistance via upregulation of SERCA2b.

PubMed

Ouyang, Zijun; Li, Wanshuai; Meng, Qianqian; Zhang, Qi; Wang, Xingqi; Elgehama, Ahmed; Wu, Xudong; Shen, Yan; Sun, Yang; Wu, Xuefeng; Xu, Qiang

2017-05-01

Increased endoplasmic reticulum (ER) stress has emerged as a vital contributor to dysregulated glucose homeostasis, and impaired function of sarco-endoplasmic reticulum Ca 2+ -ATPase 2b (SERCA2b) is one of the central mechanisms underlying ER stress. In this study, we reported that SERCA2b upregulation contributed to the amelioration of ER stress and insulin resistance by a small natural compound jaceosidin. In a model of differentiated C2C12 myotubes, jaceosidin-triggered SERCA2b upregulation enhanced insulin sensitivity and decreased ER stress. Moreover, the activity of Ca 2+ -ATPase in thapsigargin-treated myotubes was also augmented by jaceosidin. Furthermore, jaceosidin significantly suppressed blood glucose levels, improved glucose tolerance and lowered body weight, but did not alter food intake in insulin-resistant obese mice. In addition, this compound markedly reduced lipid accumulation, suppressed the expression of lipogenic genes in liver and ameliorated liver injury. The ameliorative effects of jaceosidin were due to its ability to reduce ER stress via increasing the expression of SERCA2b in the muscles of obese mice. Taken together, jaceosidin could improve ER stress and attenuate insulin resistance via SERCA2b upregulation in mice skeletal muscles. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Presence of a thapsigargin-sensitive calcium pump in Trypanosoma evansi: Immunological, physiological, molecular and structural evidences.

PubMed

Pérez-Gordones, M C; Serrano, M L; Rojas, H; Martínez, J C; Uzcanga, G; Mendoza, M

2015-12-01

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG. Copyright © 2015 Elsevier Inc. All rights reserved.

New insights into SERCA2a gene therapy in heart failure: pay attention to the negative effects of B-type natriuretic peptides.

PubMed

Zhai, Yuting; Luo, Yuanyuan; Wu, Pei; Li, Dongye

2018-05-01

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) is a target of interest in gene therapy for heart failure with reduced ejection fraction (HFrEF). However, the results of an important clinical study, the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, were controversial. Promising results were observed in the CUPID 1 trial, but the results of the CUPID 2 trial were negative. The factors that caused the controversial results remain unclear. Importantly, enrolled patients were required to have a higher plasma level of B-type natriuretic peptide (BNP) in the CUPID 2 trial. Moreover, BNP was shown to inhibit SERCA2a expression. Therefore, it is possible that high BNP levels interact with treatment effects of SERCA2a gene transfer and accordingly lead to negative results of CUPID 2 trial. From this point of view, effects of SERCA2a gene therapy should be explored in heart failure with preserved ejection fraction, which is characterised by lower BNP levels compared with HFrEF. In this review, we summarise the current knowledge of SERCA2a gene therapy for heart failure, analyse potential interaction between BNP levels and therapeutic effects of SERCA2a gene transfer and provide directions for future research to solve the identified problems. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

PubMed

Gong, Haibin; Li, Yanfei; Wang, Lei; Lv, Qian; Wang, Xiuli

2016-09-01

The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca 2+ and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca2+-ATPases in muscle and heart.

PubMed

Algenstaedt, P; Antonetti, D A; Yaffe, M B; Kahn, C R

1997-09-19

Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the lambdaEXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca2+-ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2- to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca2+-ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

PubMed Central

Silveira, C.F.S.M.P.; Campos, D.H.S.; Freire, P.P.; Deus, A.F.; Okoshi, K.; Padovani, C.R.; Cicogna, A.C.

2017-01-01

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group. PMID:28423119

Characterization of sarcoplasmic reticulum Ca{sup 2+} ATPase nucleotide binding domain mutants using NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myint, Wazo; Gong, Qingguo; Ahn, Jinwoo

2011-02-04

Research highlights: {yields} Structural consequence by substitution mutations on the isolated SERCA-nucleotide binding (SERCA-N) domain was studied. {yields} The study fills a gap between the previous clinical, physiological, and biochemical data and the molecular basis of SERCA-N. {yields} The E412G mutation, known to be seen in patients with Darier's disease, was found to maintain the active conformation but exhibit reduced protein stability. -- Abstract: Sarcoplasmic reticulum Ca{sup 2+} ATPase (SERCA) is essential for muscle function by transporting Ca{sup 2+} from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolatedmore » SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. {sup 15}N-{sup 1}H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant ({approx}2.5 mM) was similar to that of WT ({approx}3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.« less

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress.

PubMed

Ihara, Yoshito; Kageyama, Kan; Kondo, Takahito

2005-04-22

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.

REACTIVE CARBONYL SPECIES AND THEIR ROLES IN SARCOPLASMIC RETICULUM Ca2+ CYCLING DEFECT IN THE DIABETIC HEART

PubMed Central

Tian, Chengju; Alomar, Fadhel; Moore, Caronda J; Shao, Chun Hong; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

2016-01-01

Efficient and rhythmic cardiac contractions depend critically on the adequate and synchronized release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptor Ca2+ release channels (RyR2) and its reuptake via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a). It is well established that this orchestrated process becomes compromised in diabetes. What remain incompletely defined are the molecular mechanisms responsible for the dysregulation of RyR2 and SERCA2a in diabetes. Earlier, found elevated levels of carbonyl adducts on RyR2 and SERCA2a isolated from hearts of type 1 diabetic rats and showed the presence of these post-translational modifications compromised their functions. We also showed that these mono- and di-carbonyl reactive carbonyl species (RCS) do not indiscriminately react with all basic amino acid residues on RyR2 and SERCA2a; some residues are more susceptible to carbonylation (modification by RCS) than others. A key unresolved question in the field is which of the many RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a? This brief review introduces readers to the field of RCS and their roles in perturbing SR Ca2+ cycling in diabetes. It also provides new experimental evidence that not all RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a, methylglyoxal and glyoxal preferentially do. PMID:23430128

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition

PubMed Central

Stary, Victoria; Puppala, Dheeraj; Scherrer-Crosbie, Marielle; Dillmann, Wolfgang H.

2016-01-01

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca2+ ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca2+ indicators to selectively measure mitochondrial and cytosolic Ca2+ using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 − (small Ca2+ intensity)/(large Ca2+ intensity)]. Blocking of complex I and II, cytochrome-c oxidase, F0F1 synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P < 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P < 0.04). N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P < 0.001). CGP, an antagonist of the mitochondrial Na+-Ca2+ exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P < 0.0001). The major findings of this study are that impairment of mitochondrial Ca2+ cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca2+ content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca2+ signaling in myocytes from diseased hearts, leading to new therapeutic targets. PMID:26846549

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

PubMed Central

Cabello, Nuria; Llach, Anna; Vallmitjana, Alexander; Benítez, Raúl; Badimon, Lina; Cinca, Juan; Llorente-Cortés, Vicenta; Hove-Madsen, Leif

2013-01-01

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal. PMID:23516438

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

PubMed

Mortimer, Nathan T; Goecks, Jeremy; Kacsoh, Balint Z; Mobley, James A; Bowersock, Gregory J; Taylor, James; Schlenke, Todd A

2013-06-04

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

Tracing Cytoplasmic Ca2+ Ion and Water Access Points in the Ca2+-ATPase

PubMed Central

Musgaard, Maria; Thøgersen, Lea; Schiøtt, Birgit; Tajkhorshid, Emad

2012-01-01

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca2+ ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca2+-free state of the transporter. Additional molecular dynamics simulations performed on a Ca2+-bound state of SERCA reveal a water-filled pathway that may be used by the Ca2+ ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca2+ entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca2+ translocation pathway toward the transmembrane ion-binding sites. PMID:22339863

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

PubMed

Espinoza-Fonseca, L Michel

2017-03-28

Ca 2+ transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca 2+ homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca 2+ uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca 2+ and other ions across the SR. During Ca 2+ uptake by the SR Ca 2+ -ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca 2+ transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca 2+ release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca 2+ transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

Resistance Training Regulates Cardiac Function through Modulation of miRNA-214

PubMed Central

Melo, Stéphano Freitas Soares; Barauna, Valério Garrone; Júnior, Miguel Araújo Carneiro; Bozi, Luiz Henrique Marchesi; Drummond, Lucas Rios; Natali, Antônio José; de Oliveira, Edilamar Menezes

2015-01-01

Aims: To determine the effects of resistance training (RT) on the expression of microRNA (miRNA)-214 and its target in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), and on the morphological and mechanical properties of isolated left ventricular myocytes. Main methods: Male Wistar rats were divided into two groups (n = 7/group): Control (CO) or trained (TR). The exercise-training protocol consisted of: 4 × 12 bouts, 5×/week during 8 weeks, with 80% of one repetition maximum. Key findings: RT increased the left ventricular myocyte width by 15% and volume by 12%, compared with control animals (p < 0.05). The time to half relaxation and time to peak were 8.4% and 4.4% lower, respectively, in cells from TR group as compared to CO group (p < 0.05). RT decreased miRNA-214 level by 18.5% while its target SERCA2a expression were 18.5% higher (p < 0.05). Significance: Our findings showed that RT increases single left ventricular myocyte dimensions and also leads to faster cell contraction and relaxation. These mechanical adaptations may be related to the augmented expression of SERCA2a which, in turn, may be associated with the epigenetic modification of decreased miRNA-214 expression. PMID:25822872

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition.

PubMed

Stary, Victoria; Puppala, Dheeraj; Scherrer-Crosbie, Marielle; Dillmann, Wolfgang H; Armoundas, Antonis A

2016-04-15

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca(2+)ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca(2+)indicators to selectively measure mitochondrial and cytosolic Ca(2+)using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 - (small Ca(2+)intensity)/(large Ca(2+)intensity)]. Blocking of complex I and II, cytochrome-coxidase, F0F1synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P< 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P< 0.04).N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P< 0.001). CGP, an antagonist of the mitochondrial Na(+)-Ca(2+)exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P< 0.0001). The major findings of this study are that impairment of mitochondrial Ca(2+)cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca(2+)content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca(2+)signaling in myocytes from diseased hearts, leading to new therapeutic targets. Copyright © 2016 the American Physiological Society.

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production.

PubMed

Bocchi, Leonardo; Savi, Monia; Naponelli, Valeria; Vilella, Rocchina; Sgarbi, Gianluca; Baracca, Alessandra; Solaini, Giancarlo; Bettuzzi, Saverio; Rizzi, Federica; Stilli, Donatella

2018-06-15

Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2. © 2018 The Author(s). Published by S. Karger AG, Basel.

Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes

NASA Astrophysics Data System (ADS)

Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul

2000-04-01

Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

PubMed

Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

2012-05-01

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Right ventricular beneficial effects of beta adrenergic receptor kinase inhibitor (betaARKct) gene transfer in a rat model of severe pressure overload.

PubMed

Molina, Ezequiel J; Gupta, Dipin; Palma, Jon; Gaughan, John P; Macha, Mahender

2009-06-01

Heart failure is associated with abnormalities in betaAR cascade regulation, calcium cycling, expression of inflammatory mediators and apoptosis. Adenoviral mediated gene transfer of betaARKct has beneficial indirect effects on these pathologic processes upon the left ventricular myocardium. The concomitant biochemical changes that occur in the right ventricle have not been well characterized. Sprague-Dawley rats underwent aortic banding and were followed by echocardiography. After a decrease in fractional shortening of 25% from baseline, intracoronary injection of adenoviral-betaARKct (n=14) or adenoviral-beta-galactosidase (control, n=13) was performed. Rats were randomly euthanized on post-operative day 7, 14 or 21. Protein analysis including RV myocardial levels of betaARKct, betaARK1, SERCA(2a), inflammatory tissue mediators (IL-1, IL-6 and TNF-alpha), apoptotic markers (bax and bak), and MAP kinases (jnk, p38 and erk) was performed. ANOVA was employed for group comparison. Adenoviral-betaARKct treated animals showed increased expression of betaARKct and decreased levels of betaARK1 compared with controls. This treatment group also demonstrated normalization of SERCA(2a) expression and decreased levels of the inflammatory markers IL-1, IL-6 and TNF-alpha. The pro-apoptotic markers bax and bak were similarly improved. Ventricular levels of the MAP kinase jnk were increased. Differences were most significant 7 days after gene transfer, but the majority of these changes persisted at 21 days. These results suggest that attenuation of the pathologic mechanisms of beta adrenergic receptor desensitization, SERCA(2a) expression, inflammation and apoptosis, not only occur in the left ventricle but also in the right ventricular myocardium after intracoronary gene transfer of betaARKct during heart failure.

Different oxidative status and expression of calcium channel components in stress-induced dysfunctional chicken muscle.

PubMed

Xing, T; Zhao, X; Wang, P; Chen, H; Xu, X; Zhou, G

2017-04-01

The objective of this study was to assess the effects of transport stress at high ambient temperatures on the oxidation status and the expression of essential elements responsible for the Ca transport (sarco- (endo-) plasmic reticulum Ca-ATPase (SERCA1) and the ryanodine receptor (RyR) in (PM) muscles of broilers. Briefly, Arbor Acres broiler chickens ( = 112) were randomly categorized into 2 treatments: unstressed control (C) and 0.5 h transport (T). Each treatment consisted of 8 replicates of 7 birds each. Birds were transported according to a designed protocol. PM muscle samples in T group were collected and classified as normal (T-NOR) or pale, soft, and exudative-like (T-PSE) using meat quality parameters. The results indicated that production of corticosterone (CORT) and reactive oxygen species (ROS) increased significantly after transportation ( < 0.05). Thiobarbituric acid reactive substance values and carbonyl contents increased significantly in the T group ( < 0.05). Moreover, the extent of lipid peroxidation and protein oxidation was more severe in the T-PSE group compared to the T-NOR group ( < 0.05). The mRNA and protein expression of SERCA1 and αRyR increased in the T-NOR group but decreased significantly in the T-PSE group compared to the CON group ( < 0.05). The mRNA expression of βRyR was found to be enhanced in the T-NOR group compared to the CON group, whereas there was no difference in the T-PSE group ( < 0.05). The results indicate that short-distance transport of broilers affects their physiological responses and biochemical changes which may lead to different oxidative states and, importantly, to different expressions of SERCA and RyR. These induced changes in abnormal sarcoplasmic Ca homeostasis have significant implications for the development of PSE-like meat.

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

PubMed Central

Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

2016-01-01

Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the biphasic decay was replaced by slow decay. We conclude that, in the presence of adrenergic stimulation, Ca leak can produce biphasic decay; the slow phase results from the leak opposing Ca uptake by SERCA. The degree of leak determines whether decay of Ca waves, biphasic or monophasic, occurs. PMID:26537441

CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

PubMed

Neumann, Jake T; Diaz-Sylvester, Paula L; Fleischer, Sidney; Copello, Julio A

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

PubMed Central

Neumann, Jake T.; Diaz-Sylvester, Paula L.; Fleischer, Sidney

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. PMID:20923851

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

PubMed

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-04-28

The Golgi/secretory pathway Ca 2+ /Mn 2+ -transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca 2+ -dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca 2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di- tert -butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)

PubMed Central

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-01-01

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. PMID:28264934

The Evolutionary History of Sarco(endo)plasmic Calcium ATPase (SERCA)

PubMed Central

Altshuler, Ianina; Vaillant, James J.; Xu, Sen; Cristescu, Melania E.

2012-01-01

Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na+/K+ transporters, H+/K+ transporters, and plasma membrane Ca2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca2+ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain. PMID:23285113

The evolutionary history of sarco(endo)plasmic calcium ATPase (SERCA).

PubMed

Altshuler, Ianina; Vaillant, James J; Xu, Sen; Cristescu, Melania E

2012-01-01

Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+)/K(+) transporters, H(+)/K(+) transporters, and plasma membrane Ca(2+) pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca(2+) into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals.

PubMed

Evans, A Mark; Fameli, Nicola; Ogunbayo, Oluseye A; Duan, Jingxian; Navarro-Dorado, Jorge

2016-08-01

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

The Change of Left Ventricular Function in Rats with Subclinical Hypothyroid and the Effects of Thyroxine Replacement.

PubMed

Chen, Xuedi; Gao, Cuixia; Gong, Ningning; Wang, Yu; Tian, Limin

2018-01-01

The main purpose of this study was to explore the relationships between serca2a, Ryr2, adipokines, and the left ventricular function in the subclinical hypothyroidism with different TSH levels and to determine the impact of L-T4 treatment on these indexes. Sixty-five male Wistar rats were randomly divided into five groups: control group; sHT A, B, and C group; and sHT + T4 group. The sHT rats were induced by methimazole (MMI), and the sHT + T4 rats were administered with L-T4 treatment after 8 weeks of MMI administration. Serum TT4, TSH, APN, chemerin, and TNF- α were detected by radioimmunoassay kits and ELISA kits; left ventricular function was measured by PowerLab system via subclavian artery catheter. The expression of Serca2a, Ryr2, APN, chemerin, and TNF- α were detected by RT-PCR, Western blot, and immunohistochemistry. The sHT groups had significantly higher TSH, chemerin, and TNF- α and lower Serca2a, Ryr2, and APN. The left ventricular pressure and heart rate in sHT groups were significantly lower in control and sHT + T4 group. Histopathological examination revealed the pathological changes in the sHT rats' heart. L-T4 administration reduced TSH level and improved left ventricular function. TSH can impair left ventricular function by regulating several factors, and L-T4 treatment ameliorates it in sHT rats.

Human cardiomyocyte calcium handling and transverse tubules in mid-stage of post-myocardial-infarction heart failure.

PubMed

Høydal, Morten Andre; Kirkeby-Garstad, Idar; Karevold, Asbjørn; Wiseth, Rune; Haaverstad, Rune; Wahba, Alexander; Stølen, Tomas L; Contu, Riccardo; Condorelli, Gianluigi; Ellingsen, Øyvind; Smith, Godfrey L; Kemi, Ole J; Wisløff, Ulrik

2018-06-01

Cellular processes in the heart rely mainly on studies from experimental animal models or explanted hearts from patients with terminal end-stage heart failure (HF). To address this limitation, we provide data on excitation contraction coupling, cardiomyocyte contraction and relaxation, and Ca 2+ handling in post-myocardial-infarction (MI) patients at mid-stage of HF. Nine MI patients and eight control patients without MI (non-MI) were included. Biopsies were taken from the left ventricular myocardium and processed for further measurements with epifluorescence and confocal microscopy. Cardiomyocyte function was progressively impaired in MI cardiomyocytes compared with non-MI cardiomyocytes when increasing electrical stimulation towards frequencies that simulate heart rates during physical activity (2 Hz); at 3 Hz, we observed almost total breakdown of function in MI. Concurrently, we observed impaired Ca 2+ handling with more spontaneous Ca 2+ release events, increased diastolic Ca 2+ , lower Ca 2+ amplitude, and prolonged time to diastolic Ca 2+ removal in MI (P < 0.01). Significantly reduced transverse-tubule density (-35%, P < 0.01) and sarcoplasmic reticulum Ca 2+ adenosine triphosphatase 2a (SERCA2a) function (-26%, P < 0.01) in MI cardiomyocytes may explain the findings. Reduced protein phosphorylation of phospholamban (PLB) serine-16 and threonine-17 in MI provides further mechanisms to the reduced function. Depressed cardiomyocyte contraction and relaxation were associated with impaired intracellular Ca 2+ handling due to impaired SERCA2a activity caused by a combination of alteration in the PLB/SERCA2a ratio and chronic dephosphorylation of PLB as well as loss of transverse tubules, which disrupts normal intracellular Ca 2+ homeostasis and handling. This is the first study that presents these mechanisms from viable and intact cardiomyocytes isolated from the left ventricle of human hearts at mid-stage of post-MI HF. © 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes.

PubMed

Sonntag, Yonathan; Musgaard, Maria; Olesen, Claus; Schiøtt, Birgit; Møller, Jesper Vuust; Nissen, Poul; Thøgersen, Lea

2011-01-01

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benaim, Gustavo, E-mail: gbenaim@idea.gob.ve; Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela; Pimentel, Adriana A., E-mail: adriana.pimentel@ucv.ve

2016-04-29

The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support thatmore » this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.« less

Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway.

PubMed

Shi, Hongtao; Han, Qinghua; Xu, Jianrong; Liu, Wenyuan; Chu, Tingting; Zhao, Li

2016-05-25

Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca(2+) concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17-phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca(2+) were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca(2+), consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17-phosphorylation signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling

PubMed Central

De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

2016-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits. PMID:26821066

Basal and β-adrenergic cardiomyocytes contractility dysfunction induced by dietary protein restriction is associated with downregulation of SERCA2a expression and disturbance of endoplasmic reticulum Ca2+ regulation in rats.

PubMed

Penitente, Arlete R; Novaes, Rômulo D; Silva, Marcelo E; Silva, Márcia F; Quintão-Júnior, Judson F; Guatimosim, Silvia; Cruz, Jader S; Chianca, Deoclécio A; Natali, Antônio J; Neves, Clóvis A

2014-01-01

The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca(2+)sparks analysis. PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca(2+)sparks were observed in PRG cardiomyocytes. The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca(2+) intracellular kinetics. © 2014 S. Karger AG, Basel.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

PubMed

Montalvo, Dolores; Pérez-Treviño, Perla; Madrazo-Aguirre, Katheryne; González-Mondellini, Fabio A; Miranda-Roblero, Hipólito O; Ramonfaur-Gracia, Diego; Jacobo-Antonio, Mariana; Mayorga-Luna, Maritza; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

2018-06-01

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca 2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca 2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca 2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (D mit ) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca 2+ signaling, T-T remodeling, D mit , citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca 2+ (19%) and blunted Ca 2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca 2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca 2+ transients. However, dRel/dt persisted during Ca 2+ transient relaxation due to abundant late Ca 2+ sparks. Isoproterenol significantly up-regulated systolic Ca 2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca 2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca 2+ -dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca 2+ leak. Furthermore, D mit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca 2+ and contractility dysregulation. We conclude that decreased SR Ca 2+ , due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD. Copyright © 2018 Elsevier Ltd. All rights reserved.

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles.

PubMed

Dufresne, Sébastien S; Dumont, Nicolas A; Boulanger-Piette, Antoine; Fajardo, Val A; Gamu, Daniel; Kake-Guena, Sandrine-Aurélie; David, Rares Ovidiu; Bouchard, Patrice; Lavergne, Éliane; Penninger, Josef M; Pape, Paul C; Tupling, A Russell; Frenette, Jérôme

2016-04-15

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function. Copyright © 2016 the American Physiological Society.

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles

PubMed Central

Dufresne, Sébastien S.; Dumont, Nicolas A.; Boulanger-Piette, Antoine; Fajardo, Val A.; Gamu, Daniel; Kake-Guena, Sandrine-Aurélie; David, Rares Ovidiu; Bouchard, Patrice; Lavergne, Éliane; Penninger, Josef M.; Pape, Paul C.; Tupling, A. Russell

2016-01-01

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca2+ sensor, and altered activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) modulating Ca2+ storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca2+ storage and SERCA activity, ultimately affecting denervated skeletal muscle function. PMID:26825123

Motion of the Ca2+-pump captured.

PubMed

Yokokawa, Masatoshi; Takeyasu, Kunio

2011-09-01

Studies of ion pumps, such as ATP synthetase and Ca(2+)-ATPase, have a long history. The crystal structures of several kinds of ion pump have been resolved, and provide static pictures of mechanisms of ion transport. In this study, using fast-scanning atomic force microscopy, we have visualized conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in real time at the single-molecule level. The analyses of individual SERCA molecules in the presence of both ATP and free Ca(2+) revealed up-down structural changes corresponding to the Albers-Post scheme. This fluctuation was strongly affected by the ATP and Ca(2+) concentrations, and was prevented by an inhibitor, thapsigargin. Interestingly, at a physiological ATP concentrations, the up-down motion disappeared completely. These results indicate that SERCA does not transit through the shortest structure, and has a catalytic pathway different from the ordinary Albers-Post scheme under physiological conditions. © 2011 The Authors Journal compilation © 2011 FEBS.

Iron-overload injury and cardiomyopathy in acquired and genetic models is attenuated by resveratrol therapy.

PubMed

Das, Subhash K; Wang, Wang; Zhabyeyev, Pavel; Basu, Ratnadeep; McLean, Brent; Fan, Dong; Parajuli, Nirmal; DesAulniers, Jessica; Patel, Vaibhav B; Hajjar, Roger J; Dyck, Jason R B; Kassiri, Zamaneh; Oudit, Gavin Y

2015-12-07

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca(2+) homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca(2+) homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload.

Iron-overload injury and cardiomyopathy in acquired and genetic models is attenuated by resveratrol therapy

PubMed Central

Das, Subhash K.; Wang, Wang; Zhabyeyev, Pavel; Basu, Ratnadeep; McLean, Brent; Fan, Dong; Parajuli, Nirmal; DesAulniers, Jessica; Patel, Vaibhav B.; Hajjar, Roger J.; Dyck, Jason R. B.; Kassiri, Zamaneh; Oudit, Gavin Y.

2015-01-01

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca2+ homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca2+ homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload. PMID:26638758

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

PubMed Central

Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O’Hanlon; Gordon, Sandra; Mortensen, Anne Louise; Clausen, Johannes D.; Pallin, Thomas David; Hansen, John Bondo; Fuglsang, Anja Thoe; Dalby-Brown, William

2018-01-01

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents. PMID:29293507

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression

PubMed Central

Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A.; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A.; Barrett, Timothy G.

2015-01-01

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca2+ imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca2+ concentration ([Ca2+]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis. PMID:25274773

Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart

PubMed Central

Vielma, Alejandra Z.; León, Luisa; Fernández, Ignacio C.; González, Daniel R.

2016-01-01

S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (−25–30%) and basal S-nitrosylation of total proteins (−25–60%), RyR2, SERCA2 and LTCC (−60–75%). NOS-1 inhibition reduced (−25–40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (−85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect. PMID:27529477

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase C674 promotes ischemia- and hypoxia-induced angiogenesis via coordinated endothelial cell and macrophage function.

PubMed

Mei, Yu; Thompson, Melissa D; Shiraishi, Yasunaga; Cohen, Richard A; Tong, Xiaoyong

2014-11-01

Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential calcium handling in two canine models of right ventricular pressure overload.

PubMed

Moon, Marc R; Aziz, Abdulhameed; Lee, Anson M; Moon, Cynthia J; Okada, Shoichi; Kanter, Evelyn M; Yamada, Kathryn A

2012-12-01

The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding. A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4). In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change. Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle. Copyright © 2012 Elsevier Inc. All rights reserved.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

PubMed

Fechner, H; Suckau, L; Kurreck, J; Sipo, I; Wang, X; Pinkert, S; Loschen, S; Rekittke, J; Weger, S; Dekkers, D; Vetter, R; Erdmann, V A; Schultheiss, H-P; Paul, M; Lamers, J; Poller, W

2007-02-01

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

PubMed

Casás-Selves, Matias; Zhang, Andrew X; Dowling, James E; Hallén, Stefan; Kawatkar, Aarti; Pace, Nicholas J; Denz, Christopher R; Pontz, Timothy; Garahdaghi, Farzin; Cao, Qing; Sabirsh, Alan; Thakur, Kumar; O'Connell, Nichole; Hu, Jun; Cornella-Taracido, Iván; Weerapana, Eranthie; Zinda, Michael; Goodnow, Robert A; Castaldi, M Paola

2017-06-21

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca 2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Levosimendan reduces myocardial damage and improves cardiodynamics in streptozotocin induced diabetic cardiomyopathy via SERCA2a/NCX1 pathway.

PubMed

Akhtar, Md Sayeed; Pillai, Krishna Kolappa; Hassan, Md Quamrul; Dhyani, Neha; Ismail, Md Vasim; Najmi, Abul Kalam

2016-05-15

Diabetic cardiomyopathy (DCM) is one of the most common causes of mortality. Its pathophysiology is not fully understood and involve number of factors including, cardiovascular and metabolic disorders. The present study was designed to study the pathogenesis of DCM and to explore the effects of levosimendan along with either ramipril or insulin in the long term management of DCM. Streptozotocin (STZ) was used to develop DCM in Wistar rats at the dose of 25mg/kg body weight for three consecutive days. Rats were randomly divided into 9 groups and treatments were started after 2weeks of STZ administration. Persistent hyperglycemia was observed in STZ treated rats, leading to significant contractile dysfunction as evidenced by decreased left ventricular pressure (LVP), +LV (dp/dt), -LV (dp/dt) as well as elevated Tau and LVEDP. Marked myocardial damage such as fibrosis, increased wall tension, depletion of contractile proteins were observed as evidenced by increased levels of TGF-β, BNP, cTroponin-I, as well as decreased expression of SERCA2a and NCX1 proteins in diabetic rats. The levosimendan alone and also in combination with either ramipril or insulin significantly normalized the myocardial dysfunctions developed during the course of persistent hyperglycemia. The study suggests that levosimendan treatment improves cardiac dysfunction significantly. Its combined use with ramipril proves better than with insulin in correcting myocardial performance as well as reduction in myocardial damage. Copyright © 2016 Elsevier Inc. All rights reserved.

Microscopic heat pulse-induced calcium dynamics in single WI-38 fibroblasts.

PubMed

Itoh, Hideki; Oyama, Kotaro; Suzuki, Madoka; Ishiwata, Shin'ichi

2014-01-01

Temperature-sensitive Ca(2+) dynamics occur primarily through transient receptor potential channels, but also by means of Ca(2+) channels and pumps on the endoplasmic reticulum membrane. As such, cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) is re-equilibrated by changes in ambient temperature. The present study investigated the effects of heat pulses (heating duration: 2 s or 150 s) on [Ca(2+)]cyt in single WI-38 fibroblasts, which are considered as normal cells. We found that Ca(2+) burst occurred immediately after short (2 s) heat pulse, which is similar to our previous report on HeLa cells, but with less thermosensitivity. The heat pulses originated from a focused 1455-nm infrared laser light were applied in the vicinity of cells under the optical microscope. Ca(2+) bursts induced by the heat pulse were suppressed by treating cells with inhibitors for sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or inositol trisphosphate receptor (IP3R). Long (150 s) heat pulses also induced Ca(2+) bursts after the onset of heating and immediately after re-cooling. Cells were more thermosensitive at physiological (37°C) than at room (25°C) temperature; however, at 37°C, cells were responsive at a higher temperature (ambient temperature+heat pulse). These results strongly suggest that the heat pulse-induced Ca(2+) burst is caused by a transient imbalance in Ca(2+) flow between SERCA and IP3R, and offer a potential new method for thermally controlling Ca(2+)-regulated cellular functions.

Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine

PubMed Central

Wongjaikam, Suwakon; Kumfu, Sirinart; Khamseekaew, Juthamas; Chattipakorn, Siriporn C.; Chattipakorn, Nipon

2017-01-01

Intracellular calcium [Ca2+]i dysregulation plays an important role in the pathophysiology of iron overload cardiomyopathy. Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca2+]i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca2+]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca2+]i homeostasis, only DFP + NAC restored cardiac [Ca2+]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca2+]i homeostasis, leading to restored myocardial contractility in iron-overloaded rats. PMID:28287621

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes.

PubMed

Maxwell, Joshua T; Blatter, Lothar A

2012-12-01

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Mechanisms of Intracellular Calcium Homeostasis in MC3T3-E1 Cells and Bone Tissues of Sprague-Dawley Rats Exposed to Fluoride.

PubMed

Duan, Xiao-qin; Li, Yan-hui; Zhang, Xiu-yun; Zhao, Zhi-tao; Wang, Ying; Wang, Huan; Li, Guang-sheng; Jing, Ling

2016-04-01

Calcium homeostasis of osteoblasts (OBs) has an important role in the physiology and pathology of bone tissue. In order to study the mechanisms of intracellular calcium homeostasis, MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, we examined intracellular-free calcium ion ([Ca(2+)]i) in MC3T3-E1 cells as well as mRNA and protein levels of Cav1.2, the main subunit of L-type voltage-dependent calcium channels (VDCCs), Na(+)/Ca(2+) exchange carriers (NCS), and plasma membrane Ca(2+)-ATPase (PMCA), inositol 1,4,5-trisphosphate receptor (IP3R) channels, sarco/endoplasmic reticulum calcium ATPase 2b (SERCA2b)/ATP2A2 in vitro, and rat bone tissues in vivo. Our results showed that [Ca(2+)]i of fluoride-treated OBs increased in a concentration-dependent manner with an increase in the concentration of fluoride. We also found that the low dose of fluoride led to high expression levels of Cav1.2, NCS-1, and PMCA and low expression levels of IP3R and SERCA2b/ATP2A2, while the high dose of fluoride induced an increase in SERCA2b/ATP2A2 levels and decrease in Cav1.2, PMCA, NCS-1, and IP3R levels. These results demonstrate that calcium channels and calcium pumps of plasma and endoplasmic reticulum (ER) membranes keep intracellular calcium homeostasis by regulating Cav1.2, NCS-1, PMCA, IP3R, and SERCA2b/ATP2A2 expression.

A transcriptomic approach to search for novel phenotypic regulators in McArdle disease.

PubMed

Nogales-Gadea, Gisela; Consuegra-García, Inés; Rubio, Juan C; Arenas, Joaquin; Cuadros, Marc; Camara, Yolanda; Torres-Torronteras, Javier; Fiuza-Luces, Carmen; Lucia, Alejandro; Martín, Miguel A; García-Arumí, Elena; Andreu, Antoni L

2012-01-01

McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes

PubMed Central

Maxwell, Joshua T; Blatter, Lothar A

2012-01-01

The widely accepted paradigm for cytosolic Ca2+ wave propagation postulates a ‘fire-diffuse-fire’ mechanism where local Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca2+ wave. A recent challenge to this paradigm proposed the requirement for an intra-SR ‘sensitization’ Ca2+ wave that precedes the cytosolic Ca2+ wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca2+]i; rhod-2) and intra-SR ([Ca2+]SR; fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca2+]i at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca2+]SR was observed. This transient elevation of [Ca2+]SR could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μm isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca2+]SR, whereas inhibition of SERCA (3 μm cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca2+ uptake by SERCA into the SR facilitates the propagation of cytosolic Ca2+ waves via luminal sensitization of the RyR, and supports a novel paradigm of a ‘fire-diffuse-uptake-fire’ mechanism for Ca2+ wave propagation in cardiac myocytes. PMID:22988145

Detection of Sequence-Specific Tyrosine Nitration of Manganese SOD and SERCA in Cardiovascular Disease and Aging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shanqin; Ying, Jia; Jiang, Bingbing

2006-06-01

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies towards site-specific nY-modified proteins and to use histochemical and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies towards peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with angiotensin II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum withmore » known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite that chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site, but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates, but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.« less

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

PubMed

Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

PubMed

Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

2008-11-01

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.

PubMed

Henderson, Mark J; Wires, Emily S; Trychta, Kathleen A; Yan, Xiaokang; Harvey, Brandon K

2015-09-06

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Cardiomyocyte marker expression in dogs with left atrial enlargement due to dilated cardiomyopathy or myxomatous mitral valve disease.

PubMed

Janus, Izabela; Kandefer-Gola, Malgorzata; Ciaputa, Rafal; Noszczyk-Nowak, Agnieszka; Paslawska, Urszula; Tursi, Massimiliano; Nowak, Marcin

2017-01-01

Dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD) are common heart conditions in dogs. They have different etiology and pathogenesis and although other studies focused on changes in the left ventricles of the affected hearts, the aim of our study was to assess the expressions of some intrinsic proteins in the enlarged left atria. We performed an immunohistochemical analysis of left atrial specimens obtained from 15 dogs with DCM, 35 dogs with MMVD and six control dogs. We assessed the expression of following proteins: SERCA1, SERCA2, sarcomeric actinin, smooth muscle actin, and dystrophin. We noted a higher percentage of SERCA1-positive cells in the MMVD group and lower percentage of dystrophin-positive cells in the DCM group as compared to control group. The expression of other proteins was similar in the hearts of control dogs and dogs with heart diseases. The observed changes in the expression patterns of some proteins in the atria of dogs with DCM and MMVD suggest that atrial enlargement relies not only on volume overload, but also alterations of the intrinsic proteins can contribute to the pathogenesis of dilated cardiomyopathy.

Microscopic heat pulse-induced calcium dynamics in single WI-38 fibroblasts

PubMed Central

Itoh, Hideki; Oyama, Kotaro; Suzuki, Madoka; Ishiwata, Shin’ichi

2014-01-01

Temperature-sensitive Ca2+ dynamics occur primarily through transient receptor potential channels, but also by means of Ca2+ channels and pumps on the endoplasmic reticulum membrane. As such, cytoplasmic Ca2+ concentration ([Ca2+]cyt) is re-equilibrated by changes in ambient temperature. The present study investigated the effects of heat pulses (heating duration: 2 s or 150 s) on [Ca2+]cyt in single WI-38 fibroblasts, which are considered as normal cells. We found that Ca2+ burst occurred immediately after short (2 s) heat pulse, which is similar to our previous report on HeLa cells, but with less thermosensitivity. The heat pulses originated from a focused 1455-nm infrared laser light were applied in the vicinity of cells under the optical microscope. Ca2+ bursts induced by the heat pulse were suppressed by treating cells with inhibitors for sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) or inositol trisphosphate receptor (IP3R). Long (150 s) heat pulses also induced Ca2+ bursts after the onset of heating and immediately after re-cooling. Cells were more thermosensitive at physiological (37°C) than at room (25°C) temperature; however, at 37°C, cells were responsive at a higher temperature (ambient temperature+heat pulse). These results strongly suggest that the heat pulse-induced Ca2+ burst is caused by a transient imbalance in Ca2+ flow between SERCA and IP3R, and offer a potential new method for thermally controlling Ca2+-regulated cellular functions. PMID:27493505

Altered sarcoplasmic reticulum calcium cycling—targets for heart failure therapy

PubMed Central

Kho, Changwon; Lee, Ahyoung; Hajjar, Roger J.

2013-01-01

Cardiac myocyte function is dependent on the synchronized movements of Ca2+ into and out of the cell, as well as between the cytosol and sarcoplasmic reticulum. These movements determine cardiac rhythm and regulate excitation–contraction coupling. Ca2+ cycling is mediated by a number of critical Ca2+-handling proteins and transporters, such as L-type Ca2+ channels (LTCCs) and sodium/calcium exchangers in the sarcolemma, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), ryanodine receptors, and cardiac phospholamban in the sarcoplasmic reticulum. The entry of Ca2+ into the cytosol through LTCCs activates the release of Ca2+ from the sarcoplasmic reticulum through ryanodine receptor channels and initiates myocyte contraction, whereas SERCA2a and cardiac phospholamban have a key role in sarcoplasmic reticulum Ca2+ sequesteration and myocyte relaxation. Excitation–contraction coupling is regulated by phosphorylation of Ca2+-handling proteins. Abnormalities in sarcoplasmic reticulum Ca2+ cycling are hallmarks of heart failure and contribute to the pathophysiology and progression of this disease. Correcting impaired intracellular Ca2+ cycling is a promising new approach for the treatment of heart failure. Novel therapeutic strategies that enhance myocyte Ca2+ homeostasis could prevent and reverse adverse cardiac remodeling and improve clinical outcomes in patients with heart failure. PMID:23090087

Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid-supported-membrane for the investigation of membrane transporters.

PubMed

Sacconi, Alessio; Moncelli, Maria Rosa; Margheri, Giancarlo; Tadini-Buoninsegni, Francesco

2013-11-12

A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca(2+) and Mg(2+), the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

2008-01-15

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped virusesmore » indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.« less

β-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

PubMed

McCrink, Katie A; Maning, Jennifer; Vu, Angela; Jafferjee, Malika; Marrero, Christine; Brill, Ava; Bathgate-Siryk, Ashley; Dabul, Samalia; Koch, Walter J; Lymperopoulos, Anastasios

2017-11-01

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins βarr (β-arrestin)-1 and βarr-2 are functionally distinct in the heart. βarr1 is cardiotoxic, decreasing contractility by opposing β 1 AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, βarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether βarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that βarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca 2+ -ATPase) in vivo and in vitro in a β 1 AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. βarr1 lacks this effect. Moreover, βarr2 does not desensitize β 1 AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to βarr1. In vivo, post-MI heart failure mice overexpressing cardiac βarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, βarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac βAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac βarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac βarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure. © 2017 American Heart Association, Inc.

Randomized Clinical Trials of Gene Transfer for Heart Failure with Reduced Ejection Fraction.

PubMed

Penny, William F; Hammond, H Kirk

2017-05-01

Despite improvements in drug and device therapy for heart failure, hospitalization rates and mortality have changed little in the past decade. Randomized clinical trials using gene transfer to improve function of the failing heart are the focus of this review. Four randomized clinical trials of gene transfer in heart failure with reduced ejection fraction (HFrEF) have been published. Each enrolled patients with stable symptomatic HFrEF and used either intracoronary delivery of a virus vector or endocardial injection of a plasmid. The initial CUPID trial randomized 14 subjects to placebo and 25 subjects to escalating doses of adeno-associated virus type 1 encoding sarcoplasmic reticulum calcium ATPase (AAV1.SERCA2a). AAV1.SERCA2a was well tolerated, and the high-dose group met a 6 month composite endpoint. In the subsequent CUPID-2 study, 243 subjects received either placebo or the high dose of AAV1.SERCA2a. AAV1.SERCA2a administration, while safe, failed to meet the primary or any secondary endpoints. STOP-HF used plasmid endocardial injection of stromal cell-derived factor-1 to promote stem-cell recruitment. In a 93-subject trial of patients with ischemic etiology heart failure, the primary endpoint (symptoms and 6 min walk distance) failed, but subgroup analyses showed improvements in subjects with the lowest ejection fractions. A fourth trial randomized 14 subjects to placebo and 42 subjects to escalating doses of adenovirus-5 encoding adenylyl cyclase 6 (Ad5.hAC6). There were no safety concerns, and patients in the two highest dose groups (combined) showed improvements in left ventricular function (left ventricular ejection fraction and -dP/dt). The safety data from four randomized clinical trials of gene transfer in patients with symptomatic HFrEF suggest that this approach can be conducted with acceptable risk, despite invasive delivery techniques in a high-risk population. Additional trials are necessary before the approach can be endorsed for clinical practice.

Exposure to low mercury concentration in vivo impairs myocardial contractile function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro

2011-09-01

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl{sub 2} (1st dose 4.6 {mu}g/kg, subsequent dose 0.07 {mu}g/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP)more » and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and {beta}-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na{sup +}-K{sup +} ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and {alpha}-1 isoform of NKA, whereas {alpha}-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: > Unchanges blood pressure, heart rate, systolic pressure. > Increases end diastolic pressure. > Promotes cardiac contractility dysfunction. > Decreases NKA activity, NCX and SERCA, increases PLB protein expression. > Small concentrations constitutes environmental cardiovascular risk factor.« less

Finite Element Estimation of Protein-Ligand Association Rates with Post-Encounter Effects: Applications to Calcium binding in Troponin C and SERCA

PubMed Central

Kekenes-Huskey, P. M.; Gillette, A.; Hake, J.; McCammon, J. A.

2012-01-01

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca2+-binding proteins: Troponin C and the Sarcoplasmic Reticulum Ca2+ ATPase (SERCA). We find that a combination of diffusional encounter and internal ‘buried channel’ descriptions provide superior descriptions of association rates, improving estimates by orders of magnitude. PMID:23293662

Finite Element Estimation of Protein-Ligand Association Rates with Post-Encounter Effects: Applications to Calcium binding in Troponin C and SERCA.

PubMed

Kekenes-Huskey, P M; Gillette, A; Hake, J; McCammon, J A

2012-10-31

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca(2+)-binding proteins: Troponin C and the Sarcoplasmic Reticulum Ca(2+) ATPase (SERCA). We find that a combination of diffusional encounter and internal 'buried channel' descriptions provide superior descriptions of association rates, improving estimates by orders of magnitude.

Antenatal/early postnatal hypothyroidism alters arterial tone regulation in 2-week-old rats.

PubMed

Sofronova, Svetlana I; Gaynullina, Dina K; Shvetsova, Anastasia A; Borzykh, Anna A; Selivanova, Ekaterina K; Kostyunina, Daria S; Sharova, Anna P; Martyanov, Andrey A; Tarasova, Olga S

2017-11-01

The mechanisms of vascular alterations resulting from early thyroid hormones deficiency are poorly understood. We tested the hypothesis that antenatal/early postnatal hypothyroidism would alter the activity of endothelial NO pathway and Rho-kinase pathway, which are specific for developing vasculature. Dams were treated with propylthiouracil (PTU, 7 ppm) in drinking water during gestation and 2 weeks after delivery, and their progeny had normal body weight but markedly reduced blood levels of thyroid hormones (ELISA). Small arteries from 2-week-old male pups were studied using wire myography, qPCR and Western blotting. Mesenteric arteries of PTU pups, compared to controls, demonstrated smaller maximum response to α 1 -adrenergic agonist methoxamine and reduced mRNA contents of smooth muscle differentiation markers α-actin and SERCA2A. Inhibition of basal NO synthesis by l-NNA led to tonic contraction of mesenteric arteries and augmented their contractile responses to methoxamine; both l-NNA effects were impaired in PTU pups. PTU pups demonstrated lower blood level of NO metabolites compared to control group (Griess reaction). Rho-kinase inhibitor Y27632 strongly reduced mesenteric arteries responses to methoxamine in PTU pups, that was accompanied by elevated Rho-kinase content in their arteries in comparison to control ones. Unlike mesenteric, saphenous arteries of PTU pups, compared to controls, had no changes in α-actin and SERCA2A contents and in responses to l-NNA and Y27632. In conclusion, thyroid hormones deficiency suppresses the anticontractile effect of NO and potentiates the procontractile Rho-kinase effects in mesenteric arteries of 2-week-old pups. Such alterations disturb perinatal cardiovascular homeostasis and might lead to cardiovascular pathologies in adulthood. © 2017 Society for Endocrinology.

Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin

PubMed Central

Hu, Li-Yen R.; Ackermann, Maegen A.; Hecker, Peter A.; Prosser, Benjamin L.; King, Brendan; O’Connell, Kelly A.; Grogan, Alyssa; Meyer, Logan C.; Berndsen, Christopher E.; Wright, Nathan T.; Jonathan Lederer, W.; Kontrogianni-Konstantopoulos, Aikaterini

2017-01-01

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations. PMID:28630914

Increased LDL electronegativity in chronic kidney disease disrupts calcium homeostasis resulting in cardiac dysfunction.

PubMed

Chang, Kuan-Cheng; Lee, An-Sheng; Chen, Wei-Yu; Lin, Yen-Nien; Hsu, Jing-Fang; Chan, Hua-Chen; Chang, Chia-Ming; Chang, Shih-Sheng; Pan, Chia-Chi; Sawamura, Tatsuya; Chang, Chi-Tzong; Su, Ming-Jai; Chen, Chu-Huang

2015-07-01

Chronic kidney disease (CKD), an independent risk factor for cardiovascular disease, is associated with abnormal lipoprotein metabolism. We examined whether electronegative low-density lipoprotein (LDL) is mechanistically linked to cardiac dysfunction in patients with early CKD. We compared echocardiographic parameters between patients with stage 2 CKD (n = 88) and normal controls (n = 89) and found that impaired relaxation was more common in CKD patients. Reduction in estimated glomerular filtration rate was an independent predictor of left ventricular relaxation dysfunction. We then examined cardiac function in a rat model of early CKD induced by unilateral nephrectomy (UNx) by analyzing pressure-volume loop data. The time constant of isovolumic pressure decay was longer and the maximal velocity of pressure fall was slower in UNx rats than in controls. When we investigated the mechanisms underlying relaxation dysfunction, we found that LDL from CKD patients and UNx rats was more electronegative than LDL from their respective controls and that LDL from UNx rats induced intracellular calcium overload in H9c2 cardiomyocytes in vitro. Furthermore, chronic administration of electronegative LDL, which signals through lectin-like oxidized LDL receptor-1 (LOX-1), induced relaxation dysfunction in wild-type but not LOX-1(-/-) mice. In in vitro and in vivo experiments, impaired cardiac relaxation was associated with increased calcium transient resulting from nitric oxide (NO)-dependent nitrosylation of SERCA2a due to increases in inducible NO synthase expression and endothelial NO synthase uncoupling. In conclusion, LDL becomes more electronegative in early CKD. This change disrupts SERCA2a-regulated calcium homeostasis, which may be the mechanism underlying cardiorenal syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2013-11-01

Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

Heat stress responses modulate calcium regulations and electrophysiological characteristics in atrial myocytes.

PubMed

Chen, Yao-Chang; Kao, Yu-Hsun; Huang, Chun-Feng; Cheng, Chen-Chuan; Chen, Yi-Jen; Chen, Shih-Ann

2010-04-01

Heat stress-induced responses change the ionic currents and calcium homeostasis. However, the molecular insights into the heat stress responses on calcium homeostasis remain unclear. The purposes of this study were to examine the mechanisms of heat stress responses on calcium handling and electrophysiological characteristics in atrial myocytes. We used indo-1 fluorimetric ratio technique and whole-cell patch clamp to investigate the intracellular calcium, action potentials, and ionic currents in isolated rabbit single atrial cardiomyocytes with or without (control) exposure to heat stress (43 degrees C, 15 min) 5+/-1 h before experiments. The expressions of sarcoplasmic reticulum ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) in the control and heat stress-treated atrial myocytes were evaluated by Western blot and real-time PCR. As compared with control myocytes, the heat stress-treated myocytes had larger sarcoplasmic reticulum calcium content and larger intracellular calcium transient with a shorter decay portion. Heat stress-treated myocytes also had larger L-type calcium currents, transient outward potassium currents, but smaller NCX currents. Heat stress responses increased the protein expressions, SERCA2a, NCX, and heat shock protein. However, heat stress responses did not change the RNA expression of SERCA2a and NCX. In conclusion, heat stress responses change calcium handling through protein but not RNA regulation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Contributions of SERCA pump and ryanodine-sensitive stores to presynaptic residual Ca2+

PubMed Central

Scullin, Chessa S.; Partridge, L. Donald

2010-01-01

The presynaptic Ca2+ signal, which triggers vesicle release, disperses to a broadly distributed residual [Ca2+] ([Ca2+]res) that plays an important role in synaptic plasticity. We have previously reported a slowing in the decay timecourse of [Ca2+]res during the second of paired pulses. In this study, we investigated the contributions of organelle and plasma membrane Ca2+ flux pathways to the reduction of effectiveness of [Ca2+]res clearance during short-term plasticity in Schaffer collateral terminals in the CA1 field of the hippocampus. We show that the slowed decay timecourse is mainly the result of a transport-dependent Ca2+ clearance process; that presynaptic caffeine-sensitive Ca2+ stores are not functionally loaded in the unstimulated terminal, but that these stores can effectively take up Ca2+ even during high frequency trains of stimuli; and that a rate limiting step of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) kinetics following the first pulse is responsible for a large portion of the observed slowing of [Ca2+]res clearance during the second pulse. We were able to accurately fit our [Ca2+]res data with a kinetic model based on these observations and this model predicted a reduction in availability of unbound SERCA during paired pulses, but no saturation of Ca2+ buffer in the endoplasmic reticulum. PMID:20153896

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump.

PubMed

Møller, Jesper V; Nissen, Poul; Sørensen, Thomas L-M; le Maire, Marc

2005-08-01

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.

Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus.

PubMed

Jiang, M; Xu, A; Jones, D L; Narayanan, N

2004-09-01

This study investigated the effects of l-thyroxine-induced hyperthyroidism on Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca(2+) pump (Ca(2+)-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca(2+)-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30-50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1 ( approximately 150%) Ca(2+) pump isoform, unaltered levels of SERCA2 Ca(2+) pump isoform, and lower levels of PLN ( approximately 50%) and delta-, beta-, and gamma-CaM kinase II (40 approximately 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca(2+) uptake and Ca(2+)-stimulated ATPase activities compared with that from euthyroid control. The V(max) of Ca(2+) uptake (in nmol Ca(2+).mg SR protein(-1).min(-1): euthyroid, 818 +/- 73; hyperthyroid, 1,649 +/- 90) but not the apparent affinity of the Ca(2+)-ATPase for Ca(2+) (euthyroid, 0.97 +/- 0.02 microM, hyperthyroid, 1.09 +/- 0.04 microM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca(2+) uptake by soleus muscle SR was approximately 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater ( approximately 36%), and the time to peak force and relaxation time were significantly lower ( approximately 30-40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca(2+) pump.

Mitochondrial calcium handling within the interstitial cells of Cajal

PubMed Central

Cheng, Leo K.

2014-01-01

The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca2+) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca2+ dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca2+ store depletions and membrane depolarization with ER store-operated Ca2+ entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca2+ transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca2+ release and MT uptake on bulk cytosolic Ca2+ levels because persistent elevations of free intracellular Ca2+ are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca2+ and the resulting frequencies of ER Ca2+ store depletions, as well as the sarco-endoplasmic reticulum Ca2+ ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca2+ depletion frequencies than protection of the cytosol from elevated free Ca2+, whereas the SERCA pump is more relevant to containing cytosolic Ca2+ elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca2+ release, subsequent MT uptake, and eventual activation of ER store-operated Ca2+ entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types. PMID:24789203

Downregulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia.

PubMed

Makanga, Martine; Dewachter, Céline; Maruyama, Hidekazu; Vuckovic, Aline; Rondelet, Benoit; Naeije, Robert; Dewachter, Laurence

2013-08-01

Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

NASA Astrophysics Data System (ADS)

Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

The modified Yi qi decoction protects cardiac ischemia-reperfusion induced injury in rats.

PubMed

Yu, Xiao; Zhao, Xiao-Dong; Bao, Rong-Qi; Yu, Jia-Yu; Zhang, Guo-Xing; Chen, Jing-Wei

2017-06-21

To investigate the effects and involved mechanisms of the modified Yi Qi decoction (MYQ) in cardiac ischemia-reperfusion (IR) induced injury. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion, low or high dose decoction of MYQ was administrated orally for 1 week or 1 month. Both in 1 week and 1 month IR rat groups, cardiac function indexes were significantly impaired compared with sham group rats, accompanied with higher ratio of infarct size to risk size, decreased expressions of sodium calcium exchanger (NCX1) and sarcoplasmic reticulum Ca 2+ -ATPase (Serca2a), and different expressions of autophagic proteins, Beclin-1 and LC3. Treatment with MYQ (low or high dose) for 1 week showed no marked beneficial effects on cardiac function and cardiac injury (ratio of infarct size to risk size), although expressions of anti-apoptotic protein, Bcl-2, NCX1 and Serca2a were increased. Treatment with MYQ (low or high dose) for 1 month showed significantly improved effects on cardiac function and cardiac injury (ratio of infarct size to risk size), accompanied with increase of Bcl-2, NCX1 and Serca2a expressions, and decrease of Bax (a pro-apoptotic protein) and Beclin-1 expressions. The results show that MYQ have potential therapeutic effects on IR-induced cardiac injury, which may be through regulation of apoptotic proteins, cytosolic Ca 2+ handling proteins and autophagic proteins signal pathways.

Serca2a and Na(+)/Ca(2+) exchanger are involved in left ventricular function following cardiac remodelling of female rats treated with anabolic androgenic steroid.

PubMed

Nascimento, Andrews Marques do; Lima, Ewelyne Miranda de; Brasil, Girlandia Alexandre; Caliman, Izabela Facco; Silva, Josiane Fernandes da; Lemos, Virgínia Soares; Andrade, Tadeu Uggere de; Bissoli, Nazaré Souza

2016-06-15

Anabolic-androgenic steroids are misused, including by women, but little is known about the cardiovascular effects of these drugs on women. To evaluated the effects of nandrolone decanoate (ND) and resistive physical exercise on cardiac contractility in young female rats. Female Wistar rats were separated into 4 groups: C (untrained animals); E (animals were submitted to resistance exercise by jumping in water 5 times per week); ND (animals were treated with ND, 20mg/kg/week for 4weeks); and NDE (trained and treated). The haemodynamic parameters (+dP/dtmax, -dP/dtmin and Tau) were assessed in the left ventricle. The heart was collected for histological analyses and collagen deposition. The gastrocnemius muscle was weighed, and hypertrophy was assessed by the ratio of their weights to gastrocnemius/tibia length. The expression of calcium handling proteins was measured by western blot analysis. ND treatment and physical exercise increased cardiac contractility and relaxation. In addition, ND promoted increases in phospholamban phosphorylated (p-PLB) and isoforms of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, while resistance exercise increased the phosphorylation of PLB and expression of Na(+)/Ca(2+) exchangers (NCX). Cardiac hypertrophy and collagen deposition were observed after ND treatment. Regulatory components of cytosolic calcium, such as SERCA2a and p-PLB, play important roles in modulating the contractility and relaxation effects of ND in females. Copyright © 2016 Elsevier Inc. All rights reserved.

Skeletal muscle proteins: a new approach to delimitate the time since death.

PubMed

Foditsch, Elena Esra; Saenger, Alexandra Maria; Monticelli, Fabio Carlo

2016-03-01

Skeletal muscle tissue is proposed as a forensic model tissue with strong potential, as it is easily accessible and its true-to-life state structure and function is well known. Despite this strong potential, skeletal muscle degradation studies are rare. The aim of this study was to test if a skeletal muscle-based protein analysis is applicable to delimitate the time since death. Under standard conditions, two pigs were stored either at 22 °C for 5 days or 4 °C for 21 days. Their Mm. biceps femori were sampled periodically for analyses of ten skeletal muscle proteins postmortem. All analyzed proteins can serve as markers for a delimitation of the time since death. Desmin, nebulin, titin, and SERCA 1 displayed distinct protein patterns at certain points of time. The other five proteins, α-actinin, calsequestrin-1, laminin, troponin T-C, and SERCA 2, showed no degradation patterns within the analyzed postmortem time frame. Referring to specific skeletal muscle proteins, results showed short-term stabilities for just a minority of analyzed proteins, while the majority of investigated proteins displayed characteristics as long-term markers. Due to specific patterns and the possibility to determine definite constraints of the presence, absence, or pattern alterations of single proteins, the feasibility of porcine skeletal muscle as forensic model tissue is outlined and the potential of skeletal muscle as forensic model tissue is underlined, especially with respect to later postmortem phases, which so far lack feasible methods to delimitate the time since death.

Intrinsic attenuation of post-irradiation calcium and ER stress imparts significant radioprotection to lepidopteran insect cells.

PubMed

Guleria, Ayushi; Thukral, Neha; Chandna, Sudhir

2018-04-15

Sf9 lepidopteran insect cells are 100-200 times more radioresistant than mammalian cells. This distinctive feature thus makes them suitable for studies exploring radioprotective molecular mechanisms. It has been established from previous studies of our group that downstream mitochondrial apoptotic signaling pathways in Sf9 cells are quite similar to mammalian cells, implicating the upstream signaling pathways in their extensive radioresistance. In the present study, intracellular and mitochondrial calcium levels remained unaltered in Sf9 cells in response to radiation, in sharp contrast to human (HEK293T) cells. The isolated mitochondria from Sf9 cells exhibited nearly 1.5 times greater calcium retention capacity than mammalian cells, highlighting their inherent stress resilience. Importantly, UPR/ER stress marker proteins (p-eIF2α, GRP4 and SERCA) remained unaltered by radiation and suggested highly attenuated ER and calcium stress. Lack of SERCA induction further corroborates the lack of radiation-induced calcium mobilization in these cells. The expression of CaMKII, an important effector molecule of calcium signaling, did not alter in response to radiation. Inhibiting CaMKII by KN-93 or suppressing CaM by siRNA failed to alter Sf9 cells response to radiation and suggests CaM-CaMKII independent radiation signaling. Therefore, this study suggests that attenuated calcium signaling/ER stress is an important determinant of lepidopteran cell radioresistance. Copyright © 2018 Elsevier Inc. All rights reserved.

Caffeine-induced [Ca2+] oscillations in neurones of frog sympathetic ganglia

PubMed Central

Cseresnyés, Zoltán; Bustamante, Alexander I; Schneider, Martin F

1999-01-01

Single cell fluorimetry was used to monitor caffeine-induced oscillations of cytosolic [Ca2+] in frog sympathetic ganglion neurones in 2.0 mm K+ Ringer solution.[Ca2+] oscillations decreased in frequency and exhibited three different amplitude patterns after the first large peak of [Ca2+]: (a) a series of big oscillations (BOs) of constant large amplitude (300–;400 nm), (b) a series of much smaller oscillations (SOs) (40–60 nm), or (c) a series of decaying oscillations (DOs) of rapidly decreasing amplitude.A model in which the oscillation amplitude was determined by the Ca2+ content of the endoplasmic reticulum (ER) whereas the oscillation frequency was controlled by how rapidly the cytosolic [Ca2+] reached the threshold for Ca2+-induced Ca2+ release (CICR) was able to simulate each observed pattern by varying the level of activity of the ER Ca2+ pump (SERCA), CICR and release-activated Ca2+ transport (RACT). A cumulative, cytosolic Ca2+-dependent inactivation of the plasma membrane (PM) Ca2+ influx or of the Ca2+-sensitive leak coefficient of the ryanodine receptors caused the oscillation frequency to decrease in the model.Transitions between BOs and SOs and changes in [Ca2+] oscillations caused by ryanodine, thapsigargin, lanthanum and FCCP could also be simulated.We conclude that RACT, SERCA, CICR and Ca2+-dependent PM Ca2+ influx are major mechanisms underlying [Ca2+] oscillations in these neurones. PMID:9831718

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

PubMed

Pirkmajer, Sergej; Kirchner, Henriette; Lundell, Leonidas S; Zelenin, Pavel V; Zierath, Juleen R; Makarova, Kira S; Wolf, Yuri I; Chibalin, Alexander V

2017-07-15

Small transmembrane proteins such as FXYDs, which interact with Na + ,K + -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca 2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na + ,K + -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

PubMed Central

Pinto, João; Gribaldo, Simonetta; Legrand, Eric; Niang, Makhtar; Kim, Nimol; Pharath, Lim; Volnay, Béatrice; Ekala, Marie Therese; Bouchier, Christiane; Fandeur, Thierry; Berzosa, Pedro; Benito, Agustin; Ferreira, Isabel Dinis; Ferreira, Cynthia; Vieira, Pedro Paulo; Alecrim, Maria das Graças; Mercereau-Puijalon, Odile; Cravo, Pedro

2010-01-01

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte. PMID:20195531

Increased thrombospondin-4 after nerve injury mediates disruption of intracellular calcium signaling in primary sensory neurons

PubMed Central

Guo, Yuan; Zhang, Zhiyong; Wu, Hsiang-en; Luo, Z. David; Hogan, Quinn H.; Pan, Bin

2017-01-01

Painful nerve injury disrupts Ca2+ signaling in primary sensory neurons by elevating plasma membrane Ca2+-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca2+ stores and stimulates store-operated Ca2+ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca2+ current (ICa) through high-voltage–activated Ca2+ channels and increases ICa through low-voltage–activated Ca2+ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca2+ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca2+ stores, and enhances store-operated Ca2+ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca2+ signaling are attenuated in voltage-gated Ca2+ channel α2δ1 subunit (Cavα2δ1) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca2+ signaling via interacting with the Cavα2δ1 and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca2+ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain. PMID:28232180

Chronic Exposure to Low Doses of HgCl2 Avoids Calcium Handling Impairment in the Right Ventricle after Myocardial Infarction in Rats

PubMed Central

Faria, Thaís de Oliveira; Costa, Gustavo Pinto; Almenara, Camila Cruz Pereira; Angeli, Jhuli Keli; Vassallo, Dalton Valentim; Stefanon, Ivanita; Vassallo, Paula Frizera

2014-01-01

Right ventricle systolic dysfunction is a major risk factor for death and heart failure after myocardial infarction (MI). Heavy metal exposure has been associated with the development of several cardiovascular diseases, such as MI. The aim of this study was to investigate whether chronic exposure to low doses of mercury chloride (HgCl2) enhances the functional deterioration of right ventricle strips after MI. Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks- 1st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, i.m. to cover daily loss); MI surgery induced and HgCl2-MI groups. One week after MI, the morphological and hemodynamic measurements and isometric tension of right ventricle strips were investigated. The chronic HgCl2 exposure did not worsen the injury compared with MI alone in the morphological or hemodynamic parameters evaluated. At basal conditions, despite similar maximum isometric force at L-max, relaxation time was increased in the MI group but unaffected in the HgCl2-MI compared to the Control group. Impairment of the sarcoplasmic reticulum (SR) function and reduction in the sarcolemmal calcium influx were observed in MI group associated with SERCA2a reduction and increased PLB protein expression. Induction of MI in chronic HgCl2 exposed rats did not cause any alteration in the developed force at L-max, lusitropic function or −dF/dt except for a tendency of a reduction SR function. These findings could be partially explained by the normalization in the sarcolemmal calcium influx and the increase in NCX protein expression observed only in this group. These results suggest that chronic exposure to low doses of HgCl2 prevents the impaired SR function and the reduced sarcolemmal calcium influx observed in MI likely by acting on NCX, PLB and SERCA2a protein expression. PMID:24748367

Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells.

PubMed

Caddy, J; Isa, S; Mainwaring, L S; Adam, E; Roberts, A; Lang, D; Morris, R H K; Thomas, A W; Webb, R

2010-10-01

Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPARγ-independent effects alongside its insulin-sensitising PPARγ-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca(2+)](i) homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30min) brought about concentration-dependent PPARγ-independent inhibition of Ca(2+)ATPase activity (IC(50) ∼2μM). Fluo-3 fluorimetric data demonstrated in intact cells that 1h treatment with 1 or 10μM rosiglitazone caused Ca(2+) ions to leak into the cytoplasm. Gene expression analysis showed that within 4h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca(2+) depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72h. After 72h 1 or 10μM rosiglitazone treatment, microsomal Ca(2+)ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca(2+)](i) returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca(2+) sequestration within the ER. This appeared to be sufficient to replenish ER Ca(2+) stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10μM rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration.

PubMed

Leon-Aparicio, Daniel; Chavez-Reyes, Jesus; Guerrero-Hernandez, Agustin

2017-07-01

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca 2+ level ([Ca 2+ ] ER ) and we have carried out simultaneous recordings of both [Ca 2+ ] i and the [Ca 2+ ] ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca 2+ ] ER by activating an ER-resident Ca 2+ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca 2+ ] ER is auto-limited because depends on a replete ER Ca 2+ store; a condition that thapsigargin does not require to decrease the [Ca 2+ ] ER . Additionally, our data indicate that the ER Ca 2+ permeable channel activated by 2-APB does not seem to participate in the ER Ca 2+ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5μM) would lead to the reduction in the [Ca 2+ ] ER that might explain the inhibitory effect of this compound on those signals that require Ca 2+ release from the ER store. Copyright © 2017 Elsevier Ltd. All rights reserved.

Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo.

PubMed

Wires, Emily S; Henderson, Mark J; Yan, Xiaokang; Bäck, Susanne; Trychta, Kathleen A; Lutrey, Molly H; Harvey, Brandon K

2017-01-01

The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

PubMed

Gehrig, Stefan M; van der Poel, Chris; Sayer, Timothy A; Schertzer, Jonathan D; Henstridge, Darren C; Church, Jarrod E; Lamon, Severine; Russell, Aaron P; Davies, Kay E; Febbraio, Mark A; Lynch, Gordon S

2012-04-04

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Cardiac Overexpression of Antioxidant Catalase Attenuates Aging-Induced Cardiomyocyte Relaxation Dysfunction

PubMed Central

Ren, Jun; Li, Qun; Wu, Shan; Li, Shi-Yan; Babcock, Sara A.

2007-01-01

Catalase, an enzyme which detoxifies H2O2, may interfere with cardiac aging. To test this hypothesis, contractile and intracellular Ca2+ properties were evaluated in cardiomyocytes from young (3–4 mo) and old (26–28 mo) FVB and transgenic mice with cardiac overexpression of catalase. Contractile indices analyzed included peak shortening (PS), time-to-90% PS (TPS90), time-to-90% relengthening (TR90), half-width duration (HWD), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ levels or decay rate. Levels of advanced glycation endproduct (AGE), Na+/Ca2+ exchanger (NCX), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), myosin heavy chain (MHC), membrane Ca2+ and K+ channels were measured by western blot. Catalase transgene prolonged survival while did not alter myocyte function by itself. Aging depressed ± dL/dt, prolonged HWD, TR90 and intracellular Ca2+ decay without affecting other indices in FVB myocytes. Aged FVB myocytes exhibited a stepper decline in PS in response to elevated stimulus or a dampened rise in PS in response to elevated extracellular Ca2+ levels. Interestingly, aging-induced defects were nullified or significantly attenuated by catalase. AGE level was elevated by 5-fold in aged FVB compared with young FVB mice, which was reduced by catalase. Expression of SERCA2a, NCX and Kv1.2 K+ channel was significantly reduced although levels of PLB, L-type Ca2+ channel dihydropyridine receptor and β-MHC isozyme remained unchanged in aged FVB hearts. Catalase restored NCX and Kv1.2 K+ channel but not SERCA2a level in aged mice. In summary, our data suggested that catalase protects cardiomyocytes from aging-induced contractile defect possibly via improved intracellular Ca2+ handling. PMID:17250874

Ca2+-induced delayed afterdepolarizations are triggered by dyadic subspace Ca2+ affirming that increasing SERCA reduces aftercontractions

PubMed Central

Noble, Penelope J.; Noble, Denis

2011-01-01

Ca2+-induced delayed afterdepolarizations (DADs) are depolarizations that occur after full repolarization. They have been observed across multiple species and cell types. Experimental results have indicated that the main cause of DADs is Ca2+ overload. The main hypothesis as to their initiation has been Ca2+ overflow from the overloaded sarcoplasmic reticulum (SR). Our results using 37 previously published mathematical models provide evidence that Ca2+-induced DADs are initiated by the same mechanism as Ca2+-induced Ca2+ release, i.e., the modulation of the opening of ryanodine receptors (RyR) by Ca2+ in the dyadic subspace; an SR overflow mechanism was not necessary for the induction of DADs in any of the models. The SR Ca2+ level is better viewed as a modulator of the appearance of DADs and the magnitude of Ca2+ release. The threshold for the total Ca2+ level within the cell (not only the SR) at which Ca2+ oscillations arise in the models is close to their baseline level (∼1- to 3-fold). It is most sensitive to changes in the maximum sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump rate (directly proportional), the opening probability of RyRs, and the Ca2+ diffusion rate from the dyadic subspace into the cytosol (both indirectly proportional), indicating that the appearance of DADs is multifactorial. This shift in emphasis away from SR overload as the trigger for DADs toward a multifactorial analysis could explain why SERCA overexpression has been shown to suppress DADs (while increasing contractility) and why DADs appear during heart failure (at low SR Ca2+ levels). PMID:21666112

Synergistic interactions of biotic and abiotic environmental stressors on gene expression.

PubMed

Altshuler, Ianina; McLeod, Anne M; Colbourne, John K; Yan, Norman D; Cristescu, Melania E

2015-03-01

Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis - cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) - using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors.

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

PubMed

Willer, Elisabeth A; Malli, Roland; Bondarenko, Alexander I; Zahler, Stefan; Vollmar, Angelika M; Graier, Wolfgang F; Fürst, Robert

2012-10-01

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Yamei; Wang, Lingxian; Wang, Lu

2015-02-15

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872more » cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.« less

Influence of infrasound exposure on the whole L-type calcium currents in rat ventricular myocytes.

PubMed

Pei, Zhaohui; Zhuang, Zhiqiang; Xiao, Pingxi; Chen, Jingzao; Sang, Hanfei; Ren, Jun; Wu, Zhenbiao; Yan, Guangmei

2009-06-01

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca2+ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca2+](i), WLCC, mRNA expression of the a(1c) subunit of L-type Ca2+ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca2+](i) in freshly isolated ventricular myocytes. The Ca2+ fluorescence intensity (FI), denoting [Ca2+](i) in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I (Ca,L). Taken together, our data suggest that changes in [Ca2+](I) levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response.

Infrasound-induced hemodynamics, ultrastructure, and molecular changes in the rat myocardium.

PubMed

Pei, Zhaohui; Sang, Hanfei; Li, Ruiman; Xiao, Pingxi; He, Jiangui; Zhuang, Zhiqiang; Zhu, Miaozhang; Chen, Jingzao; Ma, Hong

2007-04-01

Recent interest in adverse effects of infrasound on organisms arises from health concerns. We assessed the association between infrasound exposure of 5 Hz at 130 dB and changes of cardiac ultrastructure and function in rats. Thirty-two Sprague-Dawley rats were randomized into control, 1, 7, and 14 days groups for 2 h of infrasound once daily according to planned schedules. Changes of cardiac ultrastructure, hemodynamics indices, intracellular Ca(2+) concentrations ([Ca(2+)](i)), and sarcoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) were detected. Heart rates in 1 day group were significantly increased compared with control group and no significant changes in other groups. Left ventricular systolic pressures were significantly increased with time. Left ventricular diastolic end pressure and maximum rising rates of left ventricular pressure (+dl/dt) were significantly increased in 7 and 14 days groups and not changed in 1 day group, compared with control group. Maximum dropping rates of left ventricular pressure (-dl/dt) were significantly decreased in 7 and 14 days groups and not changed in 1 day group, compared with control group. In heart cells, there were several swelled mitochondria in 1 day group, more swelled mitochondria in 7 days group, platelet aggregation in the intercellular substance in 14 days group. [Ca(2+)](i) were significantly increased with time. There was a significant increase in SERCA2 in 1 day group, while a significant decrease in 7 and 14 days groups, compared with control group. Infrasound of 5 Hz at 130 dB can damage cardiac ultrastructure and function. Changes of [Ca(2+)](i) and SERCA2 play an important role in the secondary cardiac damage. (c) 2007 Wiley Periodicals, Inc.

Serca2a and Na{sup +}/Ca{sup 2+} exchanger are involved in left ventricular function following cardiac remodelling of female rats treated with anabolic androgenic steroid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nascimento, Andrews Marques do; Lima, Ewelyne Mira

Anabolic-androgenic steroids are misused, including by women, but little is known about the cardiovascular effects of these drugs on women. Aim: To evaluated the effects of nandrolone decanoate (ND) and resistive physical exercise on cardiac contractility in young female rats. Main methods: Female Wistar rats were separated into 4 groups: C (untrained animals); E (animals were submitted to resistance exercise by jumping in water 5 times per week); ND (animals were treated with ND, 20 mg/kg/week for 4 weeks); and NDE (trained and treated). The haemodynamic parameters (+ dP/dt{sub max}, − dP/dt{sub min} and Tau) were assessed in the leftmore » ventricle. The heart was collected for histological analyses and collagen deposition. The gastrocnemius muscle was weighed, and hypertrophy was assessed by the ratio of their weights to gastrocnemius/tibia length. The expression of calcium handling proteins was measured by western blot analysis. Results: ND treatment and physical exercise increased cardiac contractility and relaxation. In addition, ND promoted increases in phospholamban phosphorylated (p-PLB) and isoforms of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, while resistance exercise increased the phosphorylation of PLB and expression of Na{sup +}/Ca{sup 2+} exchangers (NCX). Cardiac hypertrophy and collagen deposition were observed after ND treatment. Conclusion: Regulatory components of cytosolic calcium, such as SERCA2a and p-PLB, play important roles in modulating the contractility and relaxation effects of ND in females. - Highlights: • ND and resistive exercise enhanced the cardiac function and increased expression of cytosolic calcium regulatory components.« less

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

PubMed

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc; Brennen, W Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; Demarzo, Angelo M; Wilding, George; Carducci, Michael A; Dionne, Craig A; Møller, Jesper V; Nissen, Poul; Christensen, S Brøgger; Isaacs, John T

2012-06-27

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: a coupled electromechanical modeling study

PubMed Central

2013-01-01

Background Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Methods Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa,L); and the enhancement in SERCA pump activity via phosphorylation of PLB. Results Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated β-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa,L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Conclusions Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa,L as the key mechanisms underlying the aforementioned positive FFR. PMID:24020888

Store-operated Ca²⁺ entry and depolarization explain the anomalous behaviour of myometrial SR: effects of SERCA inhibition on electrical activity, Ca²⁺ and force.

PubMed

Noble, Debbie; Borysova, Lyudmyla; Wray, Susan; Burdyga, Theodor

2014-09-01

In the myometrium SR Ca(2+) depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca(2+) sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca(2+)-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca(2+) transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca(2+)]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca(2+) spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca(2+) and depolarization continued a point was reached when electrical and Ca(2+) spikes and phasic contractions ceased, and a maintained, tonic force and Ca(2+) was produced. Lanthanum, a non-selective blocker of store-operated Ca(2+) entry, but not the L-type Ca(2+) channel blocker nifedipine (1-10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca(2+) depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca(2+) release is coupled to marked Ca(2+) entry, via store operated Ca(2+) channels, leading to depolarization and enhanced electrical and mechanical activity. Copyright © 2014. Published by Elsevier Ltd.

SERCA plays a crucial role in the toxicity of a betulinic acid derivative with potential antimalarial activity.

PubMed

Diedrich, Denise; Wildner, Andreia C; Silveira, Thayse F; Silva, Gloria N S; Santos, Francine Dos; da Silva, Elenilson F; do Canto, Vanessa P; Visioli, Fernanda; Gosmann, Grace; Bergold, Ana M; Zimmer, Aline R; Netz, Paulo A; Gnoatto, Simone C B

2018-05-01

Malaria is one of the most significant infectious diseases that affect poor populations in tropical areas throughout the world. Plants have been shown to be a good source for the development of new antimalarial chemotherapeutic agents, as shown for the discovery of quinine and artemisinin derivatives. Our research group has been working with semisynthetic triterpene derivatives that show potential antimalarial activity toward different strains of Plasmodium falciparum by specifically modulating calcium pathways in the parasite. Promising results were obtained for nanomolar concentrations of the semisynthetic betulinic acid derivative LAFIS13 against the P. falciparum 3D7 strain in vitro, with a selectivity index of 18 compared to a mammalian cell line. Continuing these studies, we present here in vitro and in vivo toxicological evaluations of this compound, followed by docking studies with PfATP6, a sarco/endoplasmic reticulum Ca +2 -ATPase (SERCA) protein. LAFIS13 showed an LD 50 between 300 and 50 mg/kg, and the acute administration of 50 mg/kg (i.p.) had no negative effects on hematological, biochemical and histopathological parameters. Based on the results of the in vitro assays, LAFIS13 not exerted significant effects on coagulation parameters of human peripheral blood, but a hemolytic activity was verified at higher concentrations. According to the molecular docking study, the PfATP6 protein may be a target for LAFIS13, which corroborates its previously reported modulatory effects on calcium homeostasis in the parasite. Notably, LAFIS13 showed a higher selectivity for the mammalian SERCA protein than for PfATP6, thus impairing the selectivity between parasite and host. In summary, the direct interaction with calcium pumps and the hemolytic potential of the compound proved to be plausible mechanism of LAFIS13 toxicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Neuronal calcium wave propagation varies with changes in endoplasmic reticulum parameters: a computer model

PubMed Central

Neymotin, Samuel A.; McDougal, Robert A.; Sherif, Mohamed A.; Fall, Christopher P.; Hines, Michael L.; Lytton, William W.

2015-01-01

Calcium (Ca2+) waves provide a complement to neuronal electrical signaling, forming a key part of a neuron’s second messenger system. We developed a reaction-diffusion model of an apical dendrite with diffusible inositol triphosphate (IP3), diffusible Ca2+, IP3 receptors (IP3Rs), endoplasmic reticulum (ER) Ca2+ leak, and ER pump (SERCA) on ER. Ca2+ is released from ER stores via IP3Rs upon binding of IP3 and Ca2+. This results in Ca2+-induced-Ca2+-release (CICR) and increases Ca2+ spread. At least two modes of Ca2+ wave spread have been suggested: a continuous mode based on presumed relative homogeneity of ER within the cell; and a pseudo-saltatory model where Ca2+ regeneration occurs at discrete points with diffusion between them. We compared the effects of three patterns of hypothesized IP3R distribution: 1. continuous homogeneous ER, 2. hotspots with increased IP3R density (IP3R hotspots), 3. areas of increased ER density (ER stacks). All three modes produced Ca2+ waves with velocities similar to those measured in vitro (~50–90µm /sec). Continuous ER showed high sensitivity to IP3R density increases, with time to onset reduced and speed increased. Increases in SERCA density resulted in opposite effects. The measures were sensitive to changes in density and spacing of IP3R hotspots and stacks. Increasing the apparent diffusion coefficient of Ca2+ substantially increased wave speed. An extended electrochemical model, including voltage gated calcium channels and AMPA synapses, demonstrated that membrane priming via AMPA stimulation enhances subsequent Ca2+ wave amplitude and duration. Our modeling suggests that pharmacological targeting of IP3Rs and SERCA could allow modulation of Ca2+ wave propagation in diseases where Ca2+ dysregulation has been implicated. PMID:25734493

Memantine can improve chronic ethanol exposure-induced spatial memory impairment in male C57BL/6 mice by reducing hippocampal apoptosis.

PubMed

Wang, Xiaolong; Yu, Hao; You, Jiabin; Wang, Changliang; Feng, Chunmei; Liu, Zhaodi; Li, Ya; Wei, Rucheng; Xu, Siqi; Zhao, Rui; Wu, Xu; Zhang, Guohua

2018-05-22

Chronic ethanol intake can induce neuronal apoptosis, leading to dementia. We investigated the protective effects of memantine on spatial memory impairment induced by chronic ethanol exposure in mice. Male C57BL/6 mice were administered 10% (m/V) or 20% (m/V) ethanol as the only choice of drinking water. Mice were treated for 60 d, 90 d, or 180 d. Mice were treated with memantine for the same duration (daily 10 mg/kg oral). The Morris water maze and radial arm maze test were used to measure spatial memory. Mice were sacrificed after the behavioral tests. Brains were removed to prepare for paraffin sections, and hippocampi were isolated for protein and RNA extraction. 4',6-diamidino-2-phenylindole (DAPI) staining and immunohistochemical staining of cleaved caspase-3 were performed. Western blot analysis was used to detect the expression of cleaved caspase-3 and calcium-related proteins, including N-methyl-d-aspartic acid receptor 1 (NR1), 1,4,5-trisphosphate receptor 1 (IP3R1), and sarco/endoplasmic reticulum calcium adenosine triphosphatase 1 (SERCA1). The changes of NR1, IP3R1 and SERCA1 mRNA were detected using quantitative polymerase chain reaction (qPCR). The results revealed that chronic ethanol exposure induced spatial memory impairment in mice, as well as increasing the expression of NR1, IP3R1 and SERCA1, the activation of caspase-3 and apoptosis in hippocampus. The effect was particularly prominent in the 20% ethanol group after 180 d exposure. Memantine decreased ethanol-induced spatial memory impairment, caspase-3 activation and apoptosis in the mouse hippocampus. These results suggest that disruption of intracellular calcium balance by ethanol can induce caspase-3 activation and apoptosis, which underlies subsequent spatial memory impairment in mice. Copyright © 2018 Elsevier B.V. All rights reserved.

Rhodiola Inhibits Atrial Arrhythmogenesis in a Heart Failure Model.

PubMed

Liu, Shuen-Hsin; Hsiao, Ya-Wen; Chong, Eric; Singhal, Rahul; Fong, Man-Cai; Tsai, Yung-Nan; Hsu, Chiao-Po; Chen, Yao-Chang; Chen, Yi-Jen; Chiou, Chuen-Wang; Chiang, Shuo-Ju; Chang, Shih-Lin; Chen, Shih-Ann

2016-09-01

Rhodiola, a popular plant in Tibet, has been proven to decrease arrhythmia. The aim of this study was to elucidate the molecular mechanism and electrophysiological properties of rhodiola in the suppression of atrial fibrillation. This study consisted of 3 groups as follows: Group 1: normal control rabbits (n = 5); Group 2: rabbits with heart failure (HF) created by coronary ligation and who received 2 weeks of water orally as a placebo (n = 5); and Group 3: rabbits with HF who received 2 weeks of a rhodiola 270 mg/kg/day treatment orally (n = 5). The monophasic action potential, histology, and real-time polymerase chain reaction (RT-PCR) analysis of ionic channels and PI3K/AKT/eNOS were examined. Compared with the HF group, attenuated atrial fibrosis (35.4 ± 17.4% vs. 16.9 ± 8.4%, P = 0.05) and improved left ventricular (LV) ejection fraction (51.6 ± 3.4% vs. 68.0 ± 0.5%, P = 0.001) were observed in the rhodiola group. The rhodiola group had a shorter ERP (85.3 ± 6.8 vs. 94.3 ± 1.2, P = 0.002), APD90 (89.3 ± 1.5 vs. 112.7 ± 0.7, P < 0.001) in the left atrium (LA), and decreased AF inducibility (0.90 ± 0.04 vs. 0.42 ± 0.04, P < 0.001) compared with the HF group. The mRNA expressions of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, and SERCA2a in the HF LA were up-regulated after rhodiola treatment. The rhodiola-treated HF LA demonstrated higher mRNA expression of PI3K-AKT compared with the HF group. Rhodiola reversed LA electrical remodeling, attenuated atrial fibrosis and suppressed AF in rabbits with HF. The beneficial electrophysiological effect of rhodiola may be related to upregulation of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, SERCA2a, and activation of PI3K/AKT signaling. © 2016 Wiley Periodicals, Inc.

Arterial α2-Na+ pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific α2 mice.

PubMed

Chen, Ling; Song, Hong; Wang, Youhua; Lee, Jane C; Kotlikoff, Michael I; Pritchard, Tracy J; Paul, Richard J; Zhang, Jin; Blaustein, Mordecai P

2015-09-01

Arterial myocytes express α1-catalytic subunit isoform Na(+) pumps (75-80% of total), which are ouabain resistant in rodents, and high ouabain affinity α2-Na(+) pumps. Mice with globally reduced α2-pumps (but not α1-pumps), mice with mutant ouabain-resistant α2-pumps, and mice with a smooth muscle (SM)-specific α2-transgene (α2 (SM-Tg)) that induces overexpression all have altered blood pressure (BP) phenotypes. We generated α2 (SM-DN) mice with SM-specific α2 (not α1) reduction (>50%) using nonfunctional dominant negative (DN) α2. We compared α2 (SM-DN) and α2 (SM-Tg) mice to controls to determine how arterial SM α2-pumps affect vasoconstriction and BP. α2 (SM-DN) mice had elevated basal mean BP (mean BP by telemetry: 117 ± 4 vs. 106 ± 1 mmHg, n = 7/7, P < 0.01) and enhanced BP responses to chronic ANG II infusion (240 ng·kg(-1)·min(-1)) and high (6%) NaCl. Several arterial Ca(2+) transporters, including Na(+)/Ca(2+) exchanger 1 (NCX1) and sarcoplasmic reticulum and plasma membrane Ca(2+) pumps [sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and plasma membrane Ca(2+)-ATPase 1 (PMCA1)], were also reduced (>50%). α2 (SM-DN) mouse isolated small arteries had reduced myogenic reactivity, perhaps because of reduced Ca(2+) transporter expression. In contrast, α2 (SM-Tg) mouse aortas overexpressed α2 (>2-fold), NCX1, SERCA2, and PMCA1 (43). α2 (SM-Tg) mice had reduced basal mean BP (104 ± 1 vs. 109 ± 2 mmHg, n = 15/9, P < 0.02) and attenuated BP responses to chronic ANG II (300-400 ng·kg(-1)·min(-1)) with or without 2% NaCl but normal myogenic reactivity. NCX1 expression was inversely related to basal BP in SM-α2 engineered mice but was directly related in SM-NCX1 engineered mice. NCX1, which usually mediates arterial Ca(2+) entry, and α2-Na(+) pumps colocalize at plasma membrane-sarcoplasmic reticulum junctions and functionally couple via the local Na(+) gradient to help regulate cell Ca(2+). Altered Ca(2+) transporter expression in SM-α2 engineered mice apparently compensates to minimize Ca(2+) overload (α2 (SM-DN)) or depletion (α2 (SM-Tg)) and attenuate BP changes. In contrast, Ca(2+) transporter upregulation, observed in many rodent hypertension models, should enhance Ca(2+) entry and signaling and contribute significantly to BP elevation. Copyright © 2015 the American Physiological Society.

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

PubMed

Kronenbitter, Annette; Funk, Florian; Hackert, Katarzyna; Gorreßen, Simone; Glaser, Dennis; Boknik, Peter; Poschmann, Gereon; Stühler, Kai; Isić, Malgorzata; Krüger, Martina; Schmitt, Joachim P

2018-06-01

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca 2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca 2+ handling. The amplitude of the Ca 2+ transients as well as the kinetics of Ca 2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca 2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca 2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca 2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca 2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

AFos Dissociates Cardiac Myocyte Hypertrophy and Expression of the Pathological Gene Program

PubMed Central

Jeong, Mark Y.; Kinugawa, Koichiro; Vinson, Charles; Long, Carlin S.

2005-01-01

Background Although induction of activator protein-1 (AP-1) transcription factor activity has been observed in cardiac hypertrophy, a direct role for AP-1 in myocardial growth and gene expression remains obscure. Methods and Results Hypertrophy was induced in cultured neonatal rat cardiomyocytes with phenylephrine or overexpression of a constitutively active MAP3K, MKK6. In both treatment groups, induction of the pathological gene profile was observed, ie, expression of β-myosin heavy chain (βMHC), atrial/brain natriuretic peptides (ANP/BNP), and skeletal α-actin (sACT) was increased, whereas expression for α-myosin heavy chain (αMHC) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. The role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AFos did not change the myocyte growth response, it abrogated the gene profile to both agonists, including the upregulation of both αMHC and SERCA expression. Conclusions Although c-Fos/AP-1 is necessary for induction of the pathological/fetal gene program, it does not appear to be critical for cardiomyocyte hypertrophy. PMID:15795322

Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach.

PubMed

Calderón, Juan C; Bolaños, Pura; Caputo, Carlo

2014-12-01

One hundred and eighty six enzymatically dissociated murine muscle fibres were loaded with Mag-Fluo-4 AM, and adhered to laminin, to evaluate the effect of modulating cytosolic Ca(2+) buffers and sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), mitochondria, and Na(+)/Ca(2+) exchanger (NCX) on the differential tetanic Ca(2+) transient kinetics found in different fibre types. Tetanic Ca(2+) transients were classified as morphology type I (MT-I) or type II (MT-II) according to their shape. The first peak of the MT-I (n = 25) and MT-II (n = 23) tetanic Ca(2+) transients had an amplitude (∆F/F) of 0.41 ± 0.03 and 0.83 ± 0.05 and a rise time (ms) of 1.35 and 0.98, respectively. MT-I signals had a time constant of decay (τ1, ms) of 75.9 ± 4.2 while MT-II transients showed a double exponential decay with time constants of decay (τ1 and τ2, ms) of 18.3 ± 1.4 and 742.2 ± 130.3. Sarcoendoplasmic reticulum Ca(2+) ATPase inhibition demonstrated that the decay phase of the tetanic transients mostly rely on SERCA function. Adding Ca(2+) chelators in the AM form to MT-I fibres changed the morphology of the initial five peaks to a MT-II one, modifying the decay phase of the signal in a dose-dependent manner. Mitochondria and NCX function have a minor role in explaining differences in tetanic Ca(2+) transients among fibre types but still help in removing Ca(2+) from the cytosol in both MT-I and MT-II fibres. Cytoplasmic Ca(2+) buffering capacity and SERCA function explain most of the different kinetics found in tetanic Ca(2+) transients from different fibre types, but mitochondria and NCX have a measurable role in shaping tetanic Ca(2+) responses in both slow and fast-twitch muscle fibre types. We provided experimental evidence on the mechanisms that help understand the kinetics of tetanic Ca(2+) transients themselves and explain kinetic differences found among fibre types.

Gestational exposure to diethylstilbestrol alters cardiac structure/function, protein expression and DNA methylation in adult male mice progeny

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haddad, Rami, E-mail: rami.haddad@mail.mcgill.ca; Division of Experimental Medicine, Department of Medicine, McGill University, 850 Sherbrooke Street, Montréal, Québec, Canada H3A 1A2; Kasneci, Amanda, E-mail: amanda.kasneci@mail.mcgill.ca

2013-01-01

Pregnant women, and thus their fetuses, are exposed to many endocrine disruptor compounds (EDCs). Fetal cardiomyocytes express sex hormone receptors making them potentially susceptible to re-programming by estrogenizing EDCs. Diethylstilbestrol (DES) is a proto-typical, non-steroidal estrogen. We hypothesized that changes in adult cardiac structure/function after gestational exposure to the test compound DES would be a proof in principle for the possibility of estrogenizing environmental EDCs to also alter the fetal heart. Vehicle (peanut oil) or DES (0.1, 1.0 and 10.0 μg/kg/da.) was orally delivered to pregnant C57bl/6n dams on gestation days 11.5–14.5. At 3 months, male progeny were left sedentarymore » or were swim trained for 4 weeks. Echocardiography of isoflurane anesthetized mice revealed similar cardiac structure/function in all sedentary mice, but evidence of systolic dysfunction and increased diastolic relaxation after swim training at higher DES doses. The calcium homeostasis proteins, SERCA2a, phospholamban, phospho-serine 16 phospholamban and calsequestrin 2, are important for cardiac contraction and relaxation. Immunoblot analyses of ventricle homogenates showed increased expression of SERCA2a and calsequestrin 2 in DES mice and greater molecular remodeling of these proteins and phospho-serine 16 phospholamban in swim trained DES mice. DES increased cardiac DNA methyltransferase 3a expression and DNA methylation in the CpG island within the calsequestrin 2 promoter in heart. Thus, gestational DES epigenetically altered ventricular DNA, altered cardiac function and expression, and reduced the ability of adult progeny to cardiac remodel when physically challenged. We conclude that gestational exposure to estrogenizing EDCs may impact cardiac structure/function in adult males. -- Highlights: ► Gestational DES changes cardiac SERCA2a and CASQ2 expression. ► Echocardiography identified systolic dysfunction and increased diastolic relaxation. ► DES increased DNMT3a expression and increased CpG DNA methylation. ► DES impacts fetal heart reducing cardiac reserve on challenge in adulthood. ► Fetal heart can be re-programmed by a non-steroidal estrogen.« less

p53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

PubMed

Bittremieux, Mart; Bultynck, Geert

2015-01-01

Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury.

PubMed

Wang, Yao; Wei, Su; Wang, Yi-Lu; Liu, Miao; Shang, Man; Zhang, Qi; Wu, Yan-Na; Liu, Ming-Lin; Song, Jun-Qiu; Liu, Yan-Xia

2017-08-15

To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans).

PubMed

Morrissette, Jeffery M; Franck, Jens P G; Block, Barbara A

2003-03-01

A thermogenic organ is found beneath the brain of billfishes (Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel (Scombridae). The heater organ has been shown to warm the brain and eyes up to 14 degrees C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR) enriched in Ca(2+)-ATPase (SERCA) pumps and ryanodine receptors (RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca(2+). In this study, Ca(2+) fluxes in heater SR vesicles derived from blue marlin (Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca(2+). Uptake of Ca(2+) was thapsigargin sensitive, and maximum loading ranged between 0.8 micro mol Ca(2+) mg(-1) protein and 1.0 micro mol Ca(2+) mg(-1) protein. Upon the addition of 10 mmol l(-1) caffeine or 350 micro mol l(-1) ryanodine, heater SR vesicles released only a small fraction of the loaded Ca(2+). However, ryanodine could elicit a much larger Ca(2+) release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca(2+) is elevated, a condition that normally closes RyRs. The fast Ca(2+) sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca(2+) cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs, and, in this paper, we demonstrate that heater cells contain large 'endplate-like' clusters of acetylcholine receptors that appear to provide a mechanism for nervous control of thermogenesis.

Cardioprotective effects of early and late aerobic exercise training in experimental pulmonary arterial hypertension.

PubMed

Moreira-Gonçalves, Daniel; Ferreira, Rita; Fonseca, Hélder; Padrão, Ana Isabel; Moreno, Nuno; Silva, Ana Filipa; Vasques-Nóvoa, Francisco; Gonçalves, Nádia; Vieira, Sara; Santos, Mário; Amado, Francisco; Duarte, José Alberto; Leite-Moreira, Adelino F; Henriques-Coelho, Tiago

2015-11-01

Clinical studies suggest that aerobic exercise can exert beneficial effects in pulmonary arterial hypertension (PAH), but the underlying mechanisms are largely unknown. We compared the impact of early or late aerobic exercise training on right ventricular function, remodeling and survival in experimental PAH. Male Wistar rats were submitted to normal cage activity (SED), exercise training in early (EarlyEX) and in late stage (LateEX) of PAH induced by monocrotaline (MCT, 60 mg/kg). Both exercise interventions resulted in improved cardiac function despite persistent right pressure-overload, increased exercise tolerance and survival, with greater benefits in EarlyEX+MCT. This was accompanied by improvements in the markers of cardiac remodeling (SERCA2a), neurohumoral activation (lower endothelin-1, brain natriuretic peptide and preserved vascular endothelial growth factor mRNA), metabolism and mitochondrial oxidative stress in both exercise interventions. EarlyEX+MCT provided additional improvements in fibrosis, tumor necrosis factor-alpha/interleukin-10 and brain natriuretic peptide mRNA, and beta/alpha myosin heavy chain protein expression. The present study demonstrates important cardioprotective effects of aerobic exercise in experimental PAH, with greater benefits obtained when exercise training is initiated at an early stage of the disease.

Burst discharges in neurons of the thalamic reticular nucleus are shaped by calcium-induced calcium release.

PubMed

Coulon, Philippe; Herr, David; Kanyshkova, Tatyana; Meuth, Patrick; Budde, Thomas; Pape, Hans-Christian

2009-01-01

The nucleus reticularis thalami (NRT) is a layer of inhibitory neurons that surrounds the dorsal thalamus. It appears to be the 'pacemaker' of certain forms of slow oscillations in the thalamus and was proposed to be a key determinant of the internal attentional searchlight as well as the origin of hypersynchronous activity during absence seizures. Neurons of the NRT exhibit a transient depolarization termed low threshold spike (LTS) following sustained hyperpolarization. This is caused by the activation of low-voltage-activated Ca2+ channels (LVACC). Although the role of these channels in thalamocortical oscillations was studied in great detail, little is known about the downstream intracellular Ca2+ signalling pathways and their feedback onto the oscillations. A signalling triad consisting of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), Ca2+ activated K+ channels (SK2), and LVACC is active in dendrites of NRT neurons and shapes rhythmic oscillations. The aim of our study was to find out (i) if and how Ca2+-induced Ca2+ release (CICR) via ryanodine receptors (RyR) can be evoked in NRT neurons and (ii) how the released Ca2+ affects burst activity. Combining electrophysiological, immunohistochemical, and two-photon Ca2+ imaging techniques, we show that CICR in NRT neurons takes place by a cell-type specific coupling of LVACC and RyR. CICR could be evoked by the application of caffeine, by activation of LVACC, or by repetitive LTS generation. During the latter, CICR contributed 30% to the resulting build-up of [Ca2+]i. CICR was abolished by cyclopiazonic acid, a specific blocker for SERCA, or by high concentrations of ryanodine (50 microM). Unlike other thalamic nuclei, in the NRT the activation of high-voltage-activated Ca2+ channels failed to evoke CICR. While action potentials contributed little to the build-up of [Ca2+]i upon repetitive LTS generation, the Ca2+ released via RyR significantly reduced the number of action potentials during an LTS and reduced the neurons' low threshold activity, thus potentially reducing hypersynchronicity. This effect persisted in the presence of the SK2 channel blocker apamin. We conclude that the activation of LVACC specifically causes CICR via RyR in neurons of the NRT, thereby adding a Ca2+-dependent intracellular route to the mechanisms determining rhythmic oscillatory bursting in this nucleus.

Ca2+-pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling

PubMed Central

Shiah, Amy A.; Gandra, Paulo G.; Hogan, Michael C.

2013-01-01

The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca2+-pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca2+ pumping become slowed, possibly because of intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca2+-pumping properties has not been investigated. Therefore, we inhibited cross-bridge cycling by incubating isolated Xenopus single fibers with N-benzyl-p-toluene sulfonamide (BTS) to study the mechanisms by which SR Ca2+ pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca2+]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca2+]c transients during stimulation without cross-bridge activation. At the time point that tension was reduced to 50% in the control condition, the fall in the peak [Ca2+]c and the increase in basal [Ca2+]c did not occur with BTS incubation. The progressively slower Ca2+ pumping rate and the fall in pHi during repetitive contractions were reduced during BTS conditions. However, when mitochondrial ATP supply was blocked during contractions with BTS present (BTS + cyanide), there was no further slowing in SR Ca2+ pumping during contractions compared with the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS + cyanide condition than in the control conditions. These results demonstrate that factors related to the energetic cost of cross-bridge cycling, possibly the accumulation of metabolites, inhibit the Ca2+ pumping rate during fatiguing contractions. PMID:23678027

Frequency-dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a phosphorylation

PubMed Central

Valverde, Carlos A; Mundiña-Weilenmann, Cecilia; Said, Matilde; Ferrero, Paola; Vittone, Leticia; Salas, Margarita; Palomeque, Julieta; Petroff, Martín Vila; Mattiazzi, Alicia

2005-01-01

An increase in stimulation frequency causes an acceleration of myocardial relaxation (FDAR). Several mechanisms have been postulated to explain this effect, among which is the Ca2+–calmodulin-dependent protein kinase (CaMKII)-dependent phosphorylation of the Thr17 site of phospholamban (PLN). To gain further insights into the mechanisms of FDAR, we studied the FDAR and the phosphorylation of PLN residues in perfused rat hearts, cat papillary muscles and isolated cat myocytes. This allowed us to sweep over a wide range of frequencies, in species with either positive or negative force–frequency relationships, as well as to explore the FDAR under isometric (or isovolumic) and isotonic conditions. Results were compared with those produced by isoprenaline, an intervention known to accelerate relaxation (IDAR) via PLN phosphorylation. While IDAR occurs tightly associated with a significant increase in the phosphorylation of Ser16 and Thr17 of PLN, FDAR occurs without significant changes in the phosphorylation of PLN residues in the intact heart and cat papillary muscles. Moreover, in intact hearts, FDAR was not associated with any significant change in the CaMKII-dependent phosphorylation of sarcoplasmic/endoplasmic Ca2+ ATPase (SERCA2a), and was not affected by the presence of the CaMKII inhibitor, KN-93. In isolated myocytes, FDAR occurred associated with an increase in Thr17 phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser16 and Thr17) was significantly higher in the presence of the β-agonist. Moreover, the time course of Thr17 phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser16 phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN-93 significantly decreased the phosphorylation of Thr17 that was evoked by increasing the stimulation frequency, but failed to affect FDAR. Taken together, the results provide direct evidence indicating that CaMKII phosphorylation pathways are not involved in FDAR and that FDAR and IDAR do not share a common underlying mechanism. More likely, a CaMKII-independent mechanism could be involved, whereby increasing stimulation frequency would disrupt the SERCA2a–PLN interaction, leading to an increase in SR Ca2+ uptake and myocardial relaxation. PMID:15528241

Characterization of Beta-leptinotarsin-h and the Effects of Calcium Flux Antagonists on its Activity

DTIC Science & Technology

2005-04-07

A alone. a IP3R, IP3 receptor ; LO, ligand -operated; RyR, ryanodine receptor ; SERCA, sarcoplasmic reticulum endoplasmic reticulum Ca 2C ATPase; SO...observation eliminated non-selective cation channels such as nicotinic, glutamatergic, purinergic P2X , and serotoni- nergic 5-HT3 ligand -operated Ca 2C...nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand -operated, store-operated, and transduction-operated channels were also not

Divergent Mechanisms Leading to Signaling Dysfunction in Embryonic Muscle by Bisphenol A and Tetrabromobisphenol A

PubMed Central

Pessah, Isaac N.

2017-01-01

Bisphenol A (BPA) and its brominated derivative tetrabromobisphenol A (TBBPA) are high production volume chemicals used in the manufacture of various consumer products. Although regarded as endocrine disruptors, these chemicals are suspected to exert nongenomic actions on muscle function that are not well understood. Using skeletal muscle microsomes, we examined the effects of BPA and TBBPA on ryanodine receptor type 1 (RyR1), dihydropyridine receptor (DHPR), and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We assessed the impact of these chemicals on Ca2+ dynamics and signaling in embryonic skeletal myotubes through fluorescent Ca2+ imaging and measurement of resting membrane potential (Vm). TBBPA activated RyR1 and inhibited DHPR and SERCA, inducing a net efflux of Ca2+ from loaded microsomes, whereas BPA exhibited little or no activity at these targets. Regardless, both compounds disrupted the function of intact myotubes. TBBPA diminished and eventually abrogated Ca2+ transients, altered intracellular Ca2+ equilibrium, and caused Vm depolarization. For some cells, BPA caused rapid Ca2+ transient loss without marked changes in cytosolic and sarcoplasmic reticulum Ca2+ levels, likely owing to altered cellular excitability as a result of BPA-induced Vm hyperpolarization. BPA and TBBPA both interfere with skeletal muscle function through divergent mechanisms that impair excitation-contraction coupling and may be exemplary of their adverse outcomes in other muscle types. PMID:28143888

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

PubMed

Sepp, Mervi; Sokolova, Niina; Jugai, Svetlana; Mandel, Merle; Peterson, Pearu; Vendelin, Marko

2014-01-01

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle.

PubMed

Kramer, Philip A; Duan, Jicheng; Gaffrey, Matthew J; Shukla, Anil K; Wang, Lu; Bammler, Theo K; Qian, Wei-Jun; Marcinek, David J

2018-05-23

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia. Copyright © 2018. Published by Elsevier B.V.

A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

DTIC Science & Technology

2000-03-31

touted as inhibitors of the PMCAs, but their effects have yet to be thoroughly examined on all P-type ATPases [23, 125]. SERCAs: a family ofP·type ATPases... effective in reducing the sensitivity to TG by 1aOO-fold [151]. The potent, seledive blockade of Ca2+ accumulation in the ER by TG has made it possible...accumulation while the calcium ionophore A23187 is 100%) effective [140]. Thus, 10-150/0 of net Ca2+ uptake into brain microsomal compartments is

Glucose-6-phosphate Reduces Calcium Accumulation in Rat Brain Endoplasmic Reticulum

DTIC Science & Technology

2012-04-01

Ekman and Jager, 1993) adapted from the ammonium molybdate/ malachite green method quan- tified Pi production by SERCA activity. Colorimetric reagent was...prepared by mixing one volume of 10% (w/v) (NH4)6Mo7O24– 4 H2O in 4M HCl with three volumes of 0.2% (w/v) malachite green in 4M HCl, followed by...seryl and threonyl residues in phosphoproteins using alkaline hydrolysis and malachite green. Anal. Biochem. 214, 138. Fulceri, R., Romani, A

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node.

PubMed

Musa, Hanny; Lei, Ming; Honjo, Hauro; Jones, Sandra A; Dobrzynski, Halina; Lancaster, Mathew K; Takagishi, Yoshiko; Henderson, Zaineb; Kodama, Itsuo; Boyett, Mark R

2002-03-01

We investigated the densities of the L-type Ca(2+) current, i(Ca,L), and various Ca(2+) handling proteins in rabbit sinoatrial (SA) node. The density of i(Ca,L), recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of i(Ca,L) was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+) release channel (RYR2), and sarcoplasmic reticulum Ca(2+) pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na(+)-K(+) pump was similar in peripheral and central cells. We conclude that Ca(2+) handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).

Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca2+ ATPase

PubMed Central

Cheng, Hongwei; Smith, Godfrey L.; Hancox, Jules C.; Orchard, Clive H.

2011-01-01

The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na–Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca2+]i transients from cells isolated from the rabbit AVN. Spontaneous [Ca2+]i transients were monitored from undialysed AVN cells at 37 °C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca2+]i transients. However, in voltage clamp experiments in addition to blocking NCX current (INCX) KB-R7943 partially inhibited L-type calcium current (ICa,L). Rapid reduction of external [Na+] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca2+]i transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca2+ release influences spontaneous activity in AVN cells, and that this occurs via [Ca2+]i-activated INCX; however, the inhibitory action of KB-R7943 on ICa,L means that care is required in the interpretation of data obtained using this compound. PMID:21163524

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

PubMed

Toussay, Xavier; Morel, Jean-Luc; Biendon, Nathalie; Rotureau, Lolita; Legeron, François-Pierre; Boutonnet, Marie-Charlotte; Cho, Yoon H; Macrez, Nathalie

2017-10-01

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca 2+ ) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca 2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca 2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca 2+ -release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca 2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca 2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca 2+ signals in PS1dE9 mutant mice. Copyright © 2017 Elsevier Inc. All rights reserved.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma.

PubMed

Tang, Juan; Guo, Yun-Shan; Yu, Xiao-Ling; Huang, Wan; Zheng, Ming; Zhou, Ying-Hui; Nan, Gang; Wang, Jian-Chao; Yang, Hai-Jiao; Yu, Jing-Min; Jiang, Jian-Li; Chen, Zhi-Nan

2015-10-27

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma

PubMed Central

Zheng, Ming; Zhou, Ying-Hui; Nan, Gang; Wang, Jian-Chao; Yang, Hai-Jiao; Yu, Jing-Min; Jiang, Jian-Li; Chen, Zhi-Nan

2015-01-01

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+ refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC. PMID:26498680

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy.

PubMed

Dufresne, Sébastien S; Boulanger-Piette, Antoine; Bossé, Sabrina; Argaw, Anteneh; Hamoudi, Dounia; Marcadet, Laetitia; Gamu, Daniel; Fajardo, Val A; Yagita, Hideo; Penninger, Josef M; Russell Tupling, A; Frenette, Jérôme

2018-04-24

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Histology, composition, and quality traits of chicken Pectoralis major muscle affected by wooden breast abnormality.

PubMed

Soglia, F; Mudalal, S; Babini, E; Di Nunzio, M; Mazzoni, M; Sirri, F; Cavani, C; Petracci, M

2016-03-01

Only a few years ago, the poultry industry began to face a recent abnormality in breast meat, known as wooden breast, which frequently overlaps with white striping. This study aimed to assess the impact of wooden breast abnormality on quality traits of meat. For this purpose, 32 normal (NRM), 32 wooden (WB), and 32 wooden and white-striped (WB/WS) Pectoralis major muscles were selected from the same flock of heavy broilers (males, Ross 708, weighing around 3.7 kg) in the deboning area of a commercial processing plant at 3 h postmortem and used to assess histology, proximate (moisture, protein, fat, ash, and collagen) and mineral composition (Mg, K, P, Na and Ca), sarcoplasmic and myofibrillar protein patterns, and technological traits of breast meat. Compared to the normal group, WB/WS fillets showed more severe histological lesions characterized by fiber degeneration, fibrosis, and lipidosis, coupled with a significantly harder texture. With regard to proximate and mineral composition, abnormal samples exhibited significantly (P < 0.001) higher moisture, fat, and collagen contents coupled with lower (P < 0.001) amounts of protein and ash. Furthermore, increased calcium (131 vs. 84 mg kg(-1); P < 0.05) and sodium (741 vs. 393 mg kg(-1); P < 0.001) levels were found in WB/WS meat samples. The SDS-PAGE analysis revealed a significantly lower amount of calcium-ATPase (SERCA, 114 kDa), responsible for the translocation of Ca ions across the membrane, in normal breasts compared to abnormal ones. As for meat quality traits, fillets affected by wooden abnormality exhibited significantly (P < 0.001) higher ultimate pH and lower water-holding/water-binding capacity. In particular, compared to normal, abnormal samples showed reduced marinade uptake coupled with increased drip loss and cooking losses as well. In conclusion, this study revealed that meat affected by wooden breast or both wooden breast and white striping abnormalities exhibit poorer nutritional value, harder texture, and impaired water-holding capacity. © 2015 Poultry Science Association Inc.

Chronic Cardiac-Targeted RNA Interference for the Treatment of Heart Failure Restores Cardiac Function and Reduces Pathological Hypertrophy

PubMed Central

Suckau, Lennart; Fechner, Henry; Chemaly, Elie; Krohn, Stefanie; Hadri, Lahouaria; Kockskämper, Jens; Westermann, Dirk; Bisping, Egbert; Ly, Hung; Wang, Xiaomin; Kawase, Yoshiaki; Chen, Jiqiu; Liang, Lifan; Sipo, Isaac; Vetter, Roland; Weger, Stefan; Kurreck, Jens; Erdmann, Volker; Tschope, Carsten; Pieske, Burkert; Lebeche, Djamel; Schultheiss, Heinz-Peter; Hajjar, Roger J.; Poller, Wolfgang Ch.

2009-01-01

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease. PMID:19237664

Obesity Alters Molecular and Functional Cardiac Responses to Ischemia-Reperfusion and Glucagon-Like Peptide-1 Receptor Agonism

PubMed Central

Sassoon, Daniel J; Goodwill, Adam G; Noblet, Jillian N; Conteh, Abass M; Herring, B. Paul; McClintick, Jeanette N; Tune, Johnathan D; Mather, Kieren J

2016-01-01

This study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miR) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-min coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca2+ binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion. PMID:27234258

Dronedarone administration prevents body weight gain and increases tolerance of the heart to ischemic stress: a possible involvement of thyroid hormone receptor alpha1.

PubMed

Pantos, Constantinos; Mourouzis, Iordanis; Malliopoulou, Vassiliki; Paizis, Ioannis; Tzeis, Stylianos; Moraitis, Panagiotis; Sfakianoudis, Konstantinos; Varonos, Dennis D; Cokkinos, Dennis V

2005-01-01

Hypothyroid heart displays a phenotype of cardioprotection against ischemia and this study investigated whether administration of dronedarone, an amiodarone-like compound that has been shown to preferentially antagonize thyroid hormone binding to thyroid hormone receptor alpha1 (TRalpha1), results in a similar effect. Dronedarone was given in Wistar rats (90 mg/kg, once daily (od) for 2 weeks) (DRON), while untreated animals served as controls (CONT). Hypothyroidism (HYPO) was induced by propylthiouracil administration. Isolated rat hearts were perfused in Langendorff mode and subjected to 20 minutes of zero-flow global ischemia (I) followed by 45 minutes of reperfusion (R). 3,5,3' Triiodothyronine remained unchanged while body weight and food intake were reduced. alpha-Myosin heavy chain (alpha-MHC) decreased in DRON while beta-myosin heavy chain (beta-MHC) and sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) expression (SERCA) was similar to CONT. In HYPO, alpha-MHC and SERCA were decreased while beta-MHC was increased. Myocardial glycogen content was increased in both DRON and HYPO. In DRON, resting heart rate and contractility were reduced and ischemic contracture was significantly suppressed while postischemic left ventricular end-diastolic pressure and lactate dehydrogenase release (IU/L min) after I/R were significantly decreased. In conclusion, dronedarone treatment results in cardioprotection by selectively mimicking hypothyroidism. This is accompanied by a reduction in body weight because of the suppression of food intake. TRs might prove novel pharmacologic targets for the treatment of cardiovascular illnesses.

Muscle Non-shivering Thermogenesis and Its Role in the Evolution of Endothermy

PubMed Central

Nowack, Julia; Giroud, Sylvain; Arnold, Walter; Ruf, Thomas

2017-01-01

The development of sustained, long-term endothermy was one of the major transitions in the evolution of vertebrates. Thermogenesis in endotherms does not only occur via shivering or activity, but also via non-shivering thermogenesis (NST). Mammalian NST is mediated by the uncoupling protein 1 in the brown adipose tissue (BAT) and possibly involves an additional mechanism of NST in skeletal muscle. This alternative mechanism is based on Ca2+-slippage by a sarcoplasmatic reticulum Ca2+-ATPase (SERCA) and is controlled by the protein sarcolipin. The existence of muscle based NST has been discussed for a long time and is likely present in all mammals. However, its importance for thermoregulation was demonstrated only recently in mice. Interestingly, birds, which have evolved from a different reptilian lineage than mammals and lack UCP1-mediated NST, also exhibit muscle based NST under the involvement of SERCA, though likely without the participation of sarcolipin. In this review we summarize the current knowledge on muscle NST and discuss the efficiency of muscle NST and BAT in the context of the hypothesis that muscle NST could have been the earliest mechanism of heat generation during cold exposure in vertebrates that ultimately enabled the evolution of endothermy. We suggest that the evolution of BAT in addition to muscle NST was related to heterothermy being predominant among early endothermic mammals. Furthermore, we argue that, in contrast to small mammals, muscle NST is sufficient to maintain high body temperature in birds, which have enhanced capacities to fuel muscle NST by high rates of fatty acid import. PMID:29170642

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle.

PubMed

Kovac, Jason R; Chrones, Tom; Sims, Stephen M

2008-01-01

Following smooth muscle excitation and contraction, depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE) to replenish stores and sustain cytoplasmic Ca(2+) (Ca(2+)(i)) elevations. The objectives of the present study were to characterize CCE and the Ca(2+)(i) dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca(2+)-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca(2+)(i) as well as activation of nonselective cation currents. To deplete Ca(2+)(i) stores, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca(2+)-free bathing solution. Under these conditions, addition of extracellular Ca(2+) (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 microM) and lanthanum (100 microM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca(2+)-imaging techniques, the CCE-evoked rise of Ca(2+)(i) was associated with inward currents carrying Ca(2+) that were inhibited by SKF-96365. Regional specializations in Ca(2+) influx and handling during CCE were observed. Distinct "hotspot" regions of Ca(2+) rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca(2+) entry occurs in hotspots contributing to localized Ca(2+) elevations in human colonic smooth muscle.

Exercise training prevents the development of cardiac dysfunction in the low-dose streptozotocin diabetic rats fed a high-fat diet.

PubMed

Epp, Riley A; Susser, Shanel E; Morissette, Marc P; Kehler, D Scott; Jassal, Davinder S; Duhamel, Todd A

2013-01-01

This study tested the hypothesis that exercise training would prevent the development of diabetes-induced cardiac dysfunction and altered expression of sarcoplasmic reticulum Ca(2 +)-transport proteins in the low-dose streptozotocin-induced diabetic rats fed a high-fat diet (HFD+STZ). Male Sprague-Dawley rats (4 weeks old; 125-150 g) were made diabetic using a high-fat diet (40% fat, w/w) and a low-dose of streptozotocin (35 mg·(kg body mass)(-1)) by intravenous injection. Diabetic animals were divided among a sedentary group (Sed+HFD+STZ) or an exercise-trained group (Ex+HFD+STZ) that accumulated 3554 ± 338 m·day(-1) of voluntary wheel running (mean ± SE). Sedentary animals fed a low-fat diet served as the control (Sed+LFD). Oral glucose tolerance was impaired in the sedentary diabetic group (1179 ± 29; area under the curve (a.u.c.)) compared with that in the sedentary control animals (1447 ± 42 a.u.c.). Although left ventricular systolic function was unchanged by diabetes, impaired E/A ratios (i.e., diastolic function) and rates of pressure decay (-dP/dt) indicated the presence of diastolic dysfunction. Diabetes also reduced SERCA2a protein content and maximal SERCA2a activity (V(max)) by 21% and 32%, respectively. In contrast, the change in each parameter was attenuated by exercise training. Based on these data, it appears that exercise training prevented the development of diabetic cardiomyopathy and the dysregulation of sarcoplasmic reticulum protein content in an inducible animal model of type 2 diabetes.

Ca²⁺-pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling.

PubMed

Nogueira, Leonardo; Shiah, Amy A; Gandra, Paulo G; Hogan, Michael C

2013-07-15

The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca²⁺-pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca²⁺ pumping become slowed, possibly because of intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca²⁺-pumping properties has not been investigated. Therefore, we inhibited cross-bridge cycling by incubating isolated Xenopus single fibers with N-benzyl-p-toluene sulfonamide (BTS) to study the mechanisms by which SR Ca²⁺ pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca²⁺]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca²⁺]c transients during stimulation without cross-bridge activation. At the time point that tension was reduced to 50% in the control condition, the fall in the peak [Ca²⁺]c and the increase in basal [Ca²⁺]c did not occur with BTS incubation. The progressively slower Ca²⁺ pumping rate and the fall in pHi during repetitive contractions were reduced during BTS conditions. However, when mitochondrial ATP supply was blocked during contractions with BTS present (BTS + cyanide), there was no further slowing in SR Ca²⁺ pumping during contractions compared with the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS + cyanide condition than in the control conditions. These results demonstrate that factors related to the energetic cost of cross-bridge cycling, possibly the accumulation of metabolites, inhibit the Ca²⁺ pumping rate during fatiguing contractions.

Effects of atorvastatin and losartan on monocrotaline-induced pulmonary artery remodeling in rats.

PubMed

Xie, Liangdi; Lin, Peisen; Xie, Hong; Xu, Changsheng

2010-01-01

Structural remodeling of pulmonary artery plays an important role in maintaining sustained pulmonary arterial hypertension (PAH). The anti-remodeling effects of statins have been reported in systemic hypertension. In this study, we studied the effects of atovastatin (Ato) or losartan (Los) in monocrotaline (MCL)-induced pulmonary artery remodeling using a rat model. Forty Sprague-Dawley (SD) rats were randomly assigned into four groups (n = 10): normal control (Ctr), PAH, PAH treated with Los, and PAH treated with Ato. We found that in the Los- or Ato-treated group, the mean pulmonary arterial pressure, right heart hypertrophy index, ratio of wall/lumen thickness (WT%), as well as the wall/lumen area (WA%) were significantly reduced compared to the PAH group. Also in pulmonary arteries dissected from rats in the Ato- or Los-treated group, in both mRNA and protein levels, the expression of α1C subunit of voltage-gated calcium channel (Ca(v)α1c) was downregulated, while sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA-2a) and inositol 1,4,5 triphosphate receptor 1 (IP3R-1) upregulated. However, the mRNA level of RyR-3 subunit of calcium regulating channel was increased, whereas its protein level was reduced in the treated groups. Our results suggest that atorvastatin or losartan may regress the remodeling of the pulmonary artery in pulmonary hypertensive rats, with differential expression of calcium regulating channels.

Altered Left Ventricular Ion Channel Transcriptome in a High-Fat-Fed Rat Model of Obesity: Insight into Obesity-Induced Arrhythmogenesis

PubMed Central

Yon, Marianne; Pickavance, Lucy; Yanni Gerges, Joseph; Davis, Gershan; Wilding, John; Jian, Kun; Hart, George; Boyett, Mark

2016-01-01

Introduction. Obesity is increasingly common and is associated with an increased prevalence of cardiac arrhythmias. The aim of this study was to see whether in obesity there is proarrhythmic gene expression of ventricular ion channels and related molecules. Methods and Results. Rats were fed on a high-fat diet and compared to control rats on a normal diet (n = 8). After 8 weeks, rats on the high-fat diet showed significantly greater weight gain and higher adiposity. Left ventricle samples were removed at 8 weeks and mRNA expression of ion channels and other molecules was measured using qPCR. Obese rats had significant upregulation of Cav1.2, HCN4, Kir2.1, RYR2, NCX1, SERCA2a, and RYR2 mRNA and downregulation of ERG mRNA. In the case of HCN4, it was confirmed that there was a significant increase in protein expression. The potential effects of the mRNA changes on the ventricular action potential and intracellular Ca2+ transient were predicted using computer modelling. Modelling predicted prolongation of the ventricular action potential and an increase in the intracellular Ca2+ transient, both of which would be expected to be arrhythmogenic. Conclusion. High-fat diet causing obesity results in arrhythmogenic cardiac gene expression of ion channels and related molecules. PMID:27747100

The calcium–frequency response in the rat ventricular myocyte: an experimental and modelling study

PubMed Central

Gattoni, Sara; Røe, Åsmund Treu; Frisk, Michael; Louch, William E.; Niederer, Steven A.

2016-01-01

Key points In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force–frequency response (FFR). The majority of mammals have a positive force–frequency relationship (FFR). In rat the FFR is controversial.We derive a species‐ and temperature‐specific data‐driven model of the rat ventricular myocyte.As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium–frequency response (CFR) in our model and three altered models.The results show a biphasic peak calcium–frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency‐dependent increase in diastolic calcium.Alterations to the model reveal that inclusion of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated L‐type channel and transient outward K+ current activity enhances the positive magnitude calcium–frequency response, and the absence of CAMKII‐mediated increase in activity of the sarco/endoplasmic reticulum Ca2+‐ATPase induces a negative magnitude calcium–frequency response. Abstract An increase in heart rate affects the strength of cardiac contraction by altering the Ca2+ transient as a response to physiological demands. This is described by the force–frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat‐based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca2+ transient characteristics and showed a biphasic peak calcium–frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated increase in sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L‐type channel and transient outward K+ current activity and (3) Na+/K+ pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca2+–frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca2+. PMID:26916026

Endoplasmic reticulum Chaperon Tauroursodeoxycholic Acid Alleviates Obesity-Induced Myocardial Contractile Dysfunction

PubMed Central

Ceylan-Isik, Asli F.; Sreejayan, Nair; Ren, Jun

2010-01-01

ER stress is involved in the pathophysiology of obesity although little is known about the role of ER stress on obesity-associated cardiac dysfunction. This study was designed to examine the effect of ER chaperone tauroursodeoxycholic acid (TUDCA) on obesity-induced myocardial dysfunction. Adult lean and ob/ob obese mice were treated TUDCA (50 mg/kg/d, p.o.) or vehicle for 5 wks. Oral glucose tolerance test (OGTT) was performed. Echocardiography, cardiomyocyte contractile and intracellular Ca2+ properties were assessed. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and protein expression of intracellular Ca2+ regulatory proteins were measured using 45Ca2+ uptake and Western blot analysis, respectively. Insulin signaling, ER stress markers and HSP90 were evaluated. Our results revealed that chronic TUDCA treatment lower systolic blood pressure and lessened glucose intolerance in obese mice. Obesity led to increased diastolic diameter, cardiac hypertrophy, compromised fractional shortening, cardiomyocyte contractile (peak shortening, maximal velocity of shortening/relengthening, and duration of contraction/relaxation) and intracellular Ca2+ properties, all of which were significantly attenuated by TUDCA. TUDCA reconciled obesity-associated decreased in SERCA activity and expression, and increase in serine phosphorylation of IRS, total and phosphorylated cJun, ER stress markers Bip, peIF2α and pPERK. Obesity-induced changes in phospholamban and HSP90 were unaffected by TUDCA. In vitro finding revealed that TUDCA ablated palmitic acid-induced cardiomyocyte contractile dysfunction. In summary, these data depicted a pivotal role of ER stress in obesity-associated cardiac contractile dysfunction, suggesting the therapeutic potential of ER stress as a target in the management of cardiac dysfunction in obesity. PMID:21035453

Effect of 23-day muscle disuse on sarcoplasmic reticulum Ca2+ properties and contractility in human type I and type II skeletal muscle fibers.

PubMed

Lamboley, C R; Wyckelsma, V L; Perry, B D; McKenna, M J; Lamb, G D

2016-08-01

Inactivity negatively impacts on skeletal muscle function mainly through muscle atrophy. However, recent evidence suggests that the quality of individual muscle fibers is also altered. This study examined the effects of 23 days of unilateral lower limb suspension (ULLS) on specific force and sarcoplasmic reticulum (SR) Ca(2+) content in individual skinned muscle fibers. Muscle biopsies of the vastus lateralis were taken from six young healthy adults prior to and following ULLS. After disuse, the endogenous SR Ca(2+) content was ∼8% lower in type I fibers and maximal SR Ca(2+) capacity was lower in both type I and type II fibers (-11 and -5%, respectively). The specific force, measured in single skinned fibers from three subjects, decreased significantly after ULLS in type II fibers (-23%) but not in type I fibers (-9%). Western blot analyses showed no significant change in the amounts of myosin heavy chain (MHC) I and MHC IIa following the disuse, whereas the amounts of sarco(endo)plasmic reticulum Ca(2+)-ATPase 1 (SERCA1) and calsequestrin increased by ∼120 and ∼20%, respectively, and the amount of troponin I decreased by ∼21%. These findings suggest that the decline in force and power occurring with muscle disuse is likely to be exacerbated in part by reductions in maximum specific force in type II fibers, and in the amount of releasable SR Ca(2+) in both fiber types, the latter not being attributable to a reduced calsequestrin level. Furthermore, the ∼3-wk disuse in human elicits change in SR properties, in particular a more than twofold upregulation in SERCA1 density, before any fiber-type shift. Copyright © 2016 the American Physiological Society.

PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Min, E-mail: chenminyx@gmail.com; Yunnan Centers for Diseases Prevention and Control, Kunming 650022; Wang, Yanru

2010-06-11

Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+}more » transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.« less

Isoflurane-Induced Caspase-3 Activation Is Dependent on Cytosolic Calcium and Can Be Attenuated by Memantine

PubMed Central

Zhang, Guohua; Dong, Yuanlin; Zhang, Bin; Ichinose, Fumito; Wu, Xu; Culley, Deborah J.; Crosby, Gregory

2008-01-01

Increasing evidence indicates that caspase activation and apoptosis are associated with a variety of neurodegenerative disorders, including Alzheimer's disease. We reported that anesthetic isoflurane can induce apoptosis, alter processing of the amyloid precursor protein (APP), and increase amyloid-β protein (Aβ) generation. However, the mechanism by which isoflurane induces apoptosis is primarily unknown. We therefore set out to assess effects of extracellular calcium concentration on isoflurane-induced caspase-3 activation in H4 human neuroglioma cells stably transfected to express human full-length APP (H4-APP cells). In addition, we tested effects of RNA interference (RNAi) silencing of IP3 receptor, NMDA receptor, and endoplasmic reticulum (ER) calcium pump, sacro-/ER calcium ATPase (SERCA1). Finally, we examined the effects of the NMDA receptor partial antagonist, memantine, in H4-APP cells and brain tissue of naive mice. EDTA (10 mm), BAPTA (10 μm), and RNAi silencing of IP3 receptor, NMDA receptor, or SERCA1 attenuated capase-3 activation. Memantine (4 μm) inhibited isoflurane-induced elevations in cytosolic calcium levels and attenuated isoflurane-induced caspase-3 activation, apoptosis, and cell viability. Memantine (20 mg/kg, i.p.) reduced isoflurane-induced caspase-3 activation in brain tissue of naive mice. These results suggest that disruption of calcium homeostasis underlies isoflurane-induced caspase activation and apoptosis. We also show for the first time that the NMDA receptor partial antagonist, memantine, can prevent isoflurane-induced caspase-3 activation and apoptosis in vivo and in vitro. These findings, indicating that isoflurane-induced caspase activation and apoptosis are dependent on cytosolic calcium levels, should facilitate the provision of safer anesthesia care, especially for Alzheimer's disease and elderly patients. PMID:18434534

Cardioprotective effects of baicalein on heart failure via modulation of Ca(2+) handling proteins in vivo and in vitro.

PubMed

Zhao, Fali; Fu, Lu; Yang, Wei; Dong, Yuhui; Yang, Jing; Sun, Shoubin; Hou, Yuling

2016-01-15

Baicalein is a widely used Chinese herbal medicine extracted from Labiatae plants Scutellaria baicalensis Georgi's dry root, which has multiple pharmacological activities. However, the precise mechanism of baicalein against myocardial remodeling remains poorly understood. The aim of our study was to investigate the underlying mechanism of baicalein treatment in rats model of heart failure (HF) and rat myocardial cells (H9C2). HF model was established by abdominal aorta constriction in rats and incubation with 50μM isoproterenol for 48h in H9C2 cells. Various molecular biological experiments were performed to assess the effects of baicalein on cardiac function, myocardial remodeling, apoptosis and Ca(2+) handling proteins. In the present study, first we found that baicalein alleviated HF in vivo. Additionally, treatment with baicalein inhibited the myocardial fibrosis, restrained the expression and activity of MMP2 and MMP9, and suppressed apoptosis in heart tissue. Moreover, baicalein could inhibit the cardiac myocyte hypertrophy and apoptosis induced by isoproterenol in vitro. Finally we found that baicalein could modulate the expressions and activities of Ca(2+) handling proteins, including downregulation of phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and expression of Na(+)/Ca(2+)-exchangers (NCX1), upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and ryanodine receptor 2 (RYR2). Baicalein also restrained the decreased SERCA activity induced by aortic banding. Our studies suggested that baicalein alleviated myocardial remodeling and improved cardiac function via modulation of Ca(2+) handling proteins, which may be a potential phytochemical flavonoid for therapeutics of HF. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia

PubMed Central

Pollak, Adam J.; Haghighi, Kobra; Kunduri, Swati; Arvanitis, Demetrios A.; Liu, Guan-Sheng; Singh, Vivek P.; Gonzalez, David J.; Sanoudou, Despina; Wiley, Sandra E.; Dixon, Jack E.; Kranias, Evangelia G.

2017-01-01

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias. PMID:28784772

Store-operated Ca2+ entry supports contractile function in hearts of hibernators

PubMed Central

Nakipova, Olga V.; Averin, Alexey S.; Evdokimovskii, Edward V.; Pimenov, Oleg Yu.; Kosarski, Leonid; Ignat’ev, Dmitriy; Anufriev, Andrey; Kokoz, Yuri M.; Reyes, Santiago; Terzic, Andre; Alekseev, Alexey E.

2017-01-01

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states. PMID:28531217

Transmural Heterogeneity and Remodeling of Ventricular Excitation-Contraction Coupling in Human Heart Failure

PubMed Central

Lou, Qing; Fedorov, Vadim V.; Glukhov, Alexey V.; Moazami, Nader; Fast, Vladimir G.; Efimov, Igor R.

2011-01-01

Background Excitation-contraction (EC) coupling is altered in the end-stage heart failure (HF). However, spatial heterogeneity of this remodeling has not been established at the tissue level in failing human heart. The objective is to study functional remodeling of EC coupling and calcium handling in failing and nonfailing human hearts. Methods and Results We simultaneously optically mapped action potentials (AP) and calcium transients (CaT) in coronary-perfused left ventricular wedge preparations from nonfailing (n = 6) and failing (n = 5) human hearts. Our major findings are: (1) CaT duration minus AP duration was longer at sub-endocardium in failing compared to nonfailing hearts during bradycardia (40 beats/min). (2) The transmural gradient of CaT duration was significantly smaller in failing hearts compared with nonfailing hearts at fast pacing rates (100 beats/min). (3) CaT in failing hearts had a flattened plateau at the midmyocardium; and exhibited a “two-component” slow rise at sub-endocardium in three failing hearts. (4) CaT relaxation was slower at sub-endocardium than that at sub-epicardium in both groups. Protein expression of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) was lower at sub-endocardium than that at sub-epicardium in both nonfailing and failing hearts. SERCA2a protein expression at sub-endocardium was lower in hearts with ischemic cardiomyopathy compared with nonischemic cardiomyopathy. Conclusions For the first time, we present direct experimental evidence of transmural heterogeneity of EC coupling and calcium handling in human hearts. End-stage HF is associated with the heterogeneous remodeling of EC coupling and calcium handling. PMID:21502574

Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons.

PubMed

Lautenschläger, Janin; Prell, Tino; Ruhmer, Julia; Weidemann, Lisa; Witte, Otto W; Grosskreutz, Julian

2013-09-01

Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS. Copyright © 2013 Elsevier Inc. All rights reserved.

Abnormalities of Calcium Handling Proteins in Skeletal Muscle Mirror those of the Heart in Humans with Heart Failure: a Shared Mechanism?

PubMed Central

Middlekauff, Holly R.; Vigna, Chris; Verity, M. Anthony; Fonarow, Gregg C.; Horwich, Tamara B.; Hamilton, Michele A.; Shieh, Perry; Tupling, A. Russell

2012-01-01

Background In the failing human heart, abnormalities of Ca2+ cycling have been described, but there is scant knowledge about Ca2+ handling in the skeletal muscle of humans with HF. We tested the hypothesis that in humans with HF, Ca2+ cycling proteins in skeletal muscle are abnormal. Methods and Results Ten advanced HF patients (50.4±3.7 years), and 9 age matched controls underwent vastus lateralis biopsy. Western blot analysis showed that sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, which is responsible for Ca2+ sequestration into the sarcoplasmic reticulum(SR), was lower in HF vs controls (4.8±0.5vs7.5±0.8AU, p=0.01). Although phospholamban (PLN), which inhibits SERCA2a, was not different in HF vs controls, phosphorylation (SER16 site) of PLN, which relieves this inhibition, was reduced (0.8±0.1vs3.9±0.9AU, p=0.004). Dihydropyridine receptors were reduced in HF, (2.1±0.4vs3.6±0.5AU, p=0.04). We tested the hypothesis that these abnormalities of Ca2+ handling protein content and regulation were due to increased oxidative stress, but oxygen radical scavenger proteins were not elevated in the skeletal muscle of HF patients. Conclusion In chronic HF, marked abnormalities of Ca2+ handling proteins are present in skeletal muscle, which mirror those in failing heart tissue. This suggests a common mechanism, such as chronic augmentation of sympathetic activity and autophosphorylation of Ca2+-calmodulin-dependent-protein kinase II. PMID:22939042

Myocardial Ca2+ handling and cell-to-cell coupling, key factors in prevention of sudden cardiac death.

PubMed

Tribulova, Narcis; Seki, Shingo; Radosinska, Jana; Kaplan, Peter; Babusikova, Eva; Knezl, Vladimir; Mochizuki, Seibu

2009-12-01

Using whole-heart preparations, we tested our hypothesis that Ca(2+) handling is closely related to cell-to-cell coupling at the gap junctions and that both are critical for the development and particularly the termination of ventricular fibrillation (VF) and hence the prevention of sudden arrhythmic death. Intracellular free calcium concentration ([Ca(2+)](i)), ECG, and left ventricular pressure were continuously monitored in isolated guinea pig hearts before and during development of low K(+)-induced sustained VF and during its conversion into sinus rhythm facilitated by stobadine. We also examined myocardial ultrastructure to detect cell-to-cell coupling alterations. We demonstrated that VF occurrence was preceded by a 55.9% +/- 6.2% increase in diastolic [Ca(2+)](i), which was associated with subcellular alterations indicating Ca(2+) overload of the cardiomyocytes and disorders in coupling among the cells. Moreover, VF itself further increased [Ca(2+)](i) by 58.2% +/- 3.4% and deteriorated subcellular and cell-to-cell coupling abnormalities that were heterogeneously distributed throughout the myocardium. In contrast, termination of VF and its conversion into sinus rhythm was marked by restoration of basal [Ca(2+)](i), resulting in recovery of intercellular coupling linked with synchronous contraction. Furthermore, we have shown that hearts exhibiting lower SERCA2a (sarcoplasmic reticulum Ca(2+)-ATPase) activity and abnormal intercellular coupling (as in older guinea pigs) are more prone to develop Ca(2+) overload associated with cell-to-cell uncoupling than hearts with higher SERCA2a activity (as in young guinea pigs). Consequently, young animals are better able to terminate VF spontaneously. These findings indicate the crucial role of Ca(2+) handling in relation to cell-to-cell coupling in both the occurrence and termination of malignant arrhythmia.

Disbalance of calcium regulation-related genes in broiler hearts induced by selenium deficiency.

PubMed

Zhang, Ziwei; Liu, Man; Guan, Zhenqiong; Yang, Jie; Liu, Zhonghua; Xu, Shiwen

2017-06-01

Dietary selenium (Se) deficiency may influence the calcium (Ca) homeostasis in broilers. Our objective was to investigate the effects of Se deficiency on Ca regulation-related genes in broiler hearts. In the present study, 1-day-old broilers were fed either a commercial diet (as control group) with 0.15 mg/kg Se or a Se-deficient diet (as L group) with 0.033 mg/kg Se for 35 days. We examined the mRNA expression levels of 15 Ca regulation-related genes (ITPR 1, ITPR 2, ITPR3, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, TRPC1, TRPC3, stromal interacting molecule 1, ORAI1, calmodulin (CaLM) and calreticulin (CRT) in broiler hearts. Then, Kyoto Encyclopedia of Genes and Genomes analysis, protein-protein interactions (PPI) analysis and correlation analysis were performed to analyse the relationships between these genes. The results showed that the mRNA expression levels of ITPR 1, ITPR 2, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, CaLM and CRT were generally decreased by Se deficiency, while mRNA expression levels of TRPC1, TRPC3, stromal interacting molecule 1, ORAI1 and ITPR3 were increased by Se deficiency. Kyoto Encyclopedia of Genes and Genomes and PPI analysis showed that these Ca regulation-related genes are involved in the Ca signalling pathway and a total of 15 PPIs with a combined score of >0.4 were obtained. In conclusion, the results demonstrated that Se deficiency might cause heart injury via modulating the Ca-related pathway genes, and then induce Ca 2+ overload in the heart of broilers.

The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana.

PubMed

Urbina, Daniela C; Silva, Herman; Meisel, Lee A

2006-01-01

Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 microM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin) suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism.

(Z)3,4,5,4‧-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level

NASA Astrophysics Data System (ADS)

Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kin-Ting Kam, Richard; Liu, Liang; Lai-Han Leung, Elaine

2015-11-01

Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4‧-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.

The calcium-frequency response in the rat ventricular myocyte: an experimental and modelling study.

PubMed

Gattoni, Sara; Røe, Åsmund Treu; Frisk, Michael; Louch, William E; Niederer, Steven A; Smith, Nicolas P

2016-08-01

In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force-frequency response (FFR). The majority of mammals have a positive force-frequency relationship (FFR). In rat the FFR is controversial. We derive a species- and temperature-specific data-driven model of the rat ventricular myocyte. As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium-frequency response (CFR) in our model and three altered models. The results show a biphasic peak calcium-frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency-dependent increase in diastolic calcium. Alterations to the model reveal that inclusion of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated L-type channel and transient outward K(+) current activity enhances the positive magnitude calcium-frequency response, and the absence of CAMKII-mediated increase in activity of the sarco/endoplasmic reticulum Ca(2+) -ATPase induces a negative magnitude calcium-frequency response. An increase in heart rate affects the strength of cardiac contraction by altering the Ca(2+) transient as a response to physiological demands. This is described by the force-frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat-based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca(2+) transient characteristics and showed a biphasic peak calcium-frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated increase in sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L-type channel and transient outward K(+) current activity and (3) Na(+) /K(+) pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca(2+) -frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca(2+) . © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

PubMed Central

Cabral, Wayne A.; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N.; Sargent, Brandi M.; Weis, MaryAnn; Barnes, Aileen M.; Webb, Emma A.; Shaw, Nicholas J.; Ala-Kokko, Leena; Lacbawan, Felicitas L.; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S.; Zimmerberg, Joshua; Eyre, David R.; Yamada, Yoshihiko; Marini, Joan C.

2016-01-01

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. PMID:27441836

The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

PubMed

Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

2012-11-01

The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

Overexpression Myocardial Inducible Nitric Oxide Synthase Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in Experimental Hypothyroidism☆,☆☆

PubMed Central

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F.; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2015-01-01

Background Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cadiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca2+]i transient ([Ca2+]iT), and β-adrenergic hyporesponsiveness. Methods and Results We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca2+]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400W, 10−5 mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. In hypothyroidism, isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca2+]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca2+]iT. Conclusions Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cadiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. PMID:26681542

Overexpression myocardial inducible nitric oxide synthase exacerbates cardiac dysfunction and beta-adrenergic desensitization in experimental hypothyroidism.

PubMed

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2016-02-01

Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness. We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

PubMed Central

Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

PubMed

Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The Endoplasmic Reticulum: A Central Player in Cell Signalling and Protein Synthesis

NASA Astrophysics Data System (ADS)

Llewelyn Roderick, H.; Berridge, Michael J.; Bootman, Martin D.

In addition to being the principle intracellular Ca 2+ store, the endoplasmic reticulum (ER) is the initial site of synthesis and folding of membrane and secretory proteins. These two roles of the ER are intimately linked. First, the function of many proteins involved in Ca 2+ handling are modulated by Ca 2+ and second, ER lumenal Ca 2+ modulates protein synthesis and folding. Within the ER, Ca 2+ is stored by low affinity high capacity Ca 2+ binding proteins and is maintained at a free concentration between 0.1 and 1 μM relative to 0.1 μM cytosolic Ca 2+. This concentration gradient is maintained by the action of the Sarco-Endoplasmic Reticulum ATPases (SERCa) which hydrolyse ATP to pump Ca 2+ into the ER. Following stimulation Ca 2+ is released from the ER through several classes of ligand gated channels. The most well characterized of these being the inositol 1,4,5-trisphosphate receptor ( IP 3 R) and the Ryanodine receptor (RyR) families of proteins. This release of Ca 2+ results in a drop of ER free Ca 2+ to levels as low as 10 μM. This decrease in lumenal Ca 2+ inhibits further release through the channels and increases the rate of re-sequestration of Ca 2+ into the ER by the SERCa pumps. Under these conditions, in addition to effects on Ca 2+ handling proteins, protein synthesis is inhibited, chaperones dissociate from their substrates, secondary modifications of proteins are inhibited and the retention of many proteins within the ER is lost. Furthermore, a signalling cascade resulting in the up-regulation of many proteins involved in protein folding and Ca 2+ homeostasis is initiated. This review will focus on the proteins involved in the regulation ER lumenal Ca 2+ and the role of ER lumenal Ca 2+ in cell signalling and protein synthesis.

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

PubMed

Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

2013-10-01

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The benefits of endurance training in cardiomyocyte function in hypertensive rats are reversed within four weeks of detraining.

PubMed

Carneiro-Júnior, Miguel Araujo; Quintão-Júnior, Judson Fonseca; Drummond, Lucas Rios; Lavorato, Victor Neiva; Drummond, Filipe Rios; da Cunha, Daise Nunes Queiroz; Amadeu, Marco Aurélio; Felix, Leonardo Bonato; de Oliveira, Edilamar Menezes; Cruz, Jader Santos; Prímola-Gomes, Thales Nicolau; Mill, José Geraldo; Natali, Antonio José

2013-04-01

The aim of the present study was to verify the effects of low-intensity endurance training and detraining on the mechanical and molecular properties of cardiomyocytes from spontaneously hypertensive rats (SHRs). Male SHRs and normotensive control Wistar rats at 16-weeks of age were randomly divided into eight groups of eight animals: NC8 and HC8 (normotensive and hypertensive control for 8weeks); NT8 and HT8 (normotensive and hypertensive trained at 50-60% of maximal exercise capacity for 8weeks); NC12 and HC12 (normotensive and hypertensive control for 12weeks); NDT and HDT (normotensive and hypertensive trained for 8weeks and detrained for 4weeks). The total exercise time until fatigue (TTF) was determined by a maximal exercise capacity test. Resting heart rate (RHR) and systolic arterial pressure (SAP) were measured. After the treatments, animals were killed by cervical dislocation and left ventricular myocytes were isolated by enzymatic dispersion. Isolated cells were used to determine intracellular global Ca(2+) ([Ca(2+)]i) transient and cardiomyocyte contractility (1Hz; ~25°C). [Ca(2+)]i regulatory proteins were measured by Western blot, and the markers of pathologic cardiac hypertrophy by quantitative real-time polymerase chain reaction (q-RT-PCR). Exercise training augmented the TTF (NC8, 11.4±1.5min vs. NT8, 22.5±1.4min; HC8, 11.7±1.4min vs. HT8, 24.5±1.3min; P<0.05), reduced RHR (NT8initial, 340±8bpm vs. NT8final, 322±10bpm; HT8initial, 369±8bpm vs. HT8final, 344±10bpm; P<0.05), and SBP in SHR animals (HC8, 178±3mmHg vs. HT8, 161±4mmHg; P<0.05). HC8 rats showed a slower [Ca(2+)]i transient (Tpeak, 83.7±1.8ms vs. 71.7±2.4ms; T50%decay, 284.0±4.3ms vs. 264.0±4.1ms; P<0.05) and cell contractility (Vshortening, 86.1±6.7μm/s vs. 118.6±6.7μm/s; Vrelengthening, 57.5±7.4μm/s vs. 101.3±7.4μm/s; P<0.05), and higher expression of ANF (300%; P<0.05), skeletal α-actin (250%; P<0.05) and a decreased α/β-MHC ratio (70%; P<0.05) compared to NC8. Exercise training increased [Ca(2+)]i transient (NC8, 2.39±0.06F/F0 vs. NT8, 2.72±0.06F/F0; HC8, 2.28±0.05F/F0 vs. HT8, 2.82±0.05F/F0; P<0.05), and cell contractility (NC8, 7.4±0.3% vs. NT8, 8.4±0.3%; HC8, 6.8±0.3% vs. HT8, 7.8±0.3%; P<0.05). Furthermore, exercise normalized the expression of ANF, skeletal α-actin, and the α/β-MHC ratio in HT8 rats, augmented the expression of SERCA2a (NC8, 0.93±0.15 vs. NT8, 1.49±0.14; HC8, 0.83±0.13 vs. HT8, 1.32±0.14; P<0.05) and PLBser16 (NC8, 0.89±0.18 vs. NT8, 1.23±0.17; HC8, 0.77±0.17 vs. HT8, 1.32±0.16; P<0.05), and reduced PLBt/SERCA2a (NC8, 1.21±0.19 vs. NT8, 0.50±0.21; HC8, 1.38±0.17 vs. HT8, 0.66±0.21; P<0.05). However, all these adaptations returned to control values within 4weeks of detraining in both SHR and normotensive control animals. In conclusion, low-intensity endurance training induces positive benefits to left ventricular myocyte mechanical and molecular properties, which are reversed within 4weeks of detraining. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cardiac and peripheral adjustments induced by early exercise training intervention were associated with autonomic improvement in infarcted rats: role in functional capacity and mortality.

PubMed

Jorge, Luciana; Rodrigues, Bruno; Rosa, Kaleizu Teodoro; Malfitano, Christiane; Loureiro, Tatiana Carolina Alba; Medeiros, Alessandra; Curi, Rui; Brum, Patricia Chakur; Lacchini, Silvia; Montano, Nicola; De Angelis, Kátia; Irigoyen, Maria-Cláudia

2011-04-01

To test the effects of early exercise training (ET) on left ventricular (LV) and autonomic functions, haemodynamics, tissues blood flows (BFs), maximal oxygen consumption (VO(2) max), and mortality after myocardial infarction (MI) in rats. Male Wistar rats were divided into: control (C), sedentary-infarcted (SI), and trained-infarcted (TI). One week after MI, TI group underwent an ET protocol (90 days, 50-70% VO(2) max). Left ventricular function was evaluated non-invasively and invasively. Baroreflex sensitivity, heart rate variability, and pulse interval were measured. Cardiac output (CO) and regional BFs were determined using coloured microspheres. Infarcted area was reduced in TI (19 ± 6%) compared with SI (34 ± 5%) after ET. Exercise training improved the LV and autonomic functions, the CO and regional BF changes induced by MI, as well as increased SERCA2 expression and mRNA vascular endothelial growth factor levels. These changes brought about by ET resulted in mortality rate reduction in the TI (13%) group compared with the SI (54%) group. Early aerobic ET reduced cardiac and peripheral dysfunctions and preserved cardiovascular autonomic control after MI in trained rats. Consequently, these ET-induced changes resulted in improved functional capacity and survival after MI.

Calcium Signaling enhancement during oocyte maturation

NASA Astrophysics Data System (ADS)

Jung, Peter; Ullah, Ghanim; Machaca, Khaled

2006-03-01

A Ca2+ signal with a special spatial and temporal characteristic universally removes cell-cycle arrest after fertilization of a mature egg cell. The Ca2+ signal is characterized by a fast rise of intracellular Ca2+ and a slow decay on the time scale of minutes. We use computational modeling of Ca2+ release on the microscale (Ca2+ puffs) and cell-scale in conjunction with experimental knowledge of the changes in the Ca2+ signaling apparatus during oocyte maturation and changing signaling patterns to explore the relationship between organization and sensitivity of IP3 receptors and SERCA pumps and the resulting signaling patterns. We hypothesize that potentiation of the IP3 receptors during oocyte maturation is the main cause for the differentiation in the signaling patterns.

Finite-element estimation of protein-ligand association rates with post-encounter effects: applications to calcium binding in troponin C and SERCA

NASA Astrophysics Data System (ADS)

Kekenes-Huskey, P. M.; Gillette, A.; Hake, J.; McCammon, J. A.

2012-01-01

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca2+-binding proteins: troponin C and the sarcoplasmic reticulum Ca2+ ATPase. We find that a combination of diffusional encounter and internal ‘buried channel’ descriptions provides superior descriptions of association rates, improving estimates by orders of magnitude.

Early-onset growth hormone deficiency results in diastolic dysfunction in adult-life and is prevented by growth hormone supplementation.

PubMed

Groban, L; Lin, M; Kassik, K A; Ingram, R L; Sonntag, W E

2011-04-01

The primary goal of growth hormone (GH) replacement is to promote linear growth in children with growth hormone deficiency (GHD). GH and insulin-like growth factor-1 (IGF-1) are also known to have roles in cardiac development and as modulators of myocardial structure and function in the adult heart. However, little is known about cardiac diastolic function in young adults with childhood onset GH deficiency in which GH treatment was discontinued following puberty. The aim of the study was to evaluate the effects of long standing GHD and peri-pubertal or continuous GH replacement therapy on diastolic function in the adult dwarf rat. The dwarf rat, which possesses a mutation in a transcription factor necessary for development of the somatotroph, does not exhibit the normal peri-pubertal rise in GH around day 28 and was used to model childhood or early-onset GHD (EOGHD). In another group of male dwarfs, GH replacement therapy was initiated at 4 weeks of age when GH pulsatility normally begins. Ten weeks after initiation of injections, GH-treated dwarf rats were divided into 2 groups; continued treatment with GH for 12 weeks (GH-replete) or treatment with saline for 12 weeks. This latter group models GH supplementation during adolescence with GHD beginning in adulthood (adult-onset GHD; AOGHD). Saline-treated heterozygous (HZ) rats were used as age-matched controls. At 26 weeks of age, cardiac function was assessed using invasive or noninvasive (conventional and tissue Doppler) indices of myocardial contractility and lusitropy. Systolic function, as determined by echocardiography, was similar among groups. Compared with HZ rats and GH-replete dwarfs, the EOGHD group exhibited significant reductions in myocardial relaxation and increases in left ventricular filling pressure, indicative of moderate diastolic dysfunction. This was further associated with a decrease in the cardiac content of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), one of the important cardiac calcium regulatory proteins. Dwarfs supplemented with GH during the peri-adolescence stage, but not beyond (AOGHD), exhibited a subtle prolongation in the deceleration time to early filling. In contrast, continual GH replacement preserved diastolic function such that the cardiac phenotype of the GH-replete dwarfs resembled that of their age-matched HZ counterpart. Our data indicate that GHD during adolescence leads to overt diastolic dysfunction in early adulthood and this is prevented by continual GH replacement therapy. Since discontinuation of GH replacement following adolescence only mitigated the lusitropic deficits that were observed in untreated dwarfs, GH treatment into adulthood could be beneficial. Copyright © 2011 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

PubMed Central

Laporta, Jimena; Keil, Kimberly P.; Vezina, Chad M.; Hernandez, Laura L.

2014-01-01

Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation. PMID:25299122

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

PubMed

Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes

PubMed Central

Bernal-Ramírez, Judith; Lozano, Omar; Oropeza-Almazán, Yuriana; Castillo, Elena Cristina; Garza, Jesús Roberto; García, Noemí; Vela, Jorge; Ortega, Eduardo; Torre-Amione, Guillermo; Ornelas-Soto, Nancy

2017-01-01

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO2) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO2-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO2 cell membrane association and internalization were analyzed. SiO2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca2+ handling, which showed impaired contractility and intracellular Ca2+ transient amplitude during β-adrenergic stimulation in SiO2 treatment. The time to 50% Ca2+ decay increased 39%, and the Ca2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Moreover, SiO2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H2O2 generation were also observed. These data indicate that SiO2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca2+ release, and reduced cell shortening. This mechanism of SiO2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death. NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca2+ handling. PMID:28130337

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

PubMed Central

Linde, Cristina I.; Karashima, Eiji; Raina, Hema; Zulian, Alessandra; Wier, Withrow G.; Hamlyn, John M.; Ferrari, Patrizia; Blaustein, Mordecai P.

2012-01-01

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1–100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. PMID:22140038

Effect of speed endurance training and reduced training volume on running economy and single muscle fiber adaptations in trained runners.

PubMed

Skovgaard, Casper; Christiansen, Danny; Christensen, Peter M; Almquist, Nicki W; Thomassen, Martin; Bangsbo, Jens

2018-02-01

The aim of the present study was to examine whether improved running economy with a period of speed endurance training and reduced training volume could be related to adaptations in specific muscle fibers. Twenty trained male (n = 14) and female (n = 6) runners (maximum oxygen consumption (VO 2 -max): 56.4 ± 4.6 mL/min/kg) completed a 40-day intervention with 10 sessions of speed endurance training (5-10 × 30-sec maximal running) and a reduced (36%) volume of training. Before and after the intervention, a muscle biopsy was obtained at rest, and an incremental running test to exhaustion was performed. In addition, running at 60% vVO 2 -max, and a 10-km run was performed in a normal and a muscle slow twitch (ST) glycogen-depleted condition. After compared to before the intervention, expression of mitochondrial uncoupling protein 3 (UCP3) was lower (P < 0.05) and dystrophin was higher (P < 0.05) in ST muscle fibers, and sarcoplasmic reticulum calcium ATPase 1 (SERCA1) was lower (P < 0.05) in fast twitch muscle fibers. Running economy at 60% vVO 2 -max (11.6 ± 0.2 km/h) and at v10-km (13.7 ± 0.3 km/h) was ~2% better (P < 0.05) after the intervention in the normal condition, but unchanged in the ST glycogen-depleted condition. Ten kilometer performance was improved (P < 0.01) by 3.2% (43.7 ± 1.0 vs. 45.2 ± 1.2 min) and 3.9% (45.8 ± 1.2 vs. 47.7 ± 1.3 min) in the normal and the ST glycogen-depleted condition, respectively. VO 2 -max was the same, but vVO 2 -max was 2.0% higher (P < 0.05; 19.3 ± 0.3 vs. 18.9 ± 0.3 km/h) after than before the intervention. Thus, improved running economy with intense training may be related to changes in expression of proteins linked to energy consuming processes in primarily ST muscle fibers. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Surfactant bilayers maintain transmembrane protein activity.

PubMed

Rayan, Gamal; Adrien, Vladimir; Reffay, Myriam; Picard, Martin; Ducruix, Arnaud; Schmutz, Marc; Urbach, Wladimir; Taulier, Nicolas

2014-09-02

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca(2+)ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Re-evaluation of how artemisinins work in light of emerging evidence of in vitro resistance

PubMed Central

Krishna, Sanjeev; Woodrow, Charles J.; Staines, Henry M.; Haynes, Richard K.; Mercereau-Puijalon, Odile

2006-01-01

There are more than half a billion cases of malaria every year. Combinations of an artemisinin with other antimalarial drugs are now recommended treatments for Plasmodium falciparum malaria in most endemic areas. These treatment regimens act rapidly to relieve symptoms and effect cure. There is considerable controversy on how artemisinins work and over emerging indications of resistance to this class of antimalarial drugs. Several individual molecules have been proposed as targets for artemisinins, in addition to the idea that artemisinins might have many targets at the same time. Our suggestion that artemisinins inhibit the parasite-encoded sarco–endoplasmic reticulum Ca2+-ATPase (SERCA) PfATP6 has gained support from recent observations that a polymorphism in the gene encoding PfATP6 is associated with in vitro resistance to artemether in field isolates of P. falciparum. PMID:16616639

Effects of Electrical Stimulation on Skeletal Muscle of Old Sedentary People

PubMed Central

Mosole, Simone; Zampieri, Sandra; Furlan, Sandra; Carraro, Ugo; Löefler, Stefan; Kern, Helmut; Volpe, Pompeo

2018-01-01

Physical activity plays an important role in preventing muscle atrophy and chronic diseases in adults and in the elderly. Calcium (Ca2+) cycling and activation of specific molecular pathways are essential in contraction-induced muscle adaptation. This study attains human muscle sections and total homogenates prepared from biopsies obtained before (control) and after 9 weeks of training by electrical stimulation (ES) on a group of volunteers. The aim of the study was to investigate about the molecular mechanisms that support functional muscle improvement by ES. Evidences of kinase/phosphatase pathways activation after ES were obtained. Moreover, expression of Sarcalumenin, Calsequestrin and sarco/endoplasmic reticulum Ca2+-ATPase (Serca) isoforms was regulated by training. In conclusion, this work shows that neuromuscular ES applied to vastus lateralis muscle of sedentary seniors combines fiber remodeling with activation of Ca2+-Calmodulin molecular pathways and modulation of key Ca2+-handling proteins. PMID:29662923

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis.

PubMed

Relling, David P; Esberg, Lucy B; Fang, Cindy X; Johnson, W Thomas; Murphy, Eric J; Carlson, Edward C; Saari, Jack T; Ren, Jun

2006-03-01

Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

Modeling CICR in rat ventricular myocytes: voltage clamp studies

PubMed Central

2010-01-01

Background The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo). Methods The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo. Results Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics mediated by the luminal SR Ca2+ sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca2+ homeostasis. Conclusions We examine the role of the above Ca2+ regulating mechanisms in handling various types of induced disturbances in Ca2+ levels by quantifying cellular Ca2+ balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses. PMID:21062495

Spontaneous Ca(2+) transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine.

PubMed

Baker, Salah A; Drumm, Bernard T; Saur, Dieter; Hennig, Grant W; Ward, Sean M; Sanders, Kenton M

2016-06-15

Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Spontaneous Ca2+ transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine

PubMed Central

Baker, Salah A.; Drumm, Bernard T.; Saur, Dieter; Hennig, Grant W.; Ward, Sean M.

2016-01-01

Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC‐DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves.Ca2+ transients in ICC‐DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread.Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells.Ca2+ transients in ICC‐DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s).Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump is necessary for maintenance of spontaneity.Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent.ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Abstract Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC‐DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC‐DMP are mediated by activation of Ca2+‐activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC‐DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC‐DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca2+ transients, suggesting that ICC‐DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high‐resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC‐DMP in situ. PMID:26824875

Istaroxime, a positive inotropic agent devoid of proarrhythmic properties in sensitive chronic atrioventricular block dogs.

PubMed

Bossu, Alexandre; Kostense, Amée; Beekman, Henriette D M; Houtman, Marien J C; van der Heyden, Marcel A G; Vos, Marc A

2018-05-16

Current inotropic agents in heart failure therapy associate with low benefit and significant adverse effects, including ventricular arrhythmias. Istaroxime, a novel Na + /K + -transporting ATPase inhibitor, also stimulates SERCA2a activity, which would confer improved inotropic and lusitropic properties with less proarrhythmic effects. We investigated hemodynamic, electrophysiological and potential proarrhythmic and antiarrhythmic effects of istaroxime in control and chronic atrioventricular block (CAVB) dogs sensitive to drug-induced Torsades de Pointes arrhythmias (TdP). In isolated normal canine ventricular cardiomyocytes, istaroxime (0.3-10 μM) evoked no afterdepolarizations and significantly shortened action potential duration (APD) at 3 and 10 μM. Istaroxime at 3 μg/kg/min significantly increased left ventricular (LV) contractility (dP/dt+) and relaxation (dP/dt-) respectively by 81 and 94% in anesthetized control dogs (n = 6) and by 61 and 49% in anesthetized CAVB dogs (n = 7) sensitive to dofetilide-induced TdP. While istaroxime induced no ventricular arrhythmias in control conditions, only single ectopic beats occurred in 2/7 CAVB dogs, which were preceded by increase of short-term variability of repolarization (STV) and T wave alternans in LV unipolar electrograms. Istaroxime pre-treatment (3 μg/kg/min for 60 min) did not alleviate dofetilide-induced increase in repolarization and STV, and mildly reduced incidence of TdP from 6/6 to 4/6 CAVB dogs. In six CAVB dogs with dofetilide-induced TdP, administration of istaroxime (90 μg/kg/5 min) suppressed arrhythmic episodes in two animals. Taken together, inotropic and lusitropic properties of istaroxime in CAVB dogs were devoid of significant proarrhythmic effects in sensitive CAVB dogs, and istaroxime provides a moderate antiarrhythmic efficacy in prevention and suppression of dofetilide-induced TdP. Copyright © 2018. Published by Elsevier Ltd.

Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes

PubMed Central

Werneck-de-Castro, Joao P.; Fonseca, Tatiana L.; Ignacio, Daniele L.; Fernandes, Gustavo W.; Andrade-Feraud, Cristina M.; Lartey, Lattoya J.; Ribeiro, Marcelo B.; Ribeiro, Miriam O.; Gereben, Balazs

2015-01-01

The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers. PMID:26214036

Increased passive stiffness promotes diastolic dysfunction despite improved Ca2+ handling during left ventricular concentric hypertrophy

PubMed Central

Røe, Åsmund T.; Aronsen, Jan Magnus; Skårdal, Kristine; Hamdani, Nazha; Linke, Wolfgang A.; Danielsen, Håvard E.; Sejersted, Ole M.; Sjaastad, Ivar; Louch, William E.

2017-01-01

Abstract Aims Concentric hypertrophy following pressure-overload is linked to preserved systolic function but impaired diastolic function, and is an important substrate for heart failure with preserved ejection fraction. While increased passive stiffness of the myocardium is a suggested mechanism underlying diastolic dysfunction in these hearts, the contribution of active diastolic Ca2+ cycling in cardiomyocytes remains unclear. In this study, we sought to dissect contributions of passive and active mechanisms to diastolic dysfunction in the concentrically hypertrophied heart following pressure-overload. Methods and results Rats were subjected to aortic banding (AB), and experiments were performed 6 weeks after surgery using sham-operated rats as controls. In vivo ejection fraction and fractional shortening were normal, confirming preservation of systolic function. Left ventricular concentric hypertrophy and diastolic dysfunction following AB were indicated by thickening of the ventricular wall, reduced peak early diastolic tissue velocity, and higher E/e’ values. Slowed relaxation was also observed in left ventricular muscle strips isolated from AB hearts, during both isometric and isotonic stimulation, and accompanied by increases in passive tension, viscosity, and extracellular collagen. An altered titin phosphorylation profile was observed with hypophosphorylation of the phosphosites S4080 and S3991 sites within the N2Bus, and S12884 within the PEVK region. Increased titin-based stiffness was confirmed by salt-extraction experiments. In contrast, isolated, unloaded cardiomyocytes exhibited accelerated relaxation in AB compared to sham, and less contracture at high pacing frequencies. Parallel enhancement of diastolic Ca2+ handling was observed, with augmented NCX and SERCA2 activity and lowered resting cytosolic [Ca2+]. Conclusion In the hypertrophied heart with preserved systolic function, in vivo diastolic dysfunction develops as cardiac fibrosis and alterations in titin phosphorylation compromise left ventricular compliance, and despite compensatory changes in cardiomyocyte Ca2+ homeostasis. PMID:28472418

Torque limit of PM motors for field-weakening region operation

DOEpatents

Royak, Semyon [Beachwood, OH; Harbaugh, Mark M [Richfield, OH

2012-02-14

The invention includes a motor controller and technique for controlling a permanent magnet motor. In accordance with one aspect of the present technique, a permanent magnet motor is controlled by receiving a torque command, determining a physical torque limit based on a stator frequency, determining a theoretical torque limit based on a maximum available voltage and motor inductance ratio, and limiting the torque command to the smaller of the physical torque limit and the theoretical torque limit. Receiving the torque command may include normalizing the torque command to obtain a normalized torque command, determining the physical torque limit may include determining a normalized physical torque limit, determining a theoretical torque limit may include determining a normalized theoretical torque limit, and limiting the torque command may include limiting the normalized torque command to the smaller of the normalized physical torque limit and the normalized theoretical torque limit.

Universal adaptive torque control for PM motors for field-weakening region operation

DOEpatents

Royak, Semyon [Beachwood, OH; Harbaugh, Mark M [Richfield, OH; Breitzmann, Robert J [South Russel, OH; Nondahl, Thomas A [Wauwatosa, WI; Schmidt, Peter B [Franklin, WI; Liu, Jingbo [Milwaukee, WI

2011-03-29

The invention includes a motor controller and method for controlling a permanent magnet motor. In accordance with one aspect of the present technique, a permanent magnet motor is controlled by, among other things, receiving a torque command, determining a normalized torque command by normalizing the torque command to a characteristic current of the motor, determining a normalized maximum available voltage, determining an inductance ratio of the motor, and determining a direct-axis current based upon the normalized torque command, the normalized maximum available voltage, and the inductance ratio of the motor.

Skeletal Muscle Thermogenesis and Its Role in Whole Body Energy Metabolism

PubMed Central

Herrera, Jose Luis; Reis, Felipe C. G.

2017-01-01

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis. PMID:29086530

A robust method to screen detergents for membrane protein stabilization, revisited.

PubMed

Champeil, Philippe; Orlowski, Stéphane; Babin, Simon; Lund, Sten; le Maire, Marc; Møller, Jesper; Lenoir, Guillaume; Montigny, Cédric

2016-10-15

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents. Copyright © 2016 Elsevier Inc. All rights reserved.

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

NASA Astrophysics Data System (ADS)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

Current perspectives on the mechanism of action of artemisinins.

PubMed

Golenser, Jacob; Waknine, Judith H; Krugliak, Miriam; Hunt, Nicholas H; Grau, Georges E

2006-12-01

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.

Advances in gene therapy for heart failure.

PubMed

Fish, Kenneth M; Ishikawa, Kiyotake

2015-04-01

Chronic heart failure is expected to increase its social and economic burden as a consequence of improved survival in patients with acute cardiac events. Cardiac gene therapy holds significant promise in heart failure treatment for patients with currently very limited or no treatment options. The introduction of adeno-associated virus (AAV) gene vector changed the paradigm of cardiac gene therapy, and now it is the primary vector of choice for chronic heart failure gene therapy in clinical and preclinical studies. Recently, there has been significant progress towards clinical translation in this field spearheaded by AAV-1 mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene therapy targeting chronic advanced heart failure patients. Meanwhile, several independent laboratories are reporting successful gene therapy approaches in clinically relevant large animal models of heart failure and some of these approaches are expected to enter clinical trials in the near future. This review will focus on gene therapy approaches targeting heart failure that is in clinical trials and those close to its initial clinical trial application.

Quantification and characterization of grouped type I myofibers in human aging.

PubMed

Kelly, Neil A; Hammond, Kelley G; Stec, Michael J; Bickel, C Scott; Windham, Samuel T; Tuggle, S Craig; Bamman, Marcas M

2018-01-01

Myofiber type grouping is a histological hallmark of age-related motor unit remodeling. Despite the accepted concept that denervation-reinnervation events lead to myofiber type grouping, the completeness of those conversions remains unknown. Type I myofiber grouping was assessed in vastus lateralis biopsies from Young (26 ± 4 years; n = 27) and Older (66 ± 4 years; n = 91) adults. Grouped and ungrouped type I myofibers were evaluated for phenotypic differences. Higher type I grouping in Older versus Young was driven by more myofibers per group (i.e., larger group size) (P < 0.05). In Older only, grouped type I myofibers displayed larger cross-sectional area, more myonuclei, lower capillary supply, and more sarco(endo)plasmic reticulum calcium ATPase I (SERCA I) expression (P < 0.05) than ungrouped type I myofibers. Grouped type I myofibers retain type II characteristics suggesting that conversion during denervation-reinnervation events is either progressive or incomplete. Muscle Nerve 57: E52-E59, 2018. © 2017 Wiley Periodicals, Inc.

Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production

PubMed Central

Poksay, Karen S.; Sheffler, Douglas J.; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E.; Cosford, Nicholas D. P.; John, Varghese

2017-01-01

Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds – identified here using cells and tissues expressing wt human APP – in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects. PMID:28261092

Protein Changes Contributing to Right Ventricular Cardiomyocyte Diastolic Dysfunction in Pulmonary Arterial Hypertension

PubMed Central

Rain, Silvia; Bos, Denielli da Silva Goncalves; Handoko, M. Louis; Westerhof, Nico; Stienen, Ger; Ottenheijm, Coen; Goebel, Max; Dorfmüller, Peter; Guignabert, Christophe; Humbert, Marc; Bogaard, Harm‐Jan; dos Remedios, Cris; Saripalli, Chandra; Hidalgo, Carlos G.; Granzier, Henk L.; Vonk‐Noordegraaf, Anton; van der Velden, Jolanda; de Man, Frances S.

2014-01-01

Background Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca2+/calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamKIIδ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA‐mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca2+‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH. PMID:24895160

In vivo Ca2+ buffering capacity and microvascular oxygen pressures following muscle contractions in diabetic rat skeletal muscles: fiber-type specific effects.

PubMed

Eshima, Hiroaki; Poole, David C; Kano, Yutaka

2015-07-15

In Type 1 diabetes, skeletal muscle resting intracellular Ca(2+) concentration ([Ca(2+)]i) homeostasis is impaired following muscle contractions. It is unclear to what degree this behavior is contingent upon fiber type and muscle oxygenation conditions. We tested the hypotheses that: 1) the rise in resting [Ca(2+)]i evident in diabetic rat slow-twitch (type I) muscle would be exacerbated in fast-twitch (type II) muscle following contraction; and 2) these elevated [Ca(2+)]i levels would relate to derangement of microvascular partial pressure of oxygen (PmvO2 ) rather than sarcoplasmic reticulum dysfunction per se. Adult male Wistar rats were divided randomly into diabetic (DIA: streptozotocin ip) and healthy (CONT) groups. Four weeks later extensor digitorum longus (EDL, predominately type II fibers) and soleus (SOL, predominately type I fibers) muscle contractions were elicited by continuous electrical stimulation (120 s, 100 Hz). Ca(2+) imaging was achieved using fura 2-AM in vivo (i.e., circulation intact). DIA increased fatigability in EDL (P < 0.05) but not SOL. In recovery, SOL [Ca(2+)]i either returned to its resting baseline within 150 s (CONT 1.00 ± 0.02 at 600 s) or was not elevated in recovery at all (DIA 1.03 ± 0.02 at 600 s, P > 0.05). In recovery, EDL CONT [Ca(2+)]i also decreased to values not different from baseline (1.06 ± 0.01, P > 0.05) at 600 s. In marked contrast, EDL DIA [Ca(2+)]i remained elevated for the entire recovery period (i.e., 1.23 ± 0.03 at 600 s, P < 0.05). The inability of [Ca(2+)]i to return to baseline in EDL DIA was not associated with any reduction of SR Ca(2+)-ATPase (SERCA) 1 or SERCA2 protein levels (both increased 30-40%, P < 0.05). However, Pmv(O2) recovery kinetics were markedly slowed in EDL such that mean Pmv(O2) was substantially depressed (CONT 27.9 ± 2.0 vs. DIA 18.4 ± 2.0 Torr, P < 0.05), and this behavior was associated with the elevated [Ca(2+)]i. In contrast, this was not the case for SOL (P > 0.05) in that neither [Ca(2+)]i nor Pmv(O2) were deranged in recovery with DIA. In conclusion, recovery of [Ca(2+)]i homeostasis is impaired in diabetic rat fast-twitch but not slow-twitch muscle in concert with reduced Pmv(O2) pressures. Copyright © 2015 the American Physiological Society.

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Determination of target normal cost and funding target. 1.430(d)-1 Section 1.430(d)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Determination of target normal cost and funding target. (a) In general—(1) Overview. This section sets forth...

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Determination of target normal cost and funding target. 1.430(d)-1 Section 1.430(d)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Determination of target normal cost and funding target. (a) In general—(1) Overview. This section sets forth...

Structure and mechanism of Zn2+-transporting P-type ATPases

PubMed Central

Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele; Autzen, Henriette Elisabeth; Andersson, Magnus; Klymchuk, Tetyana; Nielsen, Anna Marie; Rees, Douglas C.; Nissen, Poul; Gourdon, Pontus

2014-01-01

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis1. In prokaryotes and photosynthetic eukaryotes Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements2,3. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.Pi) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu+-ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.Pi state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn2+ release as a built-in counter-ion, as also proposed for H+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-transport. These findings suggest a mechanistic link between PIB-type Zn2+-ATPases and PIII-type H+-ATPases, and show at the same time structural features of the extracellular release pathway that resemble the PII-type ATPases such as the sarco(endo)plasmic reticulum Ca2+-ATPase4,5 (SERCA) and Na+,K+-ATPase6. PMID:25132545

Long-term obesity promotes alterations in diastolic function induced by reduction of phospholamban phosphorylation at serine-16 without affecting calcium handling.

PubMed

Lima-Leopoldo, Ana Paula; Leopoldo, André S; da Silva, Danielle C T; do Nascimento, André F; de Campos, Dijon H S; Luvizotto, Renata A M; de Deus, Adriana F; Freire, Paula P; Medeiros, Alessandra; Okoshi, Katashi; Cicogna, Antonio C

2014-09-15

Few studies have evaluated the relationship between the duration of obesity, cardiac function, and the proteins involved in myocardial calcium (Ca(2+)) handling. We hypothesized that long-term obesity promotes cardiac dysfunction due to a reduction of expression and/or phosphorylation of myocardial Ca(2+)-handling proteins. Thirty-day-old male Wistar rats were distributed into two groups (n = 10 each): control (C; standard diet) and obese (Ob; high-fat diet) for 30 wk. Morphological and histological analyses were assessed. Left ventricular cardiac function was assessed in vivo by echocardiographic evaluation and in vitro by papillary muscle. Cardiac protein expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), calsequestrin, L-type Ca(2+) channel, and phospholamban (PLB), as well as PLB serine-16 phosphorylation (pPLB Ser(16)) and PLB threonine-17 phosphorylation (pPLB Thr(17)) were determined by Western blot. The adiposity index was higher (82%) in Ob rats than in C rats. Obesity promoted cardiac hypertrophy without alterations in interstitial collagen levels. Ob rats had increased endocardial and midwall fractional shortening, posterior wall shortening velocity, and A-wave compared with C rats. Cardiac index, early-to-late diastolic mitral inflow ratio, and isovolumetric relaxation time were lower in Ob than in C. The Ob muscles developed similar baseline data and myocardial responsiveness to increased extracellular Ca(2+). Obesity caused a reduction in cardiac pPLB Ser(16) and the pPLB Ser(16)/PLB ratio in Ob rats. Long-term obesity promotes alterations in diastolic function, most likely due to the reduction of pPLB Ser(16), but does not impair the myocardial Ca(2+) entry and recapture to SR. Copyright © 2014 the American Physiological Society.

Ca2+ regulatory mechanisms of exercise protection against coronary artery disease in metabolic syndrome and diabetes.

PubMed

Sturek, Michael

2011-08-01

Chronic exercise attenuates coronary artery disease (CAD) in humans largely independent of reductions in risk factors; thus major protective mechanisms of exercise are directly within the coronary vasculature. Further, tight control of diabetes, e.g., blood glucose, can be detrimental. Accordingly, knowledge of mechanisms by which exercise attenuates diabetic CAD could catalyze development of molecular therapies. Exercise attenuates CAD (atherosclerosis) and restenosis in miniature swine models, which enable precise control of exercise parameters (intensity, duration, and frequency) and characterization of the metabolic syndrome (MetS) and diabetic milieu. Intracellular Ca(2+) is a pivotal second messenger for coronary smooth muscle (CSM) excitation-contraction and excitation-transcription coupling that modulates CSM proliferation, migration, and calcification. CSM of diabetic dyslipidemic Yucatan swine have impaired Ca(2+) extrusion via the plasmalemma Ca(2+) ATPase (PMCA), downregulation of L-type voltage-gated Ca(2+) channels (VGCC), increased Ca(2+) sequestration by the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA), increased nuclear Ca(2+) localization, and greater activation of K channels by Ca(2+) release from the SR. Endurance exercise training prevents Ca(2+) transport changes with virtually no effect on the diabetic milieu (glucose, lipids). In MetS Ossabaw swine transient receptor potential canonical (TRPC) channels are upregulated and exercise training reverses expression and TRPC-mediated Ca(2+) influx with almost no change in the MetS milieu. Overall, exercise effects on Ca(2+) signaling modulate CSM phenotype. Future studies should 1) selectively target key Ca(2+) transporters to determine definitively their causal role in atherosclerosis and 2) combine mechanistic studies with clinical outcomes, e.g., reduction of myocardial infarction.

Heart-specific overexpression of (pro)renin receptor induces atrial fibrillation in mice.

PubMed

Lian, Hong; Wang, Xiaojian; Wang, Juan; Liu, Ning; Zhang, Li; Lu, Yingdong; Yang, Yanmin; Zhang, Lianfeng

2015-04-01

Atrial fibrillation (AF) is the most common cardiac arrhythmia, causing substantial cardiovascular morbidity and mortality. The renin-angiotensin system (RAS) has been shown to be involved in the pathophysiology of AF. The (pro)renin receptor [(p)RR] is the last identified member of RAS. However, the role of (p)RR in AF is still unknown. Circulating levels of (p)RR were determined using an immunosorbent assay in 22 patients with AF (paroxysmal or persistent) and 22 healthy individuals. The plasma levels of (p)RR increased 3.6-fold in AF patients (P<0.001), indicating a relationship between (p)RR and AF. To investigate the role of (p)RR in the regulation of cardiac arrhythmia, we generated a transgenic mouse with overexpression of human (p)RR gene specifically in the heart. Electrocardiograms from (p)RR transgenic mice showed typical atrial flutter since 2 months, then spontaneously converted to atrial fibrillation by 10 months. The atria of the transgenic mice demonstrated significant dilation and fibrosis, and exhibited a high incidence of sudden death. Additionally, the genes of SERCA and HCN4, which are involved in the electrophysiology of AF, were significantly down-regulated and up-regulated respectively in transgenic mice atria. The phosphorylation of Erk1/2 significantly increased in the atria of the transgenic mice, and the activated Erk1/2 was found predominantly in cardiac fibroblasts, suggesting that the transgenic (p)RR gene may induce atrial fibrillation by activation of Erk1/2 in the cardiac fibroblasts of the atria. (p)RR promotes atrial structural and electrical remodeling in vivo, which indicates that (p)RR plays an important role in the pathological development of AF. Copyright © 2015. Published by Elsevier Ireland Ltd.

Calcium regulation in crustaceans during the molt cycle: a review and update.

PubMed

Ahearn, Gregory A; Mandal, Prabir K; Mandal, Anita

2004-02-01

Epithelial cells of the gut, gills, antennal glands and integument regulate calcium concentrations in crustaceans during the molt cycle. A cellular calcium transport model has been proposed suggesting the presence of calcium pumps, cation antiporters and calcium channels in transporting epithelial membranes that regulate the movements of this cation across the cell layer. Basolateral calcium transport during postmolt appears mainly regulated by the low affinity NCX antiporter, while calcium regulating 'housekeeping' activities of these cells in intermolt are controlled by the high affinity calcium ATPase (PMCA). A model is proposed for the involvement of the epithelial ER in the massive transepithelial calcium fluxes that occur during premolt and postmolt. This model involves the endoplasmic reticulum SERCA and RyR proteins and proposed cytoplasmic unstirred layers adjacent to apical and basolateral plasma membranes where calcium activities may largely exceed those in the bulk cytoplasmic phase. A result of the proposed transepithelial calcium transport model is that large quantities of calcium can be moved through these cells by these processes without affecting the low, and carefully controlled, bulk cytoplasmic calcium activities.

Disorder of endoplasmic reticulum calcium channel components is associated with the increased apoptotic potential in pale, soft, exudative pork.

PubMed

Guo, Bing; Zhang, Wangang; Tume, Ron K; Hudson, Nicholas J; Huang, Feng; Yin, Yan; Zhou, Guanghong

2016-05-01

Eight pale, soft and exudative (PSE) and eight reddish-pink, firm and non-exudative (RFN) porcine longissimus muscle samples were selected based on pH and L* at 1h postmortem (PM), and drip loss at 24h PM, and used to evaluate the cellular calcium and apoptosis status. We found that SERCA1 was decreased, while IP3R was decreased in PSE meat (P<0.05), indicative of the overloaded sarcoplasmic calcium status. In PSE meat, the pro-apoptotic factor BAX was increased while the anti-apoptotic factor Bcl-2 was decreased (P<0.05). The significantly increased activity of caspase 3 and the expression of its cleavage fragment suggested higher apoptotic potential in PSE meat compared with RFN meat (P<0.05). Moreover, the significantly higher expression level of cytochrome C (P<0.05) suggests the important role of mitochondria during apoptosis appearance in PSE meat. Taken together, our data inferred that the calcium channel disorder present in PSE meat was associated with the increased apoptotic potential. Copyright © 2016 Elsevier Ltd. All rights reserved.

SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis.

PubMed

Wang, Yuanzhong; Zhou, Dujin; Phung, Sheryl; Warden, Charles; Rashid, Rumana; Chan, Nymph; Chen, Shiuan

2017-02-21

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.more » - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.« less

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false Determination of target normal cost and funding target. 1.430(d)-1 Section 1.430(d)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Certain Stock Options § 1.430(d)-1 Determination of target normal cost and fundin...

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

PubMed

Jessica, Sabourin; Angèle, Boet; Catherine, Rucker-Martin; Mélanie, Lambert; Ana-Maria, Gomez; Jean-Pierre, Benitah; Frédéric, Perros; Marc, Humbert; Fabrice, Antigny

2018-05-01

Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca 2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca 2+ remodeling. After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca 2+ ] i transients and increased sarcoplasmic reticulum (SR) Ca 2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser 16 -phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) pump abundance. Moreover, after PH induction, Ca 2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca 2+ -release-activated Ca 2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca 2+ ] i transients amplitude, the SR Ca 2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. These new findings demonstrate RV-specific cellular Ca 2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca 2+ current participates in this Ca 2+ remodeling in RVH secondary to PH. Copyright © 2018 Elsevier Ltd. All rights reserved.

Quaternion normalization in additive EKF for spacecraft attitude determination

NASA Technical Reports Server (NTRS)

Bar-Itzhack, I. Y.; Deutschmann, J.; Markley, F. L.

1991-01-01

This work introduces, examines, and compares several quaternion normalization algorithms, which are shown to be an effective stage in the application of the additive extended Kalman filter (EKF) to spacecraft attitude determination, which is based on vector measurements. Two new normalization schemes are introduced. They are compared with one another and with the known brute force normalization scheme, and their efficiency is examined. Simulated satellite data are used to demonstrate the performance of all three schemes. A fourth scheme is suggested for future research. Although the schemes were tested for spacecraft attitude determination, the conclusions are general and hold for attitude determination of any three dimensional body when based on vector measurements, and use an additive EKF for estimation, and the quaternion for specifying the attitude.

Fuel cell system logic for differentiating between rapid and normal shutdown commands

DOEpatents

Keskula, Donald H.; Doan, Tien M.; Clingerman, Bruce J.

2000-01-01

A method of controlling the operation of a fuel cell system wherein each shutdown command for the system is subjected to decision logic which determines whether the command should be a normal shutdown command or rapid shutdown command. If the logic determines that the shutdown command should be a normal shutdown command, then the system is shutdown in a normal step-by-step process in which the hydrogen stream is consumed within the system. If the logic determines that the shutdown command should be a rapid shutdown command, the hydrogen stream is removed from the system either by dumping to atmosphere or routing to storage.

Calculation of grain boundary normals directly from 3D microstructure images

DOE PAGES

Lieberman, E. J.; Rollett, A. D.; Lebensohn, R. A.; ...

2015-03-11

The determination of grain boundary normals is an integral part of the characterization of grain boundaries in polycrystalline materials. These normal vectors are difficult to quantify due to the discretized nature of available microstructure characterization techniques. The most common method to determine grain boundary normals is by generating a surface mesh from an image of the microstructure, but this process can be slow, and is subject to smoothing issues. A new technique is proposed, utilizing first order Cartesian moments of binary indicator functions, to determine grain boundary normals directly from a voxelized microstructure image. In order to validate the accuracymore » of this technique, the surface normals obtained by the proposed method are compared to those generated by a surface meshing algorithm. Specifically, the local divergence between the surface normals obtained by different variants of the proposed technique and those generated from a surface mesh of a synthetic microstructure constructed using a marching cubes algorithm followed by Laplacian smoothing is quantified. Next, surface normals obtained with the proposed method from a measured 3D microstructure image of a Ni polycrystal are used to generate grain boundary character distributions (GBCD) for Σ3 and Σ9 boundaries, and compared to the GBCD generated using a surface mesh obtained from the same image. Finally, the results show that the proposed technique is an efficient and accurate method to determine voxelized fields of grain boundary normals.« less

Quaternion normalization in additive EKF for spacecraft attitude determination. [Extended Kalman Filters

NASA Technical Reports Server (NTRS)

Bar-Itzhack, I. Y.; Deutschmann, J.; Markley, F. L.

1991-01-01

This work introduces, examines and compares several quaternion normalization algorithms, which are shown to be an effective stage in the application of the additive extended Kalman filter to spacecraft attitude determination, which is based on vector measurements. Three new normalization schemes are introduced. They are compared with one another and with the known brute force normalization scheme, and their efficiency is examined. Simulated satellite data are used to demonstate the performance of all four schemes.

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2010 CFR

2010-04-01

... funding target and target normal cost for the plan year if the plan amendment— (i) Takes effect by the... (disregarding the effect on the plan's funding shortfall resulting from changes in interest and mortality... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Determination of target normal cost and funding...

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2013 CFR

2013-04-01

... funding target and target normal cost for the plan year if the plan amendment— (i) Takes effect by the... (disregarding the effect on the plan's funding shortfall resulting from changes in interest and mortality... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Determination of target normal cost and funding...

Comprehensive non-dimensional normalization of gait data.

PubMed

Pinzone, Ornella; Schwartz, Michael H; Baker, Richard

2016-02-01

Normalizing clinical gait analysis data is required to remove variability due to physical characteristics such as leg length and weight. This is particularly important for children where both are associated with age. In most clinical centres conventional normalization (by mass only) is used whereas there is a stronger biomechanical argument for non-dimensional normalization. This study used data from 82 typically developing children to compare how the two schemes performed over a wide range of temporal-spatial and kinetic parameters by calculating the coefficients of determination with leg length, weight and height. 81% of the conventionally normalized parameters had a coefficient of determination above the threshold for a statistical association (p<0.05) compared to 23% of those normalized non-dimensionally. All the conventionally normalized parameters exceeding this threshold showed a reduced association with non-dimensional normalization. In conclusion, non-dimensional normalization is more effective that conventional normalization in reducing the effects of height, weight and age in a comprehensive range of temporal-spatial and kinetic parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

Normal stresses in shear thickening granular suspensions.

PubMed

Pan, Zhongcheng; de Cagny, Henri; Habibi, Mehdi; Bonn, Daniel

2017-05-24

When subjected to shear, granular suspensions exhibit normal stresses perpendicular to the shear plane but the magnitude and sign of the different components of the normal stresses are still under debate. By performing both oscillatory and rotational rheology measurements on shear thickening granular suspensions and systematically varying the particle diameters and the gap sizes between two parallel-plates, we show that a transition from a positive to a negative normal stress can be observed. We find that frictional interactions which determine the shear thickening behavior of suspensions contribute to the positive normal stresses. Increasing the particle diameters or decreasing the gap sizes leads to a growing importance of hydrodynamic interactions, which results in negative normal stresses. We determine a relaxation time for the system, set by both the pore and the gap sizes, that governs the fluid flow through the inter-particle space. Finally, using a two-fluid model we determine the relative contributions from the particle phase and the liquid phase.

Calcium movements during pigment aggregation in freshwater shrimp chromatophores.

PubMed

Ribeiro, Márcia; McNamara, John Campbell

2007-02-01

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.

Three-dimensional contractile muscle tissue consisting of human skeletal myocyte cell line.

PubMed

Shima, Ai; Morimoto, Yuya; Sweeney, H Lee; Takeuchi, Shoji

2018-06-18

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips. Copyright © 2018. Published by Elsevier Inc.

Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae).

PubMed

Beraldo, F H; Sartorello, R; Lanari, R D; Garcia, C R

2001-06-01

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca2+] is maintained around 20 nM and the cells contain membrane-bound Ca2+ pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca2+ ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca2+ both in the presence and in the absence of extracellular Ca2+. In addition to the ER, an acidic compartment appears to be involved in Ca2+ storage, as collapse of intracellular pHgradients by monensin, a Na+ -H+ exchanger, and nigericin, a K+ -H+ exchanger, induce the release of Ca2+ from internal pools. A vacuolar H+ pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca2+ pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores. Copyright 2001 Harcourt Publishers Ltd.

Endocrine disrupting alkylphenols: structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability.

PubMed

Michelangeli, Francesco; Ogunbayo, Oluseye A; Wootton, Laura L; Lai, Pei F; Al-Mousa, Fawaz; Harris, Robert M; Waring, Rosemary H; Kirk, Christopher J

2008-11-25

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.

Sample size determination for logistic regression on a logit-normal distribution.

PubMed

Kim, Seongho; Heath, Elisabeth; Heilbrun, Lance

2017-06-01

Although the sample size for simple logistic regression can be readily determined using currently available methods, the sample size calculation for multiple logistic regression requires some additional information, such as the coefficient of determination ([Formula: see text]) of a covariate of interest with other covariates, which is often unavailable in practice. The response variable of logistic regression follows a logit-normal distribution which can be generated from a logistic transformation of a normal distribution. Using this property of logistic regression, we propose new methods of determining the sample size for simple and multiple logistic regressions using a normal transformation of outcome measures. Simulation studies and a motivating example show several advantages of the proposed methods over the existing methods: (i) no need for [Formula: see text] for multiple logistic regression, (ii) available interim or group-sequential designs, and (iii) much smaller required sample size.

UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

PubMed Central

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L.

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca2+-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca2+imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca2+oscillations in BON. UTP evoked a biphasic concentration-dependent Ca2+response. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca2+responses were blocked with inhibitors of PLC, IP3R, SERCA Ca2+pump, La3+sensitive Ca2+channels or chelation of intracellular free Ca2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca2+pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca2+currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K+ channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca2+buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y4/P2Y6R. Zero-Ca2+buffer augmented Ca2+responses and 5-HT release. Conclusion: UTP activates a predominant P2Y4R pathway to trigger Ca2+oscillations via internal Ca2+mobilization through a PLC/IP3/IP3R/SERCA Ca2+signaling pathway to stimulate 5-HT release; Ca2+influx is inhibitory. UTP-induced Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca2+oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release. PMID:28751862

UTP - Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells.

PubMed

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca 2+ -dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca 2+ imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca 2+ oscillations in BON. UTP evoked a biphasic concentration-dependent Ca 2+ response. Cells responded in the order of UTP, ATP > UTPγS > UDP > MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y 4 , 50% cells), UDP (P2Y 6 , 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca 2+ responses were blocked with inhibitors of PLC, IP3R, SERCA Ca 2+ pump, La 3+ sensitive Ca 2+ channels or chelation of intracellular free Ca 2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca 2+ pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca 2+ currents (I Ca ), V m -depolarization and inhibited I K (not I A ) currents. An I Kv 7.2/7.3 K + channel blocker XE-991 mimicked UTP-induced V m -depolarization and blocked UTP-responses. XE-991 blocked I K and UTP caused further reduction. La 3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca 2+ buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y 4 /P2Y 6 R. Zero-Ca 2+ buffer augmented Ca 2+ responses and 5-HT release. Conclusion: UTP activates a predominant P2Y 4 R pathway to trigger Ca 2+ oscillations via internal Ca 2+ mobilization through a PLC/IP 3 /IP3R/SERCA Ca 2+ signaling pathway to stimulate 5-HT release; Ca 2+ influx is inhibitory. UTP-induced V m -depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca 2+ oscillations or I ca /VOCC). UTP-gated signaling pathways triggered by activation of P2Y 4 R stimulate 5-HT release.

Use of scintigraphy for the determination of mucociliary clearance rates in normal, sedated, diseased and exercised horses.

PubMed Central

Willoughby, R A; Ecker, G L; McKee, S L; Riddolls, L J

1991-01-01

Mucociliary clearance rates from the trachea were determined in normal, sedated, diseased and exercised horses from scintigraphs obtained after an injection of technetium-99m sulphide colloid into the tracheal lumen. The group mean tracheal clearance rate of eight clinically normal horses during 42 trials was 2.06 +/- 0.38 cm/min. Significant between horse differences were found (p less than 0.05). When six and seven of these horses were given xylazine and detomidine hydrochloride, respectively, mean group tracheal clearance rates dropped significantly (p less than 0.05). The decreases from each normal horse's mean tracheal clearance rate ranged from 18 to 54%. There did not appear to be a difference between the tracheal clearance rates (TCRs) of the normal horses and those with chronic respiratory disease. Postexercise evaluations were not significantly different from the pre-exercise TCRs in three clinically normal horses and three horses with chronic obstructive pulmonary disease (p greater than 0.05). This minimally invasive scintigraphic technique for determining TCRs has proved to be useful and reliable. Images Fig. 1. PMID:1790485

Determination of rolling resistance coefficient based on normal tyre stiffness

NASA Astrophysics Data System (ADS)

Rykov, S. P.; Tarasuyk, V. N.; Koval, V. S.; Ovchinnikova, N. I.; Fedotov, A. I.; Fedotov, K. V.

2018-03-01

The purpose of the article is to develop analytical dependence of wheel rolling resistance coefficient based on the mathematical description of normal tyre stiffness. The article uses the methods of non-holonomic mechanics and plane section methods. The article shows that the abscissa of gravity center of tyre stiffness expansion by the length of the contact area is the shift of normal road response. It can be used for determining rolling resistance coefficient. When determining rolling resistance coefficient using ellipsis and power function equations, one can reduce labor costs for testing and increase assessment accuracy.

Relationship between the blood perfusion values determined by laser speckle imaging and laser Doppler imaging in normal skin and port wine stains.

PubMed

Chen, Defu; Ren, Jie; Wang, Ying; Zhao, Hongyou; Li, Buhong; Gu, Ying

2016-03-01

Laser Doppler imaging (LDI) and laser speckle imaging (LSI) are two major optical techniques aiming at non-invasively imaging the skin blood perfusion. However, the relationship between perfusion values determined by LDI and LSI has not been fully explored. 8 healthy volunteers and 13 PWS patients were recruited. The perfusions in normal skin on the forearm of 8 healthy volunteers were simultaneously measured by both LDI and LSI during post-occlusive reactive hyperemia (PORH). Furthermore, the perfusions of port wine stains (PWS) lesions and contralateral normal skin of 10 PWS patients were also determined. In addition, the perfusions for PWS lesions from 3 PWS patients were successively monitored at 0, 10 and 20min during vascular-targeted photodynamic therapy (V-PDT). The average perfusion values determined by LSI were compared with those of LDI for each subject. In the normal skin during PORH, power function provided better fits of perfusion values than linear function: powers for individual subjects go from 1.312 to 1.942 (R(2)=0.8967-0.9951). There was a linear relationship between perfusion values determined by LDI and LSI in PWS and contralateral normal skin (R(2)=0.7308-0.9623), and in PWS during V-PDT (R(2)=0.8037-0.9968). The perfusion values determined by LDI and LSI correlate closely in normal skin and PWS over a broad range of skin perfusion. However, it still suggests that perfusion range and characteristics of the measured skin should be carefully considered if LDI and LSI measures are compared. Copyright © 2015 Elsevier B.V. All rights reserved.

Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Ancrum, Tiffany; Manevich, Yefim; Townsend, Danyelle M.

2017-01-01

Abstract Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2−/−) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2−/− cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Innovation: Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Conclusions: Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR through S-glutathionylation of a series of key interrelated proteins. Antioxid. Redox Signal. 26, 247–261. PMID:26838680

Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii.

PubMed

Ali, Muhammad Y; Pavasovic, Ana; Dammannagoda, Lalith K; Mather, Peter B; Prentis, Peter J

2017-01-01

Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na + /K + -ATPase (NKA), H + -ATPase (HAT), Na + /K + /2Cl - cotransporter (NKCC), Na + /Cl - /HCO[Formula: see text] cotransporter (NBC), Na + /H + exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca +2 -ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish , Cherax quadricarinatus, C. destructor and C. cainii , with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types.

QRS/T-wave and calcium alternans in a type I diabetic mouse model for spontaneous postmyocardial infarction ventricular tachycardia: A mechanism for the antiarrhythmic effect of statins.

PubMed

Jin, Hongwei; Welzig, Charles M; Aronovitz, Mark; Noubary, Farzad; Blanton, Robert; Wang, Bo; Rajab, Mohammad; Albano, Alfred; Link, Mark S; Noujaim, Sami F; Park, Ho-Jin; Galper, Jonas B

2017-09-01

The incidence of sudden arrhythmic death is markedly increased in diabetics. The purpose of this study was to develop a mouse model for postmyocardial infarction (post-MI) ventricular tachycardia (VT) in the diabetic heart and determine the mechanism of an antiarrhythmic effect of statins. ECG transmitters were implanted in wild-type (WT), placebo, and pravastatin-treated type I diabetic Akita mice. MIs were induced by coronary ligation, and Ca 2+ transients were studied by optical mapping, and Ca 2+ transients and sparks in left ventricular myocytes (VM) by the Ionoptix system and confocal microscopy. Burst pacing of Akita mouse hearts resulted in rate-related QRS/T-wave alternans, which was attenuated in pravastatin-treated mice. Post-MI Akita mice developed QRS/T-wave alternans and VT at 2820 ± 879 beats per mouse, which decreased to 343 ± 115 in pravastatin-treated mice (n = 13, P <.05). Optical mapping demonstrated pacing-induced VT originating in the peri-infarction zone and Ca 2+ alternans, both attenuated in hearts of statin-treated mice. Akita VM displayed Ca 2+ alternans, and triggered activity as well as increased Ca 2+ transient decay time (Tau), Ca 2+ sparks, and cytosolic Ca 2+ and decreased SR Ca 2+ stores all of which were in part reversed in cells from statin treated mice. Homogenates of Akita ventricles demonstrated decreased SERCA2a/PLB ratio and increased ratio of protein phosphatase (PP-1) to the PP-1 inhibitor PPI-1 which were reversed in homogenates of pravastatin-treated Akita mice. Pravastatin decreased the incidence of post-MI VT and Ca 2+ alternans in Akita mouse hearts in part by revering abnormalities of Ca 2+ handling via the PP-1/PPI-1 pathway. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca(2+) and proton flux dysregulation.

PubMed

Domenighetti, Andrea A; Danes, Vennetia R; Curl, Claire L; Favaloro, Jennifer M; Proietto, Joseph; Delbridge, Lea M D

2010-04-01

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, >95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca(2+) current (I(Ca)) and Na(+)-Ca(2+) exchanger (NCX) fluxes, Na(+)-H(+) exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0+/-0.4% vs. 6.2+/-0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I(Ca) density was decreased (-2.80+/-0.29 vs. -5.30+/-0.70 pA/pF), and mean I(NCX) was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H(+) flux in response to NH(4)Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca(2+) and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Combustion of Gaseous Fuels with High Temperature Air in Normal- and Micro-gravity Conditions

NASA Technical Reports Server (NTRS)

Wang, Y.; Gupta, A. K.

2001-01-01

The objective of this study is determine the effect of air preheat temperature on flame characteristics in normal and microgravity conditions. We have obtained qualitative (global flame features) and some quantitative information on the features of flames using high temperature combustion air under normal gravity conditions with propane and methane as the fuels. This data will be compared with the data under microgravity conditions. The specific focus under normal gravity conditions has been on determining the global flame features as well as the spatial distribution of OH, CH, and C2 from flames using high temperature combustion air at different equivalence ratio.

7 CFR 760.4 - Normal marketings of milk.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Normal marketings of milk. 760.4 Section 760.4... Farmers for Milk § 760.4 Normal marketings of milk. (a) The county committee shall determine the affected farmer's normal marketings which, for the purposes of this subpart, shall be the sum of the quantities of...

7 CFR 760.4 - Normal marketings of milk.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false Normal marketings of milk. 760.4 Section 760.4... Farmers for Milk § 760.4 Normal marketings of milk. (a) The county committee shall determine the affected farmer's normal marketings which, for the purposes of this subpart, shall be the sum of the quantities of...

7 CFR 760.4 - Normal marketings of milk.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Normal marketings of milk. 760.4 Section 760.4... Farmers for Milk § 760.4 Normal marketings of milk. (a) The county committee shall determine the affected farmer's normal marketings which, for the purposes of this subpart, shall be the sum of the quantities of...

Accurate determination of complex materials coefficients of piezoelectric resonators.

PubMed

Du, Xiao-Hong; Wang, Qing-Ming; Uchino, Kenji

2003-03-01

This paper presents a method of accurately determining the complex piezoelectric and elastic coefficients of piezoelectric ceramic resonators from the measurement of the normalized electric admittance, Y, which is electric admittance Y of piezoelectric resonator normalized by the angular frequency omega. The coefficients are derived from the measurements near three special frequency points that correspond to the maximum and the minimum normalized susceptance (B) and the maximum normalized conductance (G). The complex elastic coefficient is determined from the frequencies at these points, and the real and imaginary parts of the piezoelectric coefficient are related to the derivative of the susceptance with respect to the frequency and the asymmetry of the conductance, respectively, near the maximum conductance point. The measurements for some lead zirconate titanate (PZT) based ceramics are used as examples to demonstrate the calculation and experimental procedures and the comparisons with the standard methods.

Chinese life cycle impact assessment factors.

PubMed

Yang, J X; Nielsen, P H

2001-04-01

The methodological basis and procedures for determination of Chinese normalization references and weighting factors according to the EDIP-method is described. According to Chinese industrial development intensity and population density, China was divided into three regions and the normalization references for each region were calculated on the basis of an inventory of all of the region's environmental emissions in 1990. The normalization reference was determined as the total environmental impact potential for the area in question in 1990 (EP(j)90) divided by the population. The weighting factor was determined as the normalization reference (ER(j)90) divided by society's target contribution in the year 2000 based on Chinese political reduction plans, ER(j)T2000. This paper presents and discuss results obtained for eight different environmental impact categories relevant for China: global warming, stratospheric ozone depletion, acidification, nutrient enrichment, photochemical ozone formation and generation of bulk waste, hazardous waste and slag and ashes.

Computer program determines exact two-sided tolerance limits for normal distributions

NASA Technical Reports Server (NTRS)

Friedman, H. A.; Webb, S. R.

1968-01-01

Computer program determines by numerical integration the exact statistical two-sided tolerance limits, when the proportion between the limits is at least a specified number. The program is limited to situations in which the underlying probability distribution for the population sampled is the normal distribution with unknown mean and variance.

Verbal Processing Reaction Times in "Normal" and "Poor" Readers.

ERIC Educational Resources Information Center

Culbertson, Jack; And Others

After it had been determined that reaction time (RT) was a sensitive measure of hemispheric dominance in a verbal task performed by normal adult readers, the reaction times of three groups of subjects (20 normal reading college students, 12 normal reading third graders and 11 poor reading grade school students) were compared. Ss were exposed to…

Metabolic Cost, Mechanical Work, and Efficiency during Normal Walking in Obese and Normal-Weight Children

ERIC Educational Resources Information Center

Huang, Liang; Chen, Peijie; Zhuang, Jie; Zhang, Yanxin; Walt, Sharon

2013-01-01

Purpose: This study aimed to investigate the influence of childhood obesity on energetic cost during normal walking and to determine if obese children choose a walking strategy optimizing their gait pattern. Method: Sixteen obese children with no functional abnormalities were matched by age and gender with 16 normal-weight children. All…

Extending of flat normal dispersion profile in all-solid soft glass nonlinear photonic crystal fibres

NASA Astrophysics Data System (ADS)

Siwicki, Bartłomiej; Kasztelanic, Rafał; Klimczak, Mariusz; Cimek, Jarosław; Pysz, Dariusz; Stępień, Ryszard; Buczyński, Ryszard

2016-06-01

The bandwidth of coherent supercontinuum generated in optical fibres is strongly determined by the all-normal dispersion characteristic of the fibre. We investigate all-normal dispersion limitations in all-solid oxide-based soft glass photonic crystal fibres with various relative inclusion sizes and lattice constants. The influence of material dispersion on fibre dispersion characteristics for a selected pair of glasses is also examined. A relation between the material dispersion of the glasses and the fibre dispersion has been described. We determined the parameters which limit the maximum range of flattened all-normal dispersion profile achievable for the considered pair of heavy-metal-oxide soft glasses.

Three-dimensional facial recognition using passive long-wavelength infrared polarimetric imaging.

PubMed

Yuffa, Alex J; Gurton, Kristan P; Videen, Gorden

2014-12-20

We use a polarimetric camera to record the Stokes parameters and the degree of linear polarization of long-wavelength infrared radiation emitted by human faces. These Stokes images are combined with Fresnel relations to extract the surface normal at each pixel. Integrating over these surface normals yields a three-dimensional facial image. One major difficulty of this technique is that the normal vectors determined from the polarizations are not unique. We overcome this problem by introducing an additional boundary condition on the subject. The major sources of error in producing inversions are noise in the images caused by scattering of the background signal and the ambiguity in determining the surface normals from the Fresnel coefficients.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Estrogen reduces endoplasmic reticulum stress to protect against glucotoxicity induced-pancreatic β-cell death.

PubMed

Kooptiwut, Suwattanee; Mahawong, Pitchnischa; Hanchang, Wanthanee; Semprasert, Namoiy; Kaewin, Suchada; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-Thai

2014-01-01

Estrogen can improve glucose homeostasis not only in diabetic rodents but also in humans. However, the molecular mechanism by which estrogen prevents pancreatic β-cell death remains unclear. To investigate this issue, INS-1 cells, a rat insulinoma cell line, were cultured in medium with either 11.1mM or 40mM glucose in the presence or the absence of estrogen. Estrogen significantly reduced apoptotic β-cell death by decreasing nitrogen-induced oxidative stress and the expression of the ER stress markers GRP 78, ATF6, P-PERK, PERK, uXBP1, sXBP1, and CHOP in INS-1 cells after prolonged culture in medium with 40mM glucose. In contrast, estrogen increased the expression of survival proteins, including sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA-2), Bcl-2, and P-p38, in INS-1 cells after prolonged culture in medium with 40mM glucose. The cytoprotective effect of estrogen was attenuated by addition of the estrogen receptor (ERα and ERβ) antagonist ICI 182,780 and the estrogen membrane receptor inhibitor G15. We showed that estrogen decreases not only oxidative stress but also ER stress to protect against 40mM glucose-induced pancreatic β-cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Elucidating a molecular mechanism that the deterioration of porcine meat quality responds to increased cortisol based on transcriptome sequencing.

PubMed

Wan, Xuebin; Wang, Dan; Xiong, Qi; Xiang, Hong; Li, Huanan; Wang, Hongshuai; Liu, Zezhang; Niu, Hongdan; Peng, Jian; Jiang, Siwen; Chai, Jin

2016-11-11

Stress response is tightly linked to meat quality. The current understanding of the intrinsic mechanism of meat deterioration under stress is limited. Here, male piglets were randomly assigned to cortisol and control groups. Our results showed that when serum cortisol level was significantly increased, the meat color at 1 h postmortem, muscle bundle ratio, apoptosis rate, and gene expression levels of calcium channel and cell apoptosis including SERCA1, IP3R1, BAX, Bcl-2, and Caspase-3, were notably increased. However, the value of drip loss at 24 h postmortem and serum CK were significantly decreased. Additionally, a large number of differentially expressed genes (DEGs) in GC regulation mechanism were screened out using transcriptome sequencing technology. A total of 223 DEGs were found, including 80 up-regulated genes and 143 down-regulated genes. A total of 204 genes were enriched in GO terms, and 140 genes annotated into in KEGG database. Numerous genes were primarily involved in defense, inflammatory and wound responses. This study not only identifies important genes and signalling pathways that may affect the meat quality but also offers a reference for breeding and feeding management to provide consumers with better quality pork products.

N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization.

PubMed

Law, Betty Y K; Mok, Simon W F; Chen, Juan; Michelangeli, Francesco; Jiang, Zhi-Hong; Han, Yu; Qu, Yuan Q; Qiu, Alena C L; Xu, Su-Wei; Xue, Wei-Wei; Yao, Xiao-Jun; Gao, Jia Y; Javed, Masood-Ul-Hassan; Coghi, Paolo; Liu, Liang; Wong, Vincent K W

2017-01-01

Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified N -desmethyldauricine (LP-4), isolated from rhizoma of Menispermum dauricum DC as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca 2+ /Calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.

Calcitriol Prevents Cardiovascular Repercussions in Puromycin Aminonucleoside-Induced Nephrotic Syndrome

PubMed Central

Roberto, Roncon-Albuquerque

2018-01-01

Puromycin aminonucleoside-induced nephrotic syndrome (PAN-NS) is characterized by cardiac remodeling and increased local inflammatory activity. Patients with NS and animal models of NS have vitamin D3 deficiency. The aim of the present study was to evaluate the influence of calcitriol on cardiac remodeling and local inflammatory state in PAN-NS rat model. Male Sprague-Dawley rats were injected with PAN or vehicle on day 0. PAN and control rats were divided into two subgroups for the administration of calcitriol (PAN-D and Ct-D groups) or the vehicle (PAN-V and Ct-V groups) during 21 days. On day 21, the renal function, metabolic balance, calcitriol and FGF-23 plasma levels, prohypertrophy and proinflammatory markers (ET-1, TGF-β1, TNF-α, and IL-1β), and calcium signaling molecules (PLB and SERCA-2a) were evaluated. Twenty-one days after injection, PAN-V group presented cardiac hypertrophy and a modulation of proinflammatory markers local expression. Calcitriol treatment of PAN rats prevented cardiac hypertrophy and was associated with marked reduction in the cardiac expression levels of proinflammatory markers. Our results suggest that vitamin D3 deficiency in PAN-NS may contribute to cardiac remodeling and to the increase in local inflammatory activity. Calcitriol treatment prevents both cardiac repercussions and local inflammatory processes in PAN-NS. PMID:29607318

Distributions of experimental protein structures on coarse-grained free energy landscapes

PubMed Central

Liu, Jie; Jernigan, Robert L.

2015-01-01

Predicting conformational changes of proteins is needed in order to fully comprehend functional mechanisms. With the large number of available structures in sets of related proteins, it is now possible to directly visualize the clusters of conformations and their conformational transitions through the use of principal component analysis. The most striking observation about the distributions of the structures along the principal components is their highly non-uniform distributions. In this work, we use principal component analysis of experimental structures of 50 diverse proteins to extract the most important directions of their motions, sample structures along these directions, and estimate their free energy landscapes by combining knowledge-based potentials and entropy computed from elastic network models. When these resulting motions are visualized upon their coarse-grained free energy landscapes, the basis for conformational pathways becomes readily apparent. Using three well-studied proteins, T4 lysozyme, serum albumin, and sarco-endoplasmic reticular Ca2+ adenosine triphosphatase (SERCA), as examples, we show that such free energy landscapes of conformational changes provide meaningful insights into the functional dynamics and suggest transition pathways between different conformational states. As a further example, we also show that Monte Carlo simulations on the coarse-grained landscape of HIV-1 protease can directly yield pathways for force-driven conformational changes. PMID:26723638

Patient-Specific Induced Pluripotent Stem Cell as a Model for Familial Dilated Cardiomyopathy

PubMed Central

Sun, Ning; Yazawa, Masayuki; Liu, Jianwei; Han, Leng; Sanchez-Freire, Veronica; Abilez, Oscar J.; Navarrete, Enrique G.; Hu, Shijun; Wang, Li; Lee, Andrew; Pavlovic, Aleksandra; Lin, Shin; Chen, Rui; Hajjar, Roger J.; Snyder, Michael P.; Dolmetsch, Ricardo E.; Butte, Manish J.; Ashley, Euan A.; Longaker, Michael T.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Dilated cardiomyopathy (DCM) is the most common cardiomyopathy, characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific models, investigating underlying mechanisms, and optimizing therapy. Here we generated cardiomyocytes (CMs) from iPSCs derived from patients of a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to the control healthy individuals in the same family cohort, DCM iPSC-CMs exhibited altered Ca2+ handling, decreased contractility, and abnormal sarcomeric α-actinin distribution. When stimulated with β-adrenergic agonist, DCM iPSC-CMs showed characteristics of failure such as reduced beating rates, compromised contraction, and significantly more cells with abnormal sarcomeric α-actinin distribution. β-adrenergic blocker treatment and over-expression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a) improved DCM iPSC-CMs function. Our study demonstrated that human DCM iPSC-CMs recapitulated to some extent the disease phenotypes morphologically and functionally, and thus can serve as a useful platform for exploring molecular and cellular mechanisms and optimizing treatment of this particular disease. PMID:22517884

Woodpecker drumming behavior is linked to the elevated expression of genes that encode calcium handling proteins in the neck musculature.

PubMed

Schuppe, Eric R; Petersen, John O; Fuxjager, Matthew J

2018-05-31

Many animals perform elaborate physical displays for social communication. Identifying molecular mechanisms that co-evolve with these complex behavioral signals can therefore help reveal how forces of selection shape animal design. To study this issue, we examine gene expression profiles in select skeletal muscles that actuate woodpecker drum displays. This remarkable whole-body signal is produced when individuals rapidly hammer their bill against trees. We find that, compared to muscles that play no part in producing this behavior, the main muscle used to drum abundantly expresses two genes that encode proteins that support myocytic calcium (Ca 2+ ) handling dynamics-namely parvalbumin (PV) and sarcoplasmic reticulum Ca 2+ ATPase (SERCA1). Meanwhile, we find no such difference in the expression of another gene similarly vital to Ca 2+ handling, the ryanodine receptor (RYR1). These differences are not present in a non-woodpecker species, which readily produce much slower drum-like movements for foraging (but not social signaling). Our data therefore point to an association between the fast drum displays of woodpeckers and muscle-specific expression of genes whose protein products enhance select aspects of myocytic Ca 2+ handling. © 2018. Published by The Company of Biologists Ltd.

Normal values for segmental bioimpedance spectroscopy in pediatric patients.

PubMed

Avila, Maria Laura; Ward, Leigh C; Feldman, Brian M; Montoya, Madeline I; Stinson, Jennifer; Kiss, Alex; Brandão, Leonardo R

2015-01-01

Localized limb edema is a clinically relevant sign in diseases such as post-thrombotic syndrome and lymphedema. Quantitative evaluation of localized edema in children is mainly done by measuring the absolute difference in limb circumference, which includes fat and fat-free mass. Bioimpedance spectroscopy (BIS) provides information on the fluid volume of a body segment. Our objective was to determine normal ranges for segmental (arm and leg) BIS measurements in healthy children. Additionally, we determined the normal ranges for the difference in arm and ankle circumference and explored the influence of handedness and the correlation between techniques. Healthy children aged 1-18 years were recruited. The ratio of extracellular fluid content between contralateral limbs (estimated as the inter-arm and inter-leg extracellular impedance ratio), and the ratio of extracellular to intracellular fluid content for each limb (estimated as the intracellular to extracellular impedance ratio) were determined with a bioimpedance spectrometer. Arm and ankle circumference was determined with a Gulick II tape. We recruited 223 healthy children (48 infants, 54 preschoolers, 66 school-aged children, and 55 teenagers). Normal values for arm and leg BIS measurements, and for the difference in arm and ankle circumference were estimated for each age category. No influence of handedness was found. We found a statistically significant correlation between extracellular impedance ratio and circumference difference for arms among teenagers. We determined normal BIS ranges for arms and legs and for the difference in circumference between arms and between ankles in children. There was no statistically significant correlation between extracellular impedance ratio and difference in circumference, except in the case of arms in adolescents. This may indicate that limb circumference measures quantities other than fluid, challenging the adequacy of this technique to determine the presence of localized edema in most age groups.

New spatial upscaling methods for multi-point measurements: From normal to p-normal

NASA Astrophysics Data System (ADS)

Liu, Feng; Li, Xin

2017-12-01

Careful attention must be given to determining whether the geophysical variables of interest are normally distributed, since the assumption of a normal distribution may not accurately reflect the probability distribution of some variables. As a generalization of the normal distribution, the p-normal distribution and its corresponding maximum likelihood estimation (the least power estimation, LPE) were introduced in upscaling methods for multi-point measurements. Six methods, including three normal-based methods, i.e., arithmetic average, least square estimation, block kriging, and three p-normal-based methods, i.e., LPE, geostatistics LPE and inverse distance weighted LPE are compared in two types of experiments: a synthetic experiment to evaluate the performance of the upscaling methods in terms of accuracy, stability and robustness, and a real-world experiment to produce real-world upscaling estimates using soil moisture data obtained from multi-scale observations. The results show that the p-normal-based methods produced lower mean absolute errors and outperformed the other techniques due to their universality and robustness. We conclude that introducing appropriate statistical parameters into an upscaling strategy can substantially improve the estimation, especially if the raw measurements are disorganized; however, further investigation is required to determine which parameter is the most effective among variance, spatial correlation information and parameter p.

Color normalization for robust evaluation of microscopy images

NASA Astrophysics Data System (ADS)

Švihlík, Jan; Kybic, Jan; Habart, David

2015-09-01

This paper deals with color normalization of microscopy images of Langerhans islets in order to increase robustness of the islet segmentation to illumination changes. The main application is automatic quantitative evaluation of the islet parameters, useful for determining the feasibility of islet transplantation in diabetes. First, background illumination inhomogeneity is compensated and a preliminary foreground/background segmentation is performed. The color normalization itself is done in either lαβ or logarithmic RGB color spaces, by comparison with a reference image. The color-normalized images are segmented using color-based features and pixel-wise logistic regression, trained on manually labeled images. Finally, relevant statistics such as the total islet area are evaluated in order to determine the success likelihood of the transplantation.

49 CFR 192.605 - Procedural manual for operations, maintenance, and emergencies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... operations and maintenance activities are conducted. (b) Maintenance and normal operations. The manual... personnel to determine the effectiveness, and adequacy of the procedures used in normal operation and... or flow rate outside normal operating limits; (iii) Loss of communications; (iv) Operation of any...

The retest distribution of the visual field summary index mean deviation is close to normal.

PubMed

Anderson, Andrew J; Cheng, Allan C Y; Lau, Samantha; Le-Pham, Anne; Liu, Victor; Rahman, Farahnaz

2016-09-01

When modelling optimum strategies for how best to determine visual field progression in glaucoma, it is commonly assumed that the summary index mean deviation (MD) is normally distributed on repeated testing. Here we tested whether this assumption is correct. We obtained 42 reliable 24-2 Humphrey Field Analyzer SITA standard visual fields from one eye of each of five healthy young observers, with the first two fields excluded from analysis. Previous work has shown that although MD variability is higher in glaucoma, the shape of the MD distribution is similar to that found in normal visual fields. A Shapiro-Wilks test determined any deviation from normality. Kurtosis values for the distributions were also calculated. Data from each observer passed the Shapiro-Wilks normality test. Bootstrapped 95% confidence intervals for kurtosis encompassed the value for a normal distribution in four of five observers. When examined with quantile-quantile plots, distributions were close to normal and showed no consistent deviations across observers. The retest distribution of MD is not significantly different from normal in healthy observers, and so is likely also normally distributed - or nearly so - in those with glaucoma. Our results increase our confidence in the results of influential modelling studies where a normal distribution for MD was assumed. © 2016 The Authors Ophthalmic & Physiological Optics © 2016 The College of Optometrists.

A Bootstrap Based Measure Robust to the Choice of Normalization Methods for Detecting Rhythmic Features in High Dimensional Data.

PubMed

Larriba, Yolanda; Rueda, Cristina; Fernández, Miguel A; Peddada, Shyamal D

2018-01-01

Motivation: Gene-expression data obtained from high throughput technologies are subject to various sources of noise and accordingly the raw data are pre-processed before formally analyzed. Normalization of the data is a key pre-processing step, since it removes systematic variations across arrays. There are numerous normalization methods available in the literature. Based on our experience, in the context of oscillatory systems, such as cell-cycle, circadian clock, etc., the choice of the normalization method may substantially impact the determination of a gene to be rhythmic. Thus rhythmicity of a gene can purely be an artifact of how the data were normalized. Since the determination of rhythmic genes is an important component of modern toxicological and pharmacological studies, it is important to determine truly rhythmic genes that are robust to the choice of a normalization method. Results: In this paper we introduce a rhythmicity measure and a bootstrap methodology to detect rhythmic genes in an oscillatory system. Although the proposed methodology can be used for any high-throughput gene expression data, in this paper we illustrate the proposed methodology using several publicly available circadian clock microarray gene-expression datasets. We demonstrate that the choice of normalization method has very little effect on the proposed methodology. Specifically, for any pair of normalization methods considered in this paper, the resulting values of the rhythmicity measure are highly correlated. Thus it suggests that the proposed measure is robust to the choice of a normalization method. Consequently, the rhythmicity of a gene is potentially not a mere artifact of the normalization method used. Lastly, as demonstrated in the paper, the proposed bootstrap methodology can also be used for simulating data for genes participating in an oscillatory system using a reference dataset. Availability: A user friendly code implemented in R language can be downloaded from http://www.eio.uva.es/~miguel/robustdetectionprocedure.html.

A Bootstrap Based Measure Robust to the Choice of Normalization Methods for Detecting Rhythmic Features in High Dimensional Data

PubMed Central

Larriba, Yolanda; Rueda, Cristina; Fernández, Miguel A.; Peddada, Shyamal D.

2018-01-01

Motivation: Gene-expression data obtained from high throughput technologies are subject to various sources of noise and accordingly the raw data are pre-processed before formally analyzed. Normalization of the data is a key pre-processing step, since it removes systematic variations across arrays. There are numerous normalization methods available in the literature. Based on our experience, in the context of oscillatory systems, such as cell-cycle, circadian clock, etc., the choice of the normalization method may substantially impact the determination of a gene to be rhythmic. Thus rhythmicity of a gene can purely be an artifact of how the data were normalized. Since the determination of rhythmic genes is an important component of modern toxicological and pharmacological studies, it is important to determine truly rhythmic genes that are robust to the choice of a normalization method. Results: In this paper we introduce a rhythmicity measure and a bootstrap methodology to detect rhythmic genes in an oscillatory system. Although the proposed methodology can be used for any high-throughput gene expression data, in this paper we illustrate the proposed methodology using several publicly available circadian clock microarray gene-expression datasets. We demonstrate that the choice of normalization method has very little effect on the proposed methodology. Specifically, for any pair of normalization methods considered in this paper, the resulting values of the rhythmicity measure are highly correlated. Thus it suggests that the proposed measure is robust to the choice of a normalization method. Consequently, the rhythmicity of a gene is potentially not a mere artifact of the normalization method used. Lastly, as demonstrated in the paper, the proposed bootstrap methodology can also be used for simulating data for genes participating in an oscillatory system using a reference dataset. Availability: A user friendly code implemented in R language can be downloaded from http://www.eio.uva.es/~miguel/robustdetectionprocedure.html PMID:29456555

William Wales and the 1769 Transit of Venus: Puzzle Solving and the Determination of the Astronomical Unit

ERIC Educational Resources Information Center

Metz, Don

2009-01-01

According to Thomas Kuhn, a significant part of "normal science" is the fact gathering, empirical work which is intended to illustrate an existing paradigm. Some of this effort focuses on the determination of physical constants such as the astronomical unit (AU). For Kuhn, normal science is also what prepares students for membership in a…

Nasalance Scores of Children with Repaired Cleft Palate Who Exhibit Normal Velopharyngeal Closure during Aerodynamic Testing

ERIC Educational Resources Information Center

Zajac, David J.

2013-01-01

Purpose: To determine if children with repaired cleft palate and normal velopharyngeal (VP) closure as determined by aerodynamic testing exhibit greater acoustic nasalance than control children without cleft palate. Method: Pressure-flow procedures were used to identify 2 groups of children based on VP closure during the production of /p/ in the…

Low fat diet with omega-3 fatty acids increases plasma insulin-like growth factor concentration in healthy postmenopausal women

USDA-ARS?s Scientific Manuscript database

The insulin-like growth factor pathway plays a central role in the normal and abnormal growth of tissues; however, the nutritional determinants of insulin-like growth factor I (IGF-I) and its binding proteins in normal individuals are not well-defined. The purpose of this study was to determine the ...

A Skew-Normal Mixture Regression Model

ERIC Educational Resources Information Center

Liu, Min; Lin, Tsung-I

2014-01-01

A challenge associated with traditional mixture regression models (MRMs), which rest on the assumption of normally distributed errors, is determining the number of unobserved groups. Specifically, even slight deviations from normality can lead to the detection of spurious classes. The current work aims to (a) examine how sensitive the commonly…

Incidence and Determinants of Port Occlusions in Cancer Outpatients: A Prospective Cohort Study.

PubMed

Milani, Alessandra; Mazzocco, Ketti; Gandini, Sara; Pravettoni, Gabriella; Libutti, Livio; Zencovich, Claudia; Sbriglia, Ada; Pari, Chiara; Magon, Giorgio; Saiani, Luisa

Normal saline is considered a safe alternative for heparin as a locking solution in totally implantable venous access devices. The incidence rate of partial occlusion with the use of normal saline (easy injection, impossible aspiration) is estimated at 4%. The aim of this study was to investigate determinants of partial occlusions with the use of normal saline solution and the maintenance of positive pressure in the catheter. We enrolled 218 patients with different solid tumors who underwent pharmacologic treatment through the port with different frequencies: from once every week to at least once every month. The port was flushed with normal saline solution keeping a positive pressure in the catheter. We performed 4111 observations and documented normal port functioning in 99% of observations (n = 4057) and partial occlusions in 1% of observations (n = 54). Partial occlusions were significantly associated with frequency of port flushing (P < .05), chemotherapy (P < .001), and blood sample collection (P < .001). The use of positive pressure in addition to normal saline reduces the incidence rate of partial occlusions. The type of treatment, blood sample collection, and treatment schedule are important determinants of partial occlusions. Nurses play a key role in maintaining a functioning port using positive pressure during the flushing techniques. Certain risk factors must be monitored to prevent partial occlusions, and certain patients are more likely to present with port-related problems.

Quaternion normalization in spacecraft attitude determination

NASA Technical Reports Server (NTRS)

Deutschmann, Julie; Bar-Itzhack, Itzhack; Galal, Ken

1992-01-01

Methods are presented to normalize the attitude quaternion in two extended Kalman filters (EKF), namely, the multiplicative EKF (MEKF) and the additive EKF (AEKF). It is concluded that all the normalization methods work well and yield comparable results. In the AEKF, normalization is not essential, since the data chosen for the test do not have a rapidly varying attitude. In the MEKF, normalization is necessary to avoid divergence of the attitude estimate. All of the methods of the methods behave similarly when the spacecraft experiences low angular rates.

Coding of sounds in the auditory system and its relevance to signal processing and coding in cochlear implants.

PubMed

Moore, Brian C J

2003-03-01

To review how the properties of sounds are "coded" in the normal auditory system and to discuss the extent to which cochlear implants can and do represent these codes. Data are taken from published studies of the response of the cochlea and auditory nerve to simple and complex stimuli, in both the normal and the electrically stimulated ear. REVIEW CONTENT: The review describes: 1) the coding in the normal auditory system of overall level (which partly determines perceived loudness), spectral shape (which partly determines perceived timbre and the identity of speech sounds), periodicity (which partly determines pitch), and sound location; 2) the role of the active mechanism in the cochlea, and particularly the fast-acting compression associated with that mechanism; 3) the neural response patterns evoked by cochlear implants; and 4) how the response patterns evoked by implants differ from those observed in the normal auditory system in response to sound. A series of specific issues is then discussed, including: 1) how to compensate for the loss of cochlear compression; 2) the effective number of independent channels in a normal ear and in cochlear implantees; 3) the importance of independence of responses across neurons; 4) the stochastic nature of normal neural responses; 5) the possible role of across-channel coincidence detection; and 6) potential benefits of binaural implantation. Current cochlear implants do not adequately reproduce several aspects of the neural coding of sound in the normal auditory system. Improved electrode arrays and coding systems may lead to improved coding and, it is hoped, to better performance.

Exact Interval Estimation, Power Calculation, and Sample Size Determination in Normal Correlation Analysis

ERIC Educational Resources Information Center

Shieh, Gwowen

2006-01-01

This paper considers the problem of analysis of correlation coefficients from a multivariate normal population. A unified theorem is derived for the regression model with normally distributed explanatory variables and the general results are employed to provide useful expressions for the distributions of simple, multiple, and partial-multiple…

45 CFR 91.12 - Definitions of normal operation and statutory objective.

Code of Federal Regulations, 2010 CFR

2010-10-01

... objective. 91.12 Section 91.12 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION... Standards for Determining Age Discrimination § 91.12 Definitions of normal operation and statutory objective. For purposes of §§ 91.13 and 91.14, the terms normal operation and statutory objective shall have the...

Determination of glucose-6-phosphate dehydrogenase cut-off values in a Tunisian population.

PubMed

Laouini, Naouel; Sahli, Chaima Abdelhafidh; Jouini, Latifa; Haloui, Sabrine; Fredj, Sondes Hadj; Daboubi, Rym; Siala, Hajer; Ouali, Faida; Becher, Meriam; Toumi, Nourelhouda; Bibi, Amina; Messsaoud, Taieb

2017-07-26

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest enzymopathy worldwide. The incidence depends essentially on the methods used for the assessment. In this respect, we attempted in this study to set cut-off values of G6PD activity to discriminate among normal, heterozygous, and deficient individuals using the World Health Organization (WHO) classification and the receiver operating characteristics (ROC) curve analysis. Blood samples from 250 female and 302 male subjects were enrolled in this study. The G6PD activity was determined using a quantitative assay. The common G6PD mutations in Tunisia were determined using the amplification refractory mutation system (ARMS-PCR) method. The ROC curve was used to choice the best cut-off. Normal G6PD values were 7.69±2.37, 7.86±2.39, and 7.51±2.35 U/g Hb for the entire, male, and female groups, respectively. Cut-off values for the total, male, and female were determined using the WHO classification and ROC curves analysis. In the male population, both cut-offs established using ROC curve analysis (4.00 U/g Hb) and the 60% level (3.82 U/g Hb), respectively are sensitive and specific resulting in a good efficiency of discrimination between deficient and normal males. For the female group the ROC cut-off (5.84 U/g Hb) seems better than the 60% level cut-off (3.88 U/g Hb) to discriminate between normal and heterozygote or homozygote women with higher Youden Index. The establishment of the normal values for a population is important for a better evaluation of the assay result. The ROC curve analysis is an alternative method to determine the status of patients since it correlates DNA analysis and G6PD activity.

Determination of Zn/Cu ratio and oligoelements in serum samples by total reflection X-ray fluorescence spectrometry for cancer diagnosis

NASA Astrophysics Data System (ADS)

Marcó P., L. M.; Jiménez, E.; Hernández C., E. A.; Rojas, A.; Greaves, E. D.

2001-11-01

The method of quantification using the Compton peak as an internal standard, developed in a previous work, was applied to the routine determination of Fe, Cu, Zn and Se in serum samples from normal individuals and cancer patients by total reflection X-ray fluorescence spectrometry. Samples were classified according to age and sex of the donor, in order to determine reference values for normal individuals. Results indicate that the Zn/Cu ratio and the Cu concentration could prove to be useful tools for cancer diagnosis. Significant differences in these parameters between the normal and cancer group were found for all age ranges. The multielemental character of the technique, coupled with the small amounts of sample required and the short analysis time make it a valuable tool in clinical analysis.

Cerebral blood flow and red cell delivery in normal subjects and in multiple sclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swank, R.L.; Roth, J.G.; Woody, D.C. Jr.

1983-01-01

Regional cerebral blood flow (rCBF) was determined in 77 normal females and 53 normal males of different ages and in 26 men and 45 women with multiple sclerosis by the inhalation of radioactive Xe133 method. In the normal subjects the CBF was relatively high in the teens and fell, at first rapidly and then slowly in both sexes with age. During adult life the flow in females was significantly higher than in males. The delivery of packed red cells (RCD) was determined by multiplying the CBF by the percentage concentration of red cells (HCT). The RCD for both sexes wasmore » nearly the same. In the patients with multiple sclerosis there occurred a progressive generalized decrease in CBF and in RCD with age which was significantly greater than observed in normal subjects. The rate of decrease in CBF and RCD correlated directly with the rate of progress of the disease.« less

Regulation of Glucose Utilization by Estradiol in Breast Cancer

DTIC Science & Technology

2014-10-01

counts/min) weremeasured using 350 l of lysate. Protein concentration was determined using the BCA assay according to the manufacturer’s...instructions and measured on a Powerwave XS plate reader (Biotek). Counts were normalized to protein concentration. Glycolysis Assay—MCF-7 cells growing in 6...determined using the BCA assay according to the manufacturer’s instructions and mea- sured on a Powerwave XS plate reader. Counts were normal- ized to

The Effect on Non-Normal Distributions on the Integrated Moving Average Model of Time-Series Analysis.

ERIC Educational Resources Information Center

Doerann-George, Judith

The Integrated Moving Average (IMA) model of time series, and the analysis of intervention effects based on it, assume random shocks which are normally distributed. To determine the robustness of the analysis to violations of this assumption, empirical sampling methods were employed. Samples were generated from three populations; normal,…

Comparison of Reading Performance between Visually Impaired and Normally Sighted Students in Malaysia

ERIC Educational Resources Information Center

Mohammed, Zainora; Omar, Rokiah

2011-01-01

The aim of this study is to compare reading performance between visually impaired and normally sighted school children. Participants (n = 299) were divided into three groups: normal vision (NV, n = 193), visually impaired print reader (PR, n = 52), and Braille reader (BR, n = 54). Reading performance was determined by measuring reading rate and…

7 CFR 760.4 - Normal marketings of milk.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Normal marketings of milk. 760.4 Section 760.4... Farmers for Milk § 760.4 Normal marketings of milk. (a) The county committee shall determine the affected... whole milk which such farmer would have sold in the commercial market in each of the pay periods in the...

Normalized knee-extension strength or leg-press power after fast-track total knee arthroplasty: which measure is most closely associated with performance-based and self-reported function?

PubMed

Aalund, Peter K; Larsen, Kristian; Hansen, Torben B; Bandholm, Thomas

2013-02-01

To investigate which of the 2 muscle-impairment measures for the operated leg, normalized knee extension strength or leg press power, was most closely associated with performance-based and self-reported measures of function shortly after total knee arthroplasty (TKA). Cross-sectional, exploratory study. Laboratory at a regional hospital. Individuals (N=39) with an average age ± SD of 65.5±10.3 years, who all had unilateral TKA 28 days prior. None. The patients performed maximal isometric knee extensions and dynamic leg presses to determine their body-mass normalized knee extension strength and leg press power, respectively. The 10-meter fast speed walking- and 30-second chair stand tests were used to determine performance-based function, while the Western Ontario and McMaster Universities Osteoarthritis Index and Oxford Knee Scores were used to determine self-reported function. Normalized leg press power was more closely associated with both performance-based (r=.82, P<.001) and self-reported (r=.48, P=.002) measures of function compared with normalized knee extension strength (r=.51, P=.001 and r=.39, P=.015, respectively). Normalized leg press power was more closely associated with both performance-based and self-reported function early after TKA than normalized knee extension strength. It may be explained by the fact that performance-based measures of function are typically closed kinetic chain tasks, such as walking or rising from a chair, and self-reported measures of function typically include questions that address perceived difficulty with performing these same tasks. Copyright © 2013 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Glycolytic potential and activity of adenosine monophosphate kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) in steer carcasses with normal (<5.8) or high (>5.9) 24h pH determined in M. longissimus dorsi.

PubMed

Apaoblaza, A; Galaz, A; Strobel, P; Ramírez-Reveco, A; Jeréz-Timaure, N; Gallo, C

2015-03-01

Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem. Copyright © 2014 Elsevier Ltd. All rights reserved.

Roselle Polyphenols Exert Potent Negative Inotropic Effects via Modulation of Intracellular Calcium Regulatory Channels in Isolated Rat Heart.

PubMed

Lim, Yi-Cheng; Budin, Siti Balkis; Othman, Faizah; Latip, Jalifah; Zainalabidin, Satirah

2017-07-01

Roselle (Hibiscus sabdariffa Linn.) calyces have demonstrated propitious cardioprotective effects in animal and clinical studies; however, little is known about its action on cardiac mechanical function. This study was undertaken to investigate direct action of roselle polyphenols (RP) on cardiac function in Langendorff-perfused rat hearts. We utilized RP extract which consists of 12 flavonoids and seven phenolic acids (as shown by HPLC profiling) and has a safe concentration range between 125 and 500 μg/ml in this study. Direct perfusion of RP in concentration-dependent manner lowered systolic function of the heart as shown by lowered LVDP and dP/dt max , suggesting a negative inotropic effect. RP also reduced heart rate (negative chronotropic action) while simultaneously increasing maximal velocity of relaxation (positive lusitropic action). Conversely, RP perfusion increased coronary pressure, an indicator for improvement in coronary blood flow. Inotropic responses elicited by pharmacological agonists for L-type Ca 2+ channel [(±)-Bay K 8644], ryanodine receptor (4-chloro-m-cresol), β-adrenergic receptor (isoproterenol) and SERCA blocker (thapsigargin) were all abolished by RP. In conclusion, RP elicits negative inotropic, negative chronotropic and positive lusitropic responses by possibly modulating calcium entry, release and reuptake in the heart. Our findings have shown the potential use of RP as a therapeutic agent to treat conditions like arrhythmia.

Dissection of the Effects of Quercetin on Mouse Myocardium.

PubMed

Santos, Michel Santana; Oliveira, Evaleide Diniz; Santos-Miranda, Artur; Cruz, Jader Santos; Gondim, Antônio Nei Santana; Menezes-Filho, José Evaldo Rodrigues; Souza, Diego Santos; Pinho-da-Silva, Leidiane; Jesus, Itamar Couto Guedes; Roman-Campos, Danilo; Guatimosim, Silvia; Lara, Aline; Conde-Garcia, Eduardo Antônio; Vasconcelos, Carla Maria Lins

2017-06-01

Quercetin is a plant flavonoid with several biological activities. This study aimed to describe quercetin effects on contractile and electrophysiological properties of the cardiac muscle as well as on calcium handling. Quercetin elicited positive inotropism that was significantly reduced by propranolol indicating an involvement of the sympathetic nervous system. In cardiomyocytes, 30 μM quercetin increased I Ca,L at 0 mV from -0.95 ± 0.01 A/F to -1.21 ± 0.08 A/F. The membrane potential at which 50% of the channels are activated (V 0.5 ) shifted towards more negative potentials from -13.06 ± 1.52 mV to -19.26 ± 1.72 mV and did not alter the slope factor. Furthermore, quercetin increased [Ca 2+ ] i transient by 28% when compared to control. Quercetin accelerated [Ca 2+ ] i transient decay time, which could be attributed to SERCA activation. In resting cardiomyocytes, quercetin did not change amplitude or frequency of Ca 2+ sparks. In isolated heart, quercetin increased heart rate and decreased PRi, QTc and duration of the QRS complex. Thus, we showed that quercetin activates β-adrenoceptors, leading to increased L-type Ca 2+ current and cell-wide intracellular Ca 2+ transient without visible changes in Ca 2+ sparks. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Amalaki rasayana, a traditional Indian drug enhances cardiac mitochondrial and contractile functions and improves cardiac function in rats with hypertrophy.

PubMed

Kumar, Vikas; Aneesh, Kumar A; Kshemada, K; Ajith, Kumar G S; Binil, Raj S S; Deora, Neha; Sanjay, G; Jaleel, A; Muraleedharan, T S; Anandan, E M; Mony, R S; Valiathan, M S; Santhosh, Kumar T R; Kartha, C C

2017-08-17

We evaluated the cardioprotective effect of Amalaki Rasayana (AR), a rejuvenating Ayurvedic drug prepared from Phyllanthus emblica fruits in the reversal of remodeling changes in pressure overload left ventricular cardiac hypertrophy (LVH) and age-associated cardiac dysfunction in male Wistar rats. Six groups (aging groups) of 3 months old animals were given either AR or ghee and honey (GH) orally; seventh group was untreated. Ascending aorta was constricted using titanium clips in 3 months old rats (N = 24; AC groups) and after 6 months, AR or GH was given for further 12 months to two groups; one group was untreated. Histology, gene and protein expression analysis were done in heart tissues. Chemical composition of AR was analyzed by HPLC, HPTLC and LC-MS. AR intake improved (P < 0.05) cardiac function in aging rats and decreased LVH (P < 0.05) in AC rats as well as increased (P < 0.05) fatigue time in treadmill exercise in both groups. In heart tissues of AR administered rats of both the groups, SERCA2, CaM, Myh11, antioxidant, autophagy, oxidative phosphorylation and TCA cycle proteins were up regulated. ADRB1/2 and pCREB expression were increased; pAMPK, NF-kB were decreased. AR has thus a beneficial effect on myocardial energetics, muscle contractile function and exercise tolerance capacity.

Thermoregulatory and Cardiovascular Consequences of a Transient Thyrotoxicosis and Recovery in Male Mice.

PubMed

Hoefig, Carolin S; Harder, Lisbeth; Oelkrug, Rebecca; Meusel, Moritz; Vennström, Björn; Brabant, Georg; Mittag, Jens

2016-07-01

Thyroid hormones play a major role in body homeostasis, regulating energy expenditure and cardiovascular function. Given that obese people or athletes might consider rapid weight loss as beneficial, voluntary intoxication with T4 preparations is a growing cause for thyrotoxicosis. However, the long-lasting effects of transient thyrotoxicosis are poorly understood. Here we examined metabolic, thermoregulatory, and cardiovascular function upon induction and recovery from a 2-week thyrotoxicosis in male C57BL/6J mice. Our results showed that T4 treatment caused tachycardia, decreased hepatic glycogen stores, and higher body temperature as expected; however, we did not observe an increase in brown fat thermogenesis or decreased tail heat loss, suggesting that these tissues do not contribute to the hyperthermia induced by thyroid hormone. Most interestingly, when the T4 treatment was ended, a pronounced bradycardia was observed in the animals, which was likely caused by a rapid decline of T3 even below baseline levels. On the molecular level, this was accompanied by an overexpression of cardiac phospholamban and Serca2a mRNA, supporting the hypothesis that the heart depends more on T3 than T4. Our findings therefore demonstrate that a transient thyrotoxicosis can have pathological effects that even persist beyond the recovery of serum T4 levels, and in particular the observed bradycardia could be of clinical relevance when treating hyperthyroid patients.

Functional Effects of Hyperthyroidism on Cardiac Papillary Muscle in Rats.

PubMed

Vieira, Fabricio Furtado; Olivoto, Robson Ruiz; Silva, Priscyla Oliveira da; Francisco, Julio Cesar; Fogaça, Rosalvo Tadeu Hochmuller

2016-12-01

Hyperthyroidism is currently recognized to affect the cardiovascular system, leading to a series of molecular and functional changes. However, little is known about the functional influence of hyperthyroidism in the regulation of cytoplasmic calcium and on the sodium/calcium exchanger (NCX) in the cardiac muscle. To evaluate the functional changes in papillary muscles isolated from animals with induced hyperthyroidism. We divided 36 Wistar rats into a group of controls and another of animals with hyperthyroidism induced by intraperitoneal T3 injection. We measured in the animals' papillary muscles the maximum contraction force, speed of contraction (+df/dt) and relaxation (-df/dt), contraction and relaxation time, contraction force at different concentrations of extracellular sodium, post-rest potentiation (PRP), and contraction force induced by caffeine. In hyperthyroid animals, we observed decreased PRP at all rest times (p < 0.05), increased +df/dt and -df/dt (p < 0.001), low positive inotropic response to decreased concentration of extracellular sodium (p < 0.001), reduction of the maximum force in caffeine-induced contraction (p < 0.003), and decreased total contraction time (p < 0.001). The maximal contraction force did not differ significantly between groups (p = 0.973). We hypothesize that the changes observed are likely due to a decrease in calcium content in the sarcoplasmic reticulum, caused by calcium leakage, decreased expression of NCX, and increased expression of a-MHC and SERCA2.

UCT943, a next generation Plasmodium falciparum PI4K inhibitor preclinical candidate for the treatment of malaria.

PubMed

Brunschwig, Christel; Lawrence, Nina; Taylor, Dale; Abay, Efrem; Njoroge, Mathew; Basarab, Gregory S; Le Manach, Claire; Paquet, Tanya; Gonzàlez Cabrera, Diego; Nchinda, Aloysius T; de Kock, Carmen; Wiesner, Lubbe; Denti, Paolo; Waterson, David; Blasco, Benjamin; Leroy, Didier; Witty, Michael J; Donini, Cristina; Duffy, James; Wittlin, Sergio; White, Karen L; Charman, Susan A; Jiménez-Díaz, Maria Belén; Angulo-Barturen, Iñigo; Herreros, Esperanza; Gamo, Francisco Javier; Rochford, Rosemary; Mancama, Dalu; Coetzer, Theresa L; van der Watt, Mariëtte E; Reader, Janette; Birkholtz, Lyn-Marie; Marsh, Kennan C; Solapure, Suresh M; Vanaerschot, Manu; Fidock, David A; Fish, Paul V; Siegl, Peter; Smith, Dennis A; Wirjanata, Grennady; Noviyanti, Rintis; Price, Ric N; Marfurt, Jutta; Silue, Kigbafori D; Street, Leslie J; Chibale, Kelly

2018-06-25

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite lifecycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activity than MMV048 and was more potent against resistant P. falciparum and P. vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in P. berghei and humanized P. falciparum NOD- scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vitro intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next generation Plasmodium PI4K inhibitor, the combined preclinical data suggest that UCT943 has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent and block the transmission of malaria. Copyright © 2018 American Society for Microbiology.

Carbon monoxide exposure enhances arrhythmia after cardiac stress: involvement of oxidative stress.

PubMed

André, Lucas; Gouzi, Fares; Thireau, Jérôme; Meyer, Gregory; Boissiere, Julien; Delage, Martine; Abdellaoui, Aldja; Feillet-Coudray, Christine; Fouret, Gilles; Cristol, Jean-Paul; Lacampagne, Alain; Obert, Philippe; Reboul, Cyril; Fauconnier, Jérémy; Hayot, Maurice; Richard, Sylvain; Cazorla, Olivier

2011-11-01

Arrhythmias following cardiac stress are a key predictor of death in healthy population. Carbon monoxide (CO) is a ubiquitous pollutant promoting oxidative stress and associated with hospitalization for cardiovascular disease and cardiac mortality. We investigated the effect of chronic CO exposure on the occurrence of arrhythmic events after a cardiac stress test and the possible involvement of related oxidative stress. Wistar rats exposed chronically (4 weeks) to sustained urban CO pollution presented more arrhythmic events than controls during recovery after cardiac challenge with isoprenaline in vivo. Sudden death occurred in 22% of CO-exposed rats versus 0% for controls. Malondialdehyde (MDA), an end-product of lipid peroxidation, was increased in left ventricular tissue of CO-exposed rats. Cardiomyocytes isolated from CO-exposed rats showed higher reactive oxygen species (ROS) production (measured with MitoSox Red dye), higher diastolic Ca(2+) resulting from SR calcium leak and an higher occurrence of irregular Ca(2+) transients (measured with Indo-1) in comparison to control cells after a high pacing sequence. Acute treatment with a ROS scavenger (N-acetylcysteine, 20 mmol/L, 1 h) prevented this sequence of alterations and decreased the number of arrhythmic cells following high pacing. Chronic CO exposure promotes oxidative stress that alters Ca(2+) homeostasis (through RYR2 and SERCA defects) and thereby mediates the triggering of ventricular arrhythmia after cardiac stress that can lead to sudden death.

Vitamin D attenuates pressure overload-induced cardiac remodeling and dysfunction in mice.

PubMed

Zhang, Liang; Yan, Xiao; Zhang, Yun-Long; Bai, Jie; Hidru, Tesfaldet Habtemariam; Wang, Qing-Shan; Li, Hui-Hua

2018-04-01

Vitamin D (VD) and its analogues play critical roles in metabolic and cardiovascular diseases. Recent studies have demonstrated that VD exerts a protective role in cardiovascular diseases. However, the beneficial effect of VD on pressure overload-induced cardiac remodeling and dysfunction and its underlying mechanisms are not fully elucidated. In this study, cardiac dysfunction and hypertrophic remodeling in mice were induced by pressure overload. Cardiac function was evaluated by echocardiography, and myocardial histology was detected by H&E and Masson's trichrome staining. Cardiomyocyte size was detected by wheat germ agglutinin staining. The protein levels of signaling mediators were examined by western blotting while mRNA expression of hypertrophic and fibrotic markers was examined by qPCR analysis. Oxidative stress was detected by dihydroethidine staining. Our results showed that administration of VD3 significantly ameliorates pressure overload-induced contractile dysfunction, cardiac hypertrophy, fibrosis and inflammation in mice. In addition, VD3 treatment also markedly inhibited cardiac oxidative stress and apoptosis. Moreover, protein levels of calcineurin A, ERK1/2, AKT, TGF-β, GRP78, cATF6, and CHOP were significantly reduced whereas SERCA2 level was upregulated in the VD3-treated hearts compared with control. These results suggest that VD3 attenuates cardiac remodeling and dysfunction induced by pressure overload, and this protective effect is associated with inhibition of multiple signaling pathways. Copyright © 2018 Elsevier Ltd. All rights reserved.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

PubMed Central

Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O.; Emili, Andrew

2016-01-01

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function. PMID:27742792

Method for Expressing Clinical and Statistical Significance of Ocular and Corneal Wavefront Error Aberrations

PubMed Central

Smolek, Michael K.

2011-01-01

Purpose The significance of ocular or corneal aberrations may be subject to misinterpretation whenever eyes with different pupil sizes or the application of different Zernike expansion orders are compared. A method is shown that uses simple mathematical interpolation techniques based on normal data to rapidly determine the clinical significance of aberrations, without concern for pupil and expansion order. Methods Corneal topography (Tomey, Inc.; Nagoya, Japan) from 30 normal corneas was collected and the corneal wavefront error analyzed by Zernike polynomial decomposition into specific aberration types for pupil diameters of 3, 5, 7, and 10 mm and Zernike expansion orders of 6, 8, 10 and 12. Using this 4×4 matrix of pupil sizes and fitting orders, best-fitting 3-dimensional functions were determined for the mean and standard deviation of the RMS error for specific aberrations. The functions were encoded into software to determine the significance of data acquired from non-normal cases. Results The best-fitting functions for 6 types of aberrations were determined: defocus, astigmatism, prism, coma, spherical aberration, and all higher-order aberrations. A clinical screening method of color-coding the significance of aberrations in normal, postoperative LASIK, and keratoconus cases having different pupil sizes and different expansion orders is demonstrated. Conclusions A method to calibrate wavefront aberrometry devices by using a standard sample of normal cases was devised. This method could be potentially useful in clinical studies involving patients with uncontrolled pupil sizes or in studies that compare data from aberrometers that use different Zernike fitting-order algorithms. PMID:22157570

Method and apparatus for measuring irradiated fuel profiles

DOEpatents

Lee, David M.

1982-01-01

A new apparatus is used to substantially instantaneously obtain a profile of an object, for example a spent fuel assembly, which profile (when normalized) has unexpectedly been found to be substantially identical to the normalized profile of the burnup monitor Cs-137 obtained with a germanium detector. That profile can be used without normalization in a new method of identifying and monitoring in order to determine for example whether any of the fuel has been removed. Alternatively, two other new methods involve calibrating that profile so as to obtain a determination of fuel burnup (which is important for complying with safeguards requirements, for utilizing fuel to an optimal extent, and for storing spent fuel in a minimal amount of space). Using either of these two methods of determining burnup, one can reduce the required measurement time significantly (by more than an order of magnitude) over existing methods, yet retain equal or only slightly reduced accuracy.

In vivo determination of total knee arthroplasty kinematics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komistek, Richard D; Mahfouz, Mohamed R; Bertin, Kim

2008-01-01

The objective of this study was to determine if consistent posterior femoral rollback of an asymmetrical posterior cruciate retaining (PCR) total knee arthroplasty was mostly influenced by the implant design, surgical technique, or presence of a well-functioning posterior cruciate ligament (PCL). Three-dimensional femorotibial kinematics was determined for 80 subjects implanted by 3 surgeons, and each subject was evaluated under fluoroscopic surveillance during a deep knee bend. All subjects in this present study having an intact PCL had a well-functioning PCR knee and experienced normal kinematic patterns, although less in magnitude than the normal knee. In addition, a surprising finding wasmore » that, on average, subjects without a PCL still achieved posterior femoral rollback from full extension to maximum knee flexion. The findings in this study revealed that implant design did contribute to the normal kinematics demonstrated by subjects having this asymmetrical PCR total knee arthroplasty.« less

[Changes in the vitamin A status in dysplastic epithelium of the cervix].

PubMed

Volz, J; van Rissenbeck, A; Blanke, M; Melchert, F; Schneider, A; Biesalski, H K

1995-01-01

In 34 patients with HVP-infection of the cervix and in 40 patients with CIN III standardised biopsies were taken from the involved area and normal cervical epithelium for determination of the local concentration of retinylester. In all cases diagnosis was confirmed colposcopically, cytologically and by histology. HPV infection was confirmed by in situ hybridisation. Determination of retinylester was performed by HPLC. No significant difference of local retinyl-plamitate concentration was detectable in HPV infected versus normal tissue. Retinyl-palmitate concentration was extremely lower in CIN III compared with normal cervical epithelium and HPV-infected tissue. The determination of plasma level of retinol showed no significant difference between the two groups. So it can be presumed that the reduction of retinyl-palmitat in CIN III is a local process and a local supplementation of Vitamin A might contribute to the prevention of cervical neoplasia.

Early testosterone replacement attenuates intracellular calcium dyshomeostasis in the heart of testosterone-deprived male rats.

PubMed

Weerateerangkul, Punate; Shinlapawittayatorn, Krekwit; Palee, Siripong; Apaijai, Nattayaporn; Chattipakorn, Siriporn C; Chattipakorn, Nipon

2017-11-01

Testosterone deficiency in elderly men increases the risk of cardiovascular disease. In bilateral orchiectomized (ORX) animals, impaired cardiac Ca 2+ regulation was observed, and this impairment could be improved by testosterone replacement, indicating the important role of testosterone in cardiac Ca 2+ regulation. However, the temporal changes of Ca 2+ dyshomeostasis in testosterone-deprived conditions are unclear. Moreover, the effects of early vs. late testosterone replacement are unknown. We hypothesized that the longer the deprivation of testosterone, the greater the impairment of cardiac Ca 2+ homeostasis, and that early testosterone replacement can effectively reduce this adverse effect. Male Wistar rats were randomly divided into twelve groups, four sets of three. The first set were ORX for 2, 4 and 8 weeks, the second set were sham-operated groups of the same periods, the third set were ORX for 8 weeks coupled with a subcutaneous injection of vehicle (control), testosterone during weeks 1-8 (early replacement) or testosterone during weeks 5-8 (late replacement), and finally the 12-week sham-operated, ORX and ORX treated with testosterone groups. Cardiac Ca 2+ transients (n=4-5/group), L-type calcium current (I Ca-L ) (n=4/group), Ca 2+ regulatory proteins (n=6/group) and cardiac function (n=5/group) were determined. In the ORX rats, impaired cardiac Ca 2+ transients and reduced I Ca-L were observed initially 4 weeks after ORX as shown by decreased Ca 2+ transient amplitude, rising rate and maximum and average decay rates. No alteration of Ca 2+ regulatory proteins such as the L-type Ca 2+ channels, ryanodine receptor type 2, Na + -Ca 2+ exchangers and SERCA2a were observed. Early testosterone replacement markedly improved cardiac Ca 2+ transients, whereas late testosterone replacement did not. The cardiac contractility was also improved after early testosterone replacement. Impaired cardiac Ca 2+ homeostasis is time-dependent after testosterone deprivation. Early testosterone replacement improves cardiac Ca 2+ transient regulation and contractility, suggesting the necessity of early intervention in conditions of testosterone-deprivation. Copyright © 2017 Elsevier Ltd. All rights reserved.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

PubMed

Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I

2009-01-31

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii

PubMed Central

Pavasovic, Ana; Dammannagoda, Lalith K.; Mather, Peter B.; Prentis, Peter J.

2017-01-01

Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na+/K+-ATPase (NKA), H+-ATPase (HAT), Na+/K+/2Cl− cotransporter (NKCC), Na+/Cl−/HCO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3− cotransporter (NBC), Na+/H+ exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca+2-ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish, Cherax quadricarinatus, C. destructor and C. cainii, with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types. PMID:28852583

Effect of TGFβ on calcium signaling in megakaryocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Schmid, Evi; Department of Pediatric Surgery and Pediatric Urology, University Children's Hospital Tübingen, Tübingen

2015-05-22

TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entrymore » (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.« less

26 CFR 1.430(h)(2)-1 - Interest rates used to determine present value.

Code of Federal Regulations, 2013 CFR

2013-04-01

... present value of the benefits that are included in the target normal cost and the funding target for the... of the benefits that are included in the target normal cost and the funding target for the plan is... target normal cost and the funding target for the plan is the second segment rate with respect to the...

26 CFR 1.430(h)(2)-1 - Interest rates used to determine present value.

Code of Federal Regulations, 2014 CFR

2014-04-01

... present value of the benefits that are included in the target normal cost and the funding target for the... of the benefits that are included in the target normal cost and the funding target for the plan is... target normal cost and the funding target for the plan is the second segment rate with respect to the...

Determining density of maize canopy. 3: Temporal considerations

NASA Technical Reports Server (NTRS)

Stoner, E. R.; Baumgardner, M. F.; Anuta, P. E.; Cipra, J. E.

1972-01-01

Multispectral scanner data were collected in two flights over ground cover plots at an altitude of 305 m. Eight ground reflectance panels in close proximity to the ground cover plots were used to normalize the scanner data obtained on different dates. Separate prediction equations were obtained for both flight dates for all eleven reflective wavelength bands of the multispectral scanner. Ratios of normalized scanner data were related to leaf area index over time. Normalized scanner data were used to plot relative reflectance versus wavelength for the ground cover plots. Spectral response curves were similar to those for bare soil and green vegetation as determined by laboratory measurements. The spectral response curves from the normalized scanner data indicated that reflectance in the 0.72 to 1.3 micron wavelength range increased as leaf area index increased. A decrease in reflectance was observed in the 0.65 micron chlorophyll absorption band as leaf area index increased.

Applying the log-normal distribution to target detection

NASA Astrophysics Data System (ADS)

Holst, Gerald C.

1992-09-01

Holst and Pickard experimentally determined that MRT responses tend to follow a log-normal distribution. The log normal distribution appeared reasonable because nearly all visual psychological data is plotted on a logarithmic scale. It has the additional advantage that it is bounded to positive values; an important consideration since probability of detection is often plotted in linear coordinates. Review of published data suggests that the log-normal distribution may have universal applicability. Specifically, the log-normal distribution obtained from MRT tests appears to fit the target transfer function and the probability of detection of rectangular targets.

Commercial radioimmunoassay for beta subunit of human chorionic gonadotropin: falsely positive determinations due to elevated serum luteinizing hormone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, J.E. Jr.; Platoff, G.E.; Kubrock, C.A.

1982-01-01

Among 17 men who had received seemingly curative treatment for unilateral non-seminomatous germ cell tumors for the testis and who had consistently normal serum human chorionic gonadotropin (HCG) levels at a reference laboratory, 7 (41%) had at least one falsely positive commercial serum HCG determination. To investigate the cause of these falsely positive determinations the authors measured the cross reactivity of luteinizing hormone (LH) and follicle stimulating hormone (FSH) standards in the commercial HCG assay, and studied the relationships between commercial HCG levels and serum LH levels, serum FSH levels and gonadal status in men with and without normal gonadalmore » function. The falsely positive HCG determinations appeared to be due to elevated serum LH levels and cross reactivity of LH in the commercial HCG assay because: 1) there was substantial cross reactivity of the LH standards in the commercial assay, 2) the serum LH was elevated in four of six men with solitary testes, 3) there was a striking correlation between elevated serum LH levels and falsely elevated commercial HCG levels in ten men with solitary or absent testes, and 4) there were no falsely positive HCG determinations in 13 normal men but there were falsely positive HCG determinations in seven of ten anorchid men.« less

An ultrasonic method for determination of elastic moduli, density, attenuation and thickness of a polymer coating on a stiff plate.

PubMed

Lavrentyev, A I; Rokhlin, S I

2001-04-01

An ultrasonic method proposed by us for determination of the complete set of acoustical and geometrical properties of a thin isotropic layer between semispaces (J. Acoust. Soc. Am. 102 (1997) 3467) is extended to determination of the properties of a coating on a thin plate. The method allows simultaneous determination of the coating thickness, density, elastic moduli and attenuation (longitudinal and shear) from normal and oblique incidence reflection (transmission) frequency spectra. Reflection (transmission) from the coated plate is represented as a function of six nondimensional parameters of the coating which are determined from two experimentally measured spectra: one at normal and one at oblique incidence. The introduction of the set of nondimensional parameters allows one to transform the reconstruction process from one search in a six-dimensional space to two searches in three-dimensional spaces (one search for normal incidence and one for oblique). Thickness, density, and longitudinal and shear elastic moduli of the coating are calculated from the nondimensional parameters determined. The sensitivity of the method to individual properties and its stability against experimental noise are studied and the inversion algorithm is accordingly optimized. An example of the method and experimental measurement for comparison is given for a polypropylene coating on a steel foil.

Application of normalized spectra in resolving a challenging Orphenadrine and Paracetamol binary mixture

NASA Astrophysics Data System (ADS)

Yehia, Ali M.; Abd El-Rahman, Mohamed K.

2015-03-01

Normalized spectra have a great power in resolving spectral overlap of challenging Orphenadrine (ORP) and Paracetamol (PAR) binary mixture, four smart techniques utilizing the normalized spectra were used in this work, namely, amplitude modulation (AM), simultaneous area ratio subtraction (SARS), simultaneous derivative spectrophotometry (S1DD) and ratio H-point standard addition method (RHPSAM). In AM, peak amplitude at 221.6 nm of the division spectra was measured for both ORP and PAR determination, while in SARS, concentration of ORP was determined using the area under the curve from 215 nm to 222 nm of the regenerated ORP zero order absorption spectra, in S1DD, concentration of ORP was determined using the peak amplitude at 224 nm of the first derivative ratio spectra. PAR concentration was determined directly at 288 nm in the division spectra obtained during the manipulation steps in the previous three methods. The last RHPSAM is a dual wavelength method in which two calibrations were plotted at 216 nm and 226 nm. RH point is the intersection of the two calibration lines, where ORP and PAR concentrations were directly determined from coordinates of RH point. The proposed methods were applied successfully for the determination of ORP and PAR in their dosage form.

Sampling intensity and normalizations: Exploring cost-driving factors in nationwide mapping of tree canopy cover

Treesearch

John Tipton; Gretchen Moisen; Paul Patterson; Thomas A. Jackson; John Coulston

2012-01-01

There are many factors that will determine the final cost of modeling and mapping tree canopy cover nationwide. For example, applying a normalization process to Landsat data used in the models is important in standardizing reflectance values among scenes and eliminating visual seams in the final map product. However, normalization at the national scale is expensive and...

Metabolomic analysis of the longissimus from underperforming piglets suggests impaired carbohydrate metabolism, increased lipid utilization and higher protein breakdown in comparison to piglets with normal preweaning growth

USDA-ARS?s Scientific Manuscript database

The present study was designed to determine if normal birth weight pigs that grow poorly during the pre-weaning period have altered skeletal muscle metabolism, as previously reported for intrauterine growth retarded pigs relative to littermates with normal growth rates. Eight pairs of average birth...

High vascular delivery of EGF, but low receptor binding rate is observed in AsPC-1 tumors as compared to normal pancreas.

PubMed

Samkoe, Kimberley S; Sexton, Kristian; Tichauer, Kenneth M; Hextrum, Shannon K; Pardesi, Omar; Davis, Scott C; O'Hara, Julia A; Hoopes, P Jack; Hasan, Tayyaba; Pogue, Brian W

2012-08-01

Cellular receptor targeted imaging agents present the potential to target extracellular molecular expression in cancerous lesions; however, the image contrast in vivo does not reflect the magnitude of overexpression expected from in vitro data. Here, the in vivo delivery and binding kinetics of epidermal growth factor receptor (EGFR) was determined for normal pancreas and AsPC-1 orthotopic pancreatic tumors known to overexpress EGFR. EGFR in orthotopic xenograft AsPC-1 tumors was targeted with epidermal growth factor (EGF) conjugated with IRDye800CW. The transfer rate constants (k(e), K₁₂, k₂₁, k₂₃, and k₃₂) associated with a three-compartment model describing the vascular delivery, leakage rate and binding of targeted agents were determined experimentally. The plasma excretion rate, k (e), was determined from extracted blood plasma samples. K₁₂, k₂₁, and k₃₂ were determined from ex vivo tissue washing studies at time points ≥ 24 h. The measured in vivo uptake of IRDye800CW-EGF and a non-targeted tracer dye, IRDye700DX-carboxylate, injected simultaneously was used to determined k₂₃. The vascular exchange of IRDye800CW-EGF in the orthotopic tumor (K₁₂ and k₂₁) was higher than in the AsPC-1 tumor as compared to normal pancreas, suggesting that more targeted agent can be taken up in tumor tissue. However, the cellular associated (binding) rate constant (k₂₃) was slightly lower for AsPC-1 pancreatic tumor (4.1 × 10(-4) s(-1)) than the normal pancreas (5.5 × 10(-4) s(-1)), implying that less binding is occurring. Higher vascular delivery but low cellular association in the AsPC-1 tumor compared to the normal pancreas may be indicative of low receptor density due to low cellular content. This attribute of the AsPC-1 tumor may indicate one contributing cause of the difficulty in treating pancreatic tumors with cellular targeted agents.

Association of nicotine metabolism and sex with relapse following varenicline and nicotine replacement therapy.

PubMed

Glatard, Anaïs; Dobrinas, Maria; Gholamrezaee, Mehdi; Lubomirov, Rubin; Cornuz, Jacques; Csajka, Chantal; Eap, Chin B

2017-10-01

Nicotine is metabolized into cotinine and then into trans-3'-hydroxycotinine, mainly by cytochrome P450 2A6. Recent studies reported better effectiveness of varenicline in women and in nicotine normal metabolizers phenotypically determined by nicotine-metabolite ratio. Our objective was to study the influence of nicotine-metabolite ratio, CYP2A6 genotype and sex on the response to nicotine replacement therapy and varenicline. Data were extracted from a longitudinal study which included smokers participating in a smoking cessation program. Response to treatment was defined by the absence of relapse when a set threshold of reduction in cigarettes per day relative to the week before the study was no more reached. The analysis considered total and partial reduction defined by a diminution of 100% and of 90% in cigarettes per day, respectively. The hazard ratio of relapsing was estimated in multivariate Cox regression models including the sex and the nicotine metabolism determined by the phenotype or by CYP2A6 genotyping (rs1801272 and rs28399433). In the normal metabolizers determined by phenotyping and in women, the hazard ratio for relapsing was significantly lower with varenicline for a partial decrease (HR = 0.33, 95% CI [0.12, 0.89] and HR = 0.20, 95% CI [0.04, 0.91], respectively) and nonsignificantly lower for a total cessation (HR = 0.45, 95% CI [0.20, 1.0] and HR = 0.38, 95% CI [0.14, 1.0]). When compared with the normal metabolizers determined by phenotyping, the hazard ratio for a partial decrease was similar in the normal metabolizers determined by genotyping (HR = 0.42, 95% CI [0.18, 0.94]) while it was significantly lower with varenicline for a total cessation (HR = 0.50, 95% CI [0.26, 0.98]). Women and normal nicotine metabolizers may benefit more from varenicline over nicotine replacement therapy. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Method and apparatus for measuring irradiated fuel profiles

DOEpatents

Lee, D.M.

1980-03-27

A new apparatus is used to substantially instantaneously obtain a profile of an object, for example a spent fuel assembly, which profile (when normalized) has unexpectedly been found to be substantially identical to the normalized profile of the burnup monitor Cs-137 obtained with a germanium detector. That profile can be used without normalization in a new method of identifying and monitoring in order to determine for example whether any of the fuel has been removed. Alternatively, two other new methods involve calibrating that profile so as to obtain a determination of fuel burnup (which is important for complying with safeguards requirements, for utilizing fuel to an optimal extent, and for storing spent fuel in a minimal amount of space).

Normal and reverse flow injection–spectrophotometric determination of thiamine hydrochloride in pharmaceutical preparations using diazotized metoclopramide

PubMed Central

Al Abachi, Mouayed Q.; Hadi, Hind

2012-01-01

Simple and sensitive normal and reverse flow injection methods for spectrophotometric determination of thiamine hydrochloride (THC) at the microgram level were proposed and optimized. Both methods are based on the reaction between THC and diazotized metoclopramide in alkaline medium. Beer’s law was obeyed over the range of 10–300 and 2–90 μg/mL, the limits of detection were 2.118 and 0.839 μg/mL and the sampling rates were 80 and 95 injections per hour for normal and reverse flow injection methods respectively. The application of both methods to commercially available pharmaceuticals produced acceptable results. The flow system is suitable for application in quality control processes. PMID:29403765

Identification of CSF fistulas by radionuclide counting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Y.; Kunishio, K.; Sunami, N.

1990-07-01

A radionuclide counting method, performed with the patient prone and the neck flexed, was used successfully to diagnose CSF rhinorrhea in two patients. A normal radionuclide ratio (radionuclide counts in pledget/radionuclide counts in 1-ml blood sample) was obtained in 11 normal control subjects. Significance was determined to be a ratio greater than 0.37. Use of radionuclide counting method of determining CSF rhinorrhea is recommended when other methods have failed to locate a site of leakage or when posttraumatic meningitis suggests subclinical CSF rhinorrhea.

A model of the normal and null states of pulsars

NASA Astrophysics Data System (ADS)

Jones, P. B.

1981-12-01

A solvable three-dimensional polar cap model of pair creation and charged particle acceleration has been derived. There are no free parameters of significance apart from the polar surface magnetic flux density. The parameter determining the acceleration potential difference has been obtained by calculation of elementary nuclear and electromagnetic processes. Solutions of the model exist for both normal and null states of a pulsar, and the instability in the normal state leading to the normal to null transition has been identified. The predicted necessary condition for the transition is entirely consistent with observation.

A model of the normal and null states of pulsars

NASA Astrophysics Data System (ADS)

Jones, P. B.

A solvable three dimensional polar cap model of pair creation and charged particle acceleration is derived. There are no free parameters of significance apart from the polar surface magnetic flux density. The parameter CO determining the acceleration potential difference was obtained by calculation of elementary nuclear and electromagnetic processes. Solutions of the model exist for both normal and null states of a pulsar, and the instability in the normal state leading to the normal to null transition is identified. The predicted necessary condition for the transition is entirely consistent with observation.

Determination of stress intensity factors for interface cracks under mixed-mode loading

NASA Technical Reports Server (NTRS)

Naik, Rajiv A.; Crews, John H., Jr.

1992-01-01

A simple technique was developed using conventional finite element analysis to determine stress intensity factors, K1 and K2, for interface cracks under mixed-mode loading. This technique involves the calculation of crack tip stresses using non-singular finite elements. These stresses are then combined and used in a linear regression procedure to calculate K1 and K2. The technique was demonstrated by calculating three different bimaterial combinations. For the normal loading case, the K's were within 2.6 percent of an exact solution. The normalized K's under shear loading were shown to be related to the normalized K's under normal loading. Based on these relations, a simple equation was derived for calculating K1 and K2 for mixed-mode loading from knowledge of the K's under normal loading. The equation was verified by computing the K's for a mixed-mode case with equal and normal shear loading. The correlation between exact and finite element solutions is within 3.7 percent. This study provides a simple procedure to compute K2/K1 ratio which has been used to characterize the stress state at the crack tip for various combinations of materials and loadings. Tests conducted over a range of K2/K1 ratios could be used to fully characterize interface fracture toughness.

BIOCHEMISTRY OF NORMAL AND IRRADIATED STRAINS OF HYMENOLEPIS DIMINUTA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fairbairn, D.; Wertheim, G.; Harpur, R.P.

1961-09-01

An irradiated strain of H. diminuta was developed in which morphological anomalies persisted for at least 7 generations. This and the normal strain from which it was derived were corapared for biochemical differences, which might lead to the discovery of a biocheraical lesion. No significant differences were found in fresh weights between normal and irradiated strains of H. diminuta. Samples of H. diminuta were then prepared, and their composition determined. There was a notable loss of carbohydrates by tapeworms during 24 hr of in vivo fasting, amounting to 56% and 62% in the normal and irradiated strains, respectively. On themore » other hand, lipids increased by 10% and protein by 4% in both strains, which suggests that the substances were not concerned with energy metabolism during starvation. The giycogen of both normal and irradiated strains of H. diminuta obtained from fed or fasted rats, determined directly or after maintenance of the parasites in glucose-saline, accounted for 99% of the alkali-stable carbohydrates. which in turn, comprised about 96% of the total carbohydrates. In general, no notable differences in the growth, chemical composition, or gross metabolism between normal and irradiated strains of H. diminuta were recognized. Thus, the morphological changes due to irradiation previously described are the reflection of biochemical events. (H.H.D.)« less

Aerodynamic profiles of women with muscle tension dysphonia/aphonia.

PubMed

Gillespie, Amanda I; Gartner-Schmidt, Jackie; Rubinstein, Elaine N; Abbott, Katherine Verdolini

2013-04-01

In this study, the authors aimed to (a) determine whether phonatory airflows and estimated subglottal pressures (est-Psub) for women with primary muscle tension dysphonia/aphonia (MTD/A) differ from those for healthy speakers; (b) identify different aerodynamic profile patterns within the MTD/A subject group; and (c) determine whether results suggest new understanding of pathogenesis in MTD/A. Retrospective review of aerodynamic data collected from 90 women at the time of primary MTD/A diagnosis. Aerodynamic profiles were significantly different for women with MTD/A as compared with healthy speakers. Five distinct profiles were identified: (a) normal flow, normal est-Psub; (b) high flow, high est-Psub; (c) low flow, normal est-Psub; (d) normal flow, high est-Psub; and (e) high flow, normal est-Psub. This study is the first to identify distinct subgroups of aerodynamic profiles in women with MTD/A and to quantitatively identify a clinical phenomenon sometimes described in association with it-"breath holding"-that is shown by low airflow with normal est-Psub. Results were consistent with clinical claims that diverse respiratory and laryngeal functions may underlie phonatory patterns associated with MTD/A. One potential mechanism, based in psychobiological theory, is introduced to explain some of the variability in aerodynamic profiles of women with MTD/A.

Self-reported Knee Function Can Identify Athletes Who Fail Return to Activity Criteria up to 1 Year after Anterior Cruciate Ligament Reconstruction. A Delaware-Oslo ACL Cohort Study

PubMed Central

Logerstedt, David; Stasi, Stephanie Di; Grindem, Hege; Lynch, Andrew; Eitzen, Ingrid; Engebretsen, Lars; Risberg, May Arna; Axe, Michael J; Snyder-Mackler, Lynn

2014-01-01

STUDY DESIGN Cohort study, cross-sectional. OBJECTIVES To determine if self-reported knee function assessed with the International Knee Documentation Committee 2000 Subjective Knee Form (IKDC2000) could discriminate between successful and non-successful performance on return to activity criteria (RTAC) tests after anterior cruciate ligament (ACL) reconstruction. BACKGROUND Rehabilitation specialists are challenged in selecting appropriate performance-based and patient-reported tests that can detect side-to-side asymmetries, assess global knee function, and determine a participant's readiness to return to activity after ACL reconstruction. A simple tool or questionnaire that could identify athletes with neuromuscular impairments or activity limitations could provide rehabilitation specialists crucial data pertinent to their current knee function and their readiness to return to higher level activities. METHODS One hundred fifty-eight Level I/II athletes 6 months after ACL reconstruction and 141 athletes 12 months after ACL reconstruction completed a functional test battery to determine readiness to return to activity and the IKDC2000 to determine self-reported knee function. For each athlete, status on return to activity tests criteria was dichotomized as “Passed” or ”Failed” and status on the IKDC2000 scores was dichotomized as being “within” or “below age- and sex-matched normal ranges”. Comparisons were made between status on RTAC and IKDC2000 using Chi-square tests. Accuracy statistics were also calculated. RESULTS Six months after ACL reconstruction, 112 athletes (70.9%) failed RTAC and 76 (48.1%) were classified as having self-reported knee function below normal ranges. Among the 76 participants with IKDC2000 scores below normal ranges, 69 (90.8%) failed RTAC test battery (P<.001). However, among the 82 participants whose IKDC2000 scores were within normal limits at 6 months, only 39 (47.6%) passed RTAC test battery (P=.74). Twelve months after ACL reconstruction, 67 athletes (47.5%) failed RTAC and 31 (78.0%) had knee function below normal ranges. Among the 31 participants with IKDC2000 scores below normal ranges, 25 (80.6%) failed RTAC test battery (P<.001). However, among the 110 participants whose IKDC2000 scores were within normal limits at 12 months, only 68 (61.8%) passed RTAC test battery (P=.017). CONCLUSION The IKDC2000 may be a clinically relevant tool to determine the timeliness or necessity of RTAC testing. For scores obtained 6 and 12 months after ACL reconstruction, low IKDC2000 scores were reasonably indicative of failure on RTAC test battery, whereas normal IKDC2000 scores were not predictive of passing scores on the RTAC test battery. PMID:25347228

Compensatory Hypertrophy of Skeletal Muscle: Contractile Characteristics

ERIC Educational Resources Information Center

Ianuzzo, C. D.; Chen, V.

1977-01-01

Describes an experiment using rats that demonstrates contractile characteristics of normal and hypertrophied muscle. Compensatory hypertrophy of the plantaris muscle is induced by surgical removal of the synergistic gastrocnemium muscle. Includes methods for determination of contractile properties of normal and hypertrophied muscle and…

How do distracted and normal driving differ : an analysis of the ACAS naturalistic driving data

DOT National Transportation Integrated Search

2007-05-01

To determine how distracted and normal driving differ, this report re-examines : driving performance data from the advanced collision avoidance system (ACAS) field : operational test (FOT), a naturalistic driving study (96 drivers, 136,792 miles). : ...

Analysis of 777 cases with obstruction of the ureter or extrahepatic bile duct by ultrasonography after normal saline retention enema

PubMed Central

2012-01-01

Background Conventional transabdominal ultrasound usually fails to visualize parts of the ureter or extrahepatic bile duct covered by bowel gas. In this study, we propose a new method for gaining acoustic access to the ureters and extrahepatic bile duct to help determine the nature of obstruction to these structures when conventional transabdominal ultrasound fails. Methods The normal saline retention enema method, that is, using normal saline-filled colons to gain acoustic access to the bilateral ureters and extrahepatic bile duct and detecting the lesions with transabdominal ultrasonic diagnostic apparatus, was applied to 777 patients with obstructive lesions, including 603 with hydroureter and 174 with dilated common bile duct, which were not visualized by conventional ultrasonography. The follow-up data of all the patients were collected to verify the results obtained by this method. Results Of the 755 patients who successfully finished the examination after normal saline retention enema (the success rate of the enema is about 98%), the nature of obstruction in 718 patients was determined (the visualizing rate is approximately 95%), including 533 with ureteral calculus, 23 with ureteral stricture, 129 with extrahepatic bile duct calculus, and 33 with common bile duct tumor. Conclusions Colons filled fully with normal saline can surely give acoustic access to the bilateral ureters and extrahepatic bile duct so as to determine the nature of obstruction of these structures when conventional transabdominal ultrasound fails. PMID:22871226

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma.

PubMed

Meier, Jeremy D; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D Gregory

2010-06-01

The objectives of this study were to 1) determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract, and 2) evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. University-based medical center. Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700-picosecond pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared with the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than that of normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360 to approximately 660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7 +/- 0.06 ns [not significant] for normal and 1.3 +/- 0.06 ns for tumor, P = 0.0025) and the second-order Laguerre expansion coefficient (LEC-2) (i.e., at 460 nm: 0.135 +/- 0.001 for normal and 0.155 +/- 0.007 for tumor, P < 0.05). These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a noninvasive diagnostic technique for HNSCC. Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma

PubMed Central

Meier, Jeremy D.; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D. Gregory

2011-01-01

OBJECTIVE 1) Determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract. 2) Evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN Cross-sectional study. SETTING University-based medical center. SUBJECTS AND METHODS Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700 ps pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared to the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. RESULTS Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than the normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360~660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7±0.06 ns for normal and 1.3±0.06 ns for tumor, P=0.0025), and the Laguerre coefficients, LEC-2 (i.e., at 460 nm: 0.135±0.001 for normal and 0.155±0.007 for tumor, P<0.05). CONCLUSION These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a non-invasive diagnostic technique for HNSCC. PMID:20493355

Manipulation of near-wall turbulence by surface slip and permeability

NASA Astrophysics Data System (ADS)

Gómez-de-Segura, G.; Fairhall, C. T.; MacDonald, M.; Chung, D.; García-Mayoral, R.

2018-04-01

We study the effect on near-wall turbulence of tangential slip and wall-normal transpiration, typically produced by textured surfaces and other surface manipulations. For this, we conduct direct numerical simulations (DNSs) with different virtual origins for the different velocity components. The different origins result in a relative wall-normal displacement of the near-wall, quasi-streamwise vortices with respect to the mean flow, which in turn produces a change in drag. The objective of this work is to extend the existing understanding on how these virtual origins affect the flow. In the literature, the virtual origins for the tangential velocities are typically characterised by slip boundary conditions, while the wall-normal velocity is assumed to be zero at the boundary plane. Here we explore different techniques to define and implement the three virtual origins, with special emphasis on the wall-normal one. We investigate impedance conditions relating the wall-normal velocity to the pressure, and linear relations between the velocity components and their wall-normal gradients, as is typically done to impose slip conditions. These models are first tested to represent a smooth wall below the boundary plane, with all virtual origins equal, and later for different tangential and wall-normal origins. Our results confirm that the change in drag is determined by the offset between the origins perceived by mean flow and the quasi-streamwise vortices or, more generally, the near-wall turbulent cycle. The origin for the latter, however, is not set by the spanwise virtual origin alone, as previously proposed, but by a combination of the spanwise and wall-normal origins, and mainly determined by the shallowest of the two. These observations allow us to extend the existing expression to predict the change in drag, accounting for the wall-normal effect when the transpiration is not negligible.

Interleukin 1 increases thymidine labeling index of normal tissues of mic but not the tumor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaghloul, M.S.; Dorie, M.J.; Kallman, R.F.

1994-07-01

This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14more » days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation. 25 refs., 3 figs., 1 tab.« less

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

PubMed

Bradaric, Michael J; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L; Yu, Yi; Abramowicz, Jacques S; Bahr, Janice M; Luborsky, Judith L

2013-01-01

Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

PubMed Central

Bradaric, Michael J.; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L.; Yu, Yi; Abramowicz, Jacques S.; Bahr, Janice M.; Luborsky, Judith L.

2013-01-01

Background Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Methods Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. Results T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. Conclusions The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials. PMID:24040191

TGF-Alpha Expression During Breast Tumorigenesis

DTIC Science & Technology

1999-07-01

contributors to breast tumorigenesis. Therefore, we examined TGFa expression in normal and transformed epithelium. We also examined TOFa and EGFR protein in...patient samples it is difficult to determine what effect tissue culture has on normal T(3Fa protein synthesis and processing. TOFa protein expression

NORMAL GENE EXPRESSION IN MALE F344 RAT NASAL TRANSITIONAL/RESPIRATORY EPITHELIUM

EPA Science Inventory

Abstract

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity in rodents. Gene expression profiles were determined in order to provide normal baseline data for nasal transitional/respiratory epithelium from healthy rats. Ce...

Affective Interchange in Families with a Schizophrenic Son.

ERIC Educational Resources Information Center

Angermeyer, Matthias C.; And Others

Sixty half-hour family discussions generated by the "revealed differences technique" were analyzed to determine the emotional intensity and quality (friendliness/attacking) of messages between individuals in families with schizophrenic and "normal" sons. Thirty families in each situation (schizophrenic/normal) were matched for comparison. Both…

10 CFR 71.71 - Normal conditions of transport.

Code of Federal Regulations, 2014 CFR

2014-01-01

... NUCLEAR REGULATORY COMMISSION (CONTINUED) PACKAGING AND TRANSPORTATION OF RADIOACTIVE MATERIAL Package... each package design under normal conditions of transport must include a determination of the effect on... following table: Insolation Data Form and location of surface Total insolation for a 12-hour period(g cal...

10 CFR 71.71 - Normal conditions of transport.

Code of Federal Regulations, 2012 CFR

2012-01-01

... NUCLEAR REGULATORY COMMISSION (CONTINUED) PACKAGING AND TRANSPORTATION OF RADIOACTIVE MATERIAL Package... each package design under normal conditions of transport must include a determination of the effect on... following table: Insolation Data Form and location of surface Total insolation for a 12-hour period(g cal...

10 CFR 71.71 - Normal conditions of transport.

Code of Federal Regulations, 2011 CFR

2011-01-01

... NUCLEAR REGULATORY COMMISSION (CONTINUED) PACKAGING AND TRANSPORTATION OF RADIOACTIVE MATERIAL Package... each package design under normal conditions of transport must include a determination of the effect on... following table: Insolation Data Form and location of surface Total insolation for a 12-hour period(g cal...

10 CFR 71.71 - Normal conditions of transport.

Code of Federal Regulations, 2010 CFR

2010-01-01

... NUCLEAR REGULATORY COMMISSION (CONTINUED) PACKAGING AND TRANSPORTATION OF RADIOACTIVE MATERIAL Package... each package design under normal conditions of transport must include a determination of the effect on... following table: Insolation Data Form and location of surface Total insolation for a 12-hour period(g cal...

10 CFR 71.71 - Normal conditions of transport.

Code of Federal Regulations, 2013 CFR

2013-01-01

... NUCLEAR REGULATORY COMMISSION (CONTINUED) PACKAGING AND TRANSPORTATION OF RADIOACTIVE MATERIAL Package... each package design under normal conditions of transport must include a determination of the effect on... following table: Insolation Data Form and location of surface Total insolation for a 12-hour period(g cal...

CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion

PubMed Central

Lee, J.-M.; Ramos, E.M.; Lee, J.-H.; Gillis, T.; Mysore, J.S.; Hayden, M.R.; Warby, S.C.; Morrison, P.; Nance, M.; Ross, C.A.; Margolis, R.L.; Squitieri, F.; Orobello, S.; Di Donato, S.; Gomez-Tortosa, E.; Ayuso, C.; Suchowersky, O.; Trent, R.J.A.; McCusker, E.; Novelletto, A.; Frontali, M.; Jones, R.; Ashizawa, T.; Frank, S.; Saint-Hilaire, M.H.; Hersch, S.M.; Rosas, H.D.; Lucente, D.; Harrison, M.B.; Zanko, A.; Abramson, R.K.; Marder, K.; Sequeiros, J.; Paulsen, J.S.; Landwehrmeyer, G.B.; Myers, R.H.; MacDonald, M.E.; Durr, Alexandra; Rosenblatt, Adam; Frati, Luigi; Perlman, Susan; Conneally, Patrick M.; Klimek, Mary Lou; Diggin, Melissa; Hadzi, Tiffany; Duckett, Ayana; Ahmed, Anwar; Allen, Paul; Ames, David; Anderson, Christine; Anderson, Karla; Anderson, Karen; Andrews, Thomasin; Ashburner, John; Axelson, Eric; Aylward, Elizabeth; Barker, Roger A.; Barth, Katrin; Barton, Stacey; Baynes, Kathleen; Bea, Alexandra; Beall, Erik; Beg, Mirza Faisal; Beglinger, Leigh J.; Biglan, Kevin; Bjork, Kristine; Blanchard, Steve; Bockholt, Jeremy; Bommu, Sudharshan Reddy; Brossman, Bradley; Burrows, Maggie; Calhoun, Vince; Carlozzi, Noelle; Chesire, Amy; Chiu, Edmond; Chua, Phyllis; Connell, R.J.; Connor, Carmela; Corey-Bloom, Jody; Craufurd, David; Cross, Stephen; Cysique, Lucette; Santos, Rachelle Dar; Davis, Jennifer; Decolongon, Joji; DiPietro, Anna; Doucette, Nicholas; Downing, Nancy; Dudler, Ann; Dunn, Steve; Ecker, Daniel; Epping, Eric A.; Erickson, Diane; Erwin, Cheryl; Evans, Ken; Factor, Stewart A.; Farias, Sarah; Fatas, Marta; Fiedorowicz, Jess; Fullam, Ruth; Furtado, Sarah; Garde, Monica Bascunana; Gehl, Carissa; Geschwind, Michael D.; Goh, Anita; Gooblar, Jon; Goodman, Anna; Griffith, Jane; Groves, Mark; Guttman, Mark; Hamilton, Joanne; Harrington, Deborah; Harris, Greg; Heaton, Robert K.; Helmer, Karl; Henneberry, Machelle; Hershey, Tamara; Herwig, Kelly; Howard, Elizabeth; Hunter, Christine; Jankovic, Joseph; Johnson, Hans; Johnson, Arik; Jones, Kathy; Juhl, Andrew; Kim, Eun Young; Kimble, Mycah; King, Pamela; Klimek, Mary Lou; Klöppel, Stefan; Koenig, Katherine; Komiti, Angela; Kumar, Rajeev; Langbehn, Douglas; Leavitt, Blair; Leserman, Anne; Lim, Kelvin; Lipe, Hillary; Lowe, Mark; Magnotta, Vincent A.; Mallonee, William M.; Mans, Nicole; Marietta, Jacquie; Marshall, Frederick; Martin, Wayne; Mason, Sarah; Matheson, Kirsty; Matson, Wayne; Mazzoni, Pietro; McDowell, William; Miedzybrodzka, Zosia; Miller, Michael; Mills, James; Miracle, Dawn; Montross, Kelsey; Moore, David; Mori, Sasumu; Moser, David J.; Moskowitz, Carol; Newman, Emily; Nopoulos, Peg; Novak, Marianne; O'Rourke, Justin; Oakes, David; Ondo, William; Orth, Michael; Panegyres, Peter; Pease, Karen; Perlman, Susan; Perlmutter, Joel; Peterson, Asa; Phillips, Michael; Pierson, Ron; Potkin, Steve; Preston, Joy; Quaid, Kimberly; Radtke, Dawn; Rae, Daniela; Rao, Stephen; Raymond, Lynn; Reading, Sarah; Ready, Rebecca; Reece, Christine; Reilmann, Ralf; Reynolds, Norm; Richardson, Kylie; Rickards, Hugh; Ro, Eunyoe; Robinson, Robert; Rodnitzky, Robert; Rogers, Ben; Rosenblatt, Adam; Rosser, Elisabeth; Rosser, Anne; Price, Kathy; Price, Kathy; Ryan, Pat; Salmon, David; Samii, Ali; Schumacher, Jamy; Schumacher, Jessica; Sendon, Jose Luis Lópenz; Shear, Paula; Sheinberg, Alanna; Shpritz, Barnett; Siedlecki, Karen; Simpson, Sheila A.; Singer, Adam; Smith, Jim; Smith, Megan; Smith, Glenn; Snyder, Pete; Song, Allen; Sran, Satwinder; Stephan, Klaas; Stober, Janice; Sü?muth, Sigurd; Suter, Greg; Tabrizi, Sarah; Tempkin, Terry; Testa, Claudia; Thompson, Sean; Thomsen, Teri; Thumma, Kelli; Toga, Arthur; Trautmann, Sonja; Tremont, Geoff; Turner, Jessica; Uc, Ergun; Vaccarino, Anthony; van Duijn, Eric; Van Walsem, Marleen; Vik, Stacie; Vonsattel, Jean Paul; Vuletich, Elizabeth; Warner, Tom; Wasserman, Paula; Wassink, Thomas; Waterman, Elijah; Weaver, Kurt; Weir, David; Welsh, Claire; Werling-Witkoske, Chris; Wesson, Melissa; Westervelt, Holly; Weydt, Patrick; Wheelock, Vicki; Williams, Kent; Williams, Janet; Wodarski, Mary; Wojcieszek, Joanne; Wood, Jessica; Wood-Siverio, Cathy; Wu, Shuhua; Yastrubetskaya, Olga; de Yebenes, Justo Garcia; Zhao, Yong Qiang; Zimbelman, Janice; Zschiegner, Roland; Aaserud, Olaf; Abbruzzese, Giovanni; Andrews, Thomasin; Andrich, Jurgin; Antczak, Jakub; Arran, Natalie; Artiga, Maria J. Saiz; Bachoud-Lévi, Anne-Catherine; Banaszkiewicz, Krysztof; di Poggio, Monica Bandettini; Bandmann, Oliver; Barbera, Miguel A.; Barker, Roger A.; Barrero, Francisco; Barth, Katrin; Bas, Jordi; Beister, Antoine; Bentivoglio, Anna Rita; Bertini, Elisabetta; Biunno, Ida; Bjørgo, Kathrine; Bjørnevoll, Inga; Bohlen, Stefan; Bonelli, Raphael M.; Bos, Reineke; Bourne, Colin; Bradbury, Alyson; Brockie, Peter; Brown, Felicity; Bruno, Stefania; Bryl, Anna; Buck, Andrea; Burg, Sabrina; Burgunder, Jean-Marc; Burns, Peter; Burrows, Liz; Busquets, Nuria; Busse, Monica; Calopa, Matilde; Carruesco, Gemma T.; Casado, Ana Gonzalez; Catena, Judit López; Chu, Carol; Ciesielska, Anna; Clapton, Jackie; Clayton, Carole; Clenaghan, Catherine; Coelho, Miguel; Connemann, Julia; Craufurd, David; Crooks, Jenny; Cubillo, Patricia Trigo; Cubo, Esther; Curtis, Adrienne; De Michele, Giuseppe; De Nicola, A.; de Souza, Jenny; de Weert, A. Marit; de Yébenes, Justo Garcia; Dekker, M.; Descals, A. Martínez; Di Maio, Luigi; Di Pietro, Anna; Dipple, Heather; Dose, Matthias; Dumas, Eve M.; Dunnett, Stephen; Ecker, Daniel; Elifani, F.; Ellison-Rose, Lynda; Elorza, Marina D.; Eschenbach, Carolin; Evans, Carole; Fairtlough, Helen; Fannemel, Madelein; Fasano, Alfonso; Fenollar, Maria; Ferrandes, Giovanna; Ferreira, Jaoquim J.; Fillingham, Kay; Finisterra, Ana Maria; Fisher, K.; Fletcher, Amy; Foster, Jillian; Foustanos, Isabella; Frech, Fernando A.; Fullam, Robert; Fullham, Ruth; Gago, Miguel; García, RocioGarcía-Ramos; García, Socorro S.; Garrett, Carolina; Gellera, Cinzia; Gill, Paul; Ginestroni, Andrea; Golding, Charlotte; Goodman, Anna; Gørvell, Per; Grant, Janet; Griguoli, A.; Gross, Diana; Guedes, Leonor; BascuñanaGuerra, Monica; Guerra, Maria Rosalia; Guerrero, Rosa; Guia, Dolores B.; Guidubaldi, Arianna; Hallam, Caroline; Hamer, Stephanie; Hammer, Kathrin; Handley, Olivia J.; Harding, Alison; Hasholt, Lis; Hedge, Reikha; Heiberg, Arvid; Heinicke, Walburgis; Held, Christine; Hernanz, Laura Casas; Herranhof, Briggitte; Herrera, Carmen Durán; Hidding, Ute; Hiivola, Heli; Hill, Susan; Hjermind, Lena. E.; Hobson, Emma; Hoffmann, Rainer; Holl, Anna Hödl; Howard, Liz; Hunt, Sarah; Huson, Susan; Ialongo, Tamara; Idiago, Jesus Miguel R.; Illmann, Torsten; Jachinska, Katarzyna; Jacopini, Gioia; Jakobsen, Oda; Jamieson, Stuart; Jamrozik, Zygmunt; Janik, Piotr; Johns, Nicola; Jones, Lesley; Jones, Una; Jurgens, Caroline K.; Kaelin, Alain; Kalbarczyk, Anna; Kershaw, Ann; Khalil, Hanan; Kieni, Janina; Klimberg, Aneta; Koivisto, Susana P.; Koppers, Kerstin; Kosinski, Christoph Michael; Krawczyk, Malgorzata; Kremer, Berry; Krysa, Wioletta; Kwiecinski, Hubert; Lahiri, Nayana; Lambeck, Johann; Lange, Herwig; Laver, Fiona; Leenders, K.L.; Levey, Jamie; Leythaeuser, Gabriele; Lezius, Franziska; Llesoy, Joan Roig; Löhle, Matthias; López, Cristobal Diez-Aja; Lorenza, Fortuna; Loria, Giovanna; Magnet, Markus; Mandich, Paola; Marchese, Roberta; Marcinkowski, Jerzy; Mariotti, Caterina; Mariscal, Natividad; Markova, Ivana; Marquard, Ralf; Martikainen, Kirsti; Martínez, Isabel Haro; Martínez-Descals, Asuncion; Martino, T.; Mason, Sarah; McKenzie, Sue; Mechi, Claudia; Mendes, Tiago; Mestre, Tiago; Middleton, Julia; Milkereit, Eva; Miller, Joanne; Miller, Julie; Minster, Sara; Möller, Jens Carsten; Monza, Daniela; Morales, Blas; Moreau, Laura V.; Moreno, Jose L. López-Sendón; Münchau, Alexander; Murch, Ann; Nielsen, Jørgen E.; Niess, Anke; Nørremølle, Anne; Novak, Marianne; O'Donovan, Kristy; Orth, Michael; Otti, Daniela; Owen, Michael; Padieu, Helene; Paganini, Marco; Painold, Annamaria; Päivärinta, Markku; Partington-Jones, Lucy; Paterski, Laurent; Paterson, Nicole; Patino, Dawn; Patton, Michael; Peinemann, Alexander; Peppa, Nadia; Perea, Maria Fuensanta Noguera; Peterson, Maria; Piacentini, Silvia; Piano, Carla; Càrdenas, Regina Pons i; Prehn, Christian; Price, Kathleen; Probst, Daniela; Quarrell, Oliver; Quiroga, Purificacion Pin; Raab, Tina; Rakowicz, Maryla; Raman, Ashok; Raymond, Lucy; Reilmann, Ralf; Reinante, Gema; Reisinger, Karin; Retterstol, Lars; Ribaï, Pascale; Riballo, Antonio V.; Ribas, Guillermo G.; Richter, Sven; Rickards, Hugh; Rinaldi, Carlo; Rissling, Ida; Ritchie, Stuart; Rivera, Susana Vázquez; Robert, Misericordia Floriach; Roca, Elvira; Romano, Silvia; Romoli, Anna Maria; Roos, Raymond A.C.; Røren, Niini; Rose, Sarah; Rosser, Elisabeth; Rosser, Anne; Rossi, Fabiana; Rothery, Jean; Rudzinska, Monika; Ruíz, Pedro J. García; Ruíz, Belan Garzon; Russo, Cinzia Valeria; Ryglewicz, Danuta; Saft, Carston; Salvatore, Elena; Sánchez, Vicenta; Sando, Sigrid Botne; Šašinková, Pavla; Sass, Christian; Scheibl, Monika; Schiefer, Johannes; Schlangen, Christiane; Schmidt, Simone; Schöggl, Helmut; Schrenk, Caroline; Schüpbach, Michael; Schuierer, Michele; Sebastián, Ana Rojo; Selimbegovic-Turkovic, Amina; Sempolowicz, Justyna; Silva, Mark; Sitek, Emilia; Slawek, Jaroslaw; Snowden, Julie; Soleti, Francesco; Soliveri, Paola; Sollom, Andrea; Soltan, Witold; Sorbi, Sandro; Sorensen, Sven Asger; Spadaro, Maria; Städtler, Michael; Stamm, Christiane; Steiner, Tanja; Stokholm, Jette; Stokke, Bodil; Stopford, Cheryl; Storch, Alexander; Straßburger, Katrin; Stubbe, Lars; Sulek, Anna; Szczudlik, Andrzej; Tabrizi, Sarah; Taylor, Rachel; Terol, Santiago Duran-Sindreu; Thomas, Gareth; Thompson, Jennifer; Thomson, Aileen; Tidswell, Katherine; Torres, Maria M. Antequera; Toscano, Jean; Townhill, Jenny; Trautmann, Sonja; Tucci, Tecla; Tuuha, Katri; Uhrova, Tereza; Valadas, Anabela; van Hout, Monique S.E.; van Oostrom, J.C.H.; van Vugt, Jeroen P.P.; vanm, Walsem Marleen R.; Vandenberghe, Wim; Verellen-Dumoulin, Christine; Vergara, Mar Ruiz; Verstappen, C.C.P.; Verstraelen, Nichola; Viladrich, Celia Mareca; Villanueva, Clara; Wahlström, Jan; Warner, Thomas; Wehus, Raghild; Weindl, Adolf; Werner, Cornelius J.; Westmoreland, Leann; Weydt, Patrick; Wiedemann, Alexandra; Wild, Edward; Wild, Sue; Witjes-Ané, Marie-Noelle; Witkowski, Grzegorz; Wójcik, Magdalena; Wolz, Martin; Wolz, Annett; Wright, Jan; Yardumian, Pam; Yates, Shona; Yudina, Elizaveta; Zaremba, Jacek; Zaugg, Sabine W.; Zdzienicka, Elzbieta; Zielonka, Daniel; Zielonka, Euginiusz; Zinzi, Paola; Zittel, Simone; Zucker, Birgrit; Adams, John; Agarwal, Pinky; Antonijevic, Irina; Beck, Christopher; Chiu, Edmond; Churchyard, Andrew; Colcher, Amy; Corey-Bloom, Jody; Dorsey, Ray; Drazinic, Carolyn; Dubinsky, Richard; Duff, Kevin; Factor, Stewart; Foroud, Tatiana; Furtado, Sarah; Giuliano, Joe; Greenamyre, Timothy; Higgins, Don; Jankovic, Joseph; Jennings, Dana; Kang, Un Jung; Kostyk, Sandra; Kumar, Rajeev; Leavitt, Blair; LeDoux, Mark; Mallonee, William; Marshall, Frederick; Mohlo, Eric; Morgan, John; Oakes, David; Panegyres, Peter; Panisset, Michel; Perlman, Susan; Perlmutter, Joel; Quaid, Kimberly; Raymond, Lynn; Revilla, Fredy; Robertson, Suzanne; Robottom, Bradley; Sanchez-Ramos, Juan; Scott, Burton; Shannon, Kathleen; Shoulson, Ira; Singer, Carlos; Tabbal, Samer; Testa, Claudia; van, Kammen Dan; Vetter, Louise; Walker, Francis; Warner, John; Weiner, illiam; Wheelock, Vicki; Yastrubetskaya, Olga; Barton, Stacey; Broyles, Janice; Clouse, Ronda; Coleman, Allison; Davis, Robert; Decolongon, Joji; DeLaRosa, Jeanene; Deuel, Lisa; Dietrich, Susan; Dubinsky, Hilary; Eaton, Ken; Erickson, Diane; Fitzpatrick, Mary Jane; Frucht, Steven; Gartner, Maureen; Goldstein, Jody; Griffith, Jane; Hickey, Charlyne; Hunt, Victoria; Jaglin, Jeana; Klimek, Mary Lou; Lindsay, Pat; Louis, Elan; Loy, Clemet; Lucarelli, Nancy; Malarick, Keith; Martin, Amanda; McInnis, Robert; Moskowitz, Carol; Muratori, Lisa; Nucifora, Frederick; O'Neill, Christine; Palao, Alicia; Peavy, Guerry; Quesada, Monica; Schmidt, Amy; Segro, Vicki; Sperin, Elaine; Suter, Greg; Tanev, Kalo; Tempkin, Teresa; Thiede, Curtis; Wasserman, Paula; Welsh, Claire; Wesson, Melissa; Zauber, Elizabeth

2012-01-01

Objective: Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs. Methods: We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression. Results: An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele. Conclusions: Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors. Neurology® 2012;78:690–695 PMID:22323755

Effect of the callipyge phenotype and cooking method on tenderness of several major lamb muscles.

PubMed

Shackelford, S D; Wheeler, T L; Koohmaraie, M

1997-08-01

We conducted three experiments to determine the effects of the callipyge phenotype on the tenderness of several major lamb muscles and to determine the effect of method of cookery on the tenderness of callipyge lamb at 7 d postmortem. In Exp. 1, chops from normal (n = 23) and callipyge (n = 16) carcasses were open-hearth-broiled. Warner-Bratzler shear force values of longissimus, gluteus medius, semimembranosus, biceps femoris, semitendinosus, adductor, and quadriceps femoris were 123, 44, 28, 26, 19, 16, and 13% greater, respectively, for callipyge (P < .05). In Exp. 2, muscles from normal (n = 18) and callipyge (n = 18) carcasses were oven-roasted. Shear force of triceps brachii was 11% greater for callipyge (P < .001); however, phenotype did not affect shear force of supraspinatus (P = .87) or psoas major (P = .64). In Exp. 3, a trained sensory panel evaluated leg roasts and open-hearth-broiled leg chops from normal (n = 60) and callipyge lamb carcasses (n = 60). Callipyge chops were less tender than normal chops (P < .05). Regardless of callipyge phenotype, muscles were more (P < .05) tender when roasted; however, the effect of method of cookery on tenderness scores was greater for callipyge muscles than for normal muscles. Callipyge roasts and normal roasts had similar tenderness (P = .58), and callipyge roasts were more tender than normal chops (P < .05). Regardless of cooking method, callipyge samples were less juicy than normal samples (P < .05). These data demonstrate that the callipyge phenotype will likely reduce consumer satisfaction due to reduced tenderness and juiciness; however, reduced tenderness in callipyge leg muscles could be prevented by ovenroasting.

Evaluating Material Flammability in Microgravity and Martian Gravity Compared to the NASA Standard Normal Gravity Test

NASA Technical Reports Server (NTRS)

Oslon, Sandra. L.; Ferkul, Paul

2012-01-01

Drop tower tests are conducted at Martian gravity to determine the flammability of three materials compared to previous tests in other normal gravity and reduced gravity environments. The comparison is made with consideration of a modified NASA standard test protocol. Material flammability limits in the different gravity and flow environments are tabulated to determine the factor of safety associated with normal gravity flammability screening. Previous testing at microgravity and Lunar gravity indicated that some materials burned to lower oxygen concentrations in low gravity than in normal gravity, although the low g extinction limit criteria are not the same as 1g due to time constraints in drop testing. Similarly, the data presented in this paper for Martian gravity suggest that there is a gravity level below Earth s at which materials burn more readily than on Earth. If proven for more materials, this may indicate the need to include a factor of safety on 1g flammability limits.

Economics of Future Growth in Photovoltaics Manufacturing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basore, Paul A.; Chung, Donald; Buonassisi, Tonio

2015-06-14

The past decade's record of growth in the photovoltaics manufacturing industry indicates that global investment in manufacturing capacity for photovoltaic modules tends to increase in proportion to the size of the industry. The slope of this proportionality determines how fast the industry will grow in the future. Two key parameters determine this slope. One is the annual global investment in manufacturing capacity normalized to the manufacturing capacity for the previous year (capacity-normalized capital investment rate, CapIR, units $/W). The other is how much capital investment is required for each watt of annual manufacturing capacity, normalized to the service life ofmore » the assets (capacity-normalized capital demand rate, CapDR, units $/W). If these two parameters remain unchanged from the values they have held for the past few years, global manufacturing capacity will peak in the next few years and then decline. However, it only takes a small improvement in CapIR to ensure future growth in photovoltaics. Any accompanying improvement in CapDR will accelerate that growth.« less

Thorium normalization as a hydrocarbon accumulation indicator for Lower Miocene rocks in Ras Ghara area, Gulf of Suez, Egypt

NASA Astrophysics Data System (ADS)

El-Khadragy, A. A.; Shazly, T. F.; AlAlfy, I. M.; Ramadan, M.; El-Sawy, M. Z.

2018-06-01

An exploration method has been developed using surface and aerial gamma-ray spectral measurements in prospecting petroleum in stratigraphic and structural traps. The Gulf of Suez is an important region for studying hydrocarbon potentiality in Egypt. Thorium normalization technique was applied on the sandstone reservoirs in the region to determine the hydrocarbon potentialities zones using the three spectrometric radioactive gamma ray-logs (eU, eTh and K% logs). This method was applied on the recorded gamma-ray spectrometric logs for Rudeis and Kareem Formations in Ras Ghara oil Field, Gulf of Suez, Egypt. The conventional well logs (gamma-ray, resistivity, neutron, density and sonic logs) were analyzed to determine the net pay zones in the study area. The agreement ratios between the thorium normalization technique and the results of the well log analyses are high, so the application of thorium normalization technique can be used as a guide for hydrocarbon accumulation in the study reservoir rocks.

Normalized stiffness ratios for mechanical characterization of isotropic acoustic foams.

PubMed

Sahraoui, Sohbi; Brouard, Bruno; Benyahia, Lazhar; Parmentier, Damien; Geslain, Alan

2013-12-01

This paper presents a method for the mechanical characterization of isotropic foams at low frequency. The objective of this study is to determine the Young's modulus, the Poisson's ratio, and the loss factor of commercially available foam plates. The method is applied on porous samples having square and circular sections. The main idea of this work is to perform quasi-static compression tests of a single foam sample followed by two juxtaposed samples having the same dimensions. The load and displacement measurements lead to a direct extraction of the elastic constants by means of normalized stiffness and normalized stiffness ratio which depend on Poisson's ratio and shape factor. The normalized stiffness is calculated by the finite element method for different Poisson ratios. The no-slip boundary conditions imposed by the loading rigid plates create interfaces with a complex strain distribution. Beforehand, compression tests were performed by means of a standard tensile machine in order to determine the appropriate pre-compression rate for quasi-static tests.

Discourse changes in early Alzheimer disease, mild cognitive impairment, and normal aging.

PubMed

Chapman, Sandra Bond; Zientz, Jennifer; Weiner, Myron; Rosenberg, Roger; Frawley, William; Burns, Mary Hope

2002-01-01

The purpose of this study was to determine the sensitivity of discourse gist measures to the early cognitive-linguistic changes in Alzheimer disease (AD) and in the preclinical stages. Differences in discourse abilities were examined in 25 cognitively normal adults, 24 adults with mild probable AD, and 20 adults with mild cognitive impairment (MCI) at gist and detail levels of discourse processing. The authors found that gist and detail levels of discourse processing were significantly impaired in persons with AD and MCI as compared with normal control subjects. Gist-level discourse processing abilities showed minimal overlap between cognitively normal control subjects and those with mild AD. Moreover, the majority of the persons with MCI performed in the range of AD on gist measures. These findings indicate that discourse gist measures hold promise as a diagnostic complement to enhance early detection of AD. Further studies are needed to determine how early the discourse gist deficits arise in AD.

Normalization of a collimated 14.7 MeV neutron source in a neutron spectrometry system for benchmark experiments

NASA Astrophysics Data System (ADS)

Ofek, R.; Tsechanski, A.; Shani, G.

1988-05-01

In the present study a method used to normalize a collimated 14.7 MeV neutron beam is introduced. It combined a measurement of the fast neutron scalar flux passing through the collimator, using a copper foil activation, with a neutron transport calculation of the foil activation per unit source neutron, carried out by the discrete-ordinates transport code DOT 4.2. The geometry of the collimated neutron beam is composed of a D-T neutron source positioned 30 cm in front of a 6 cm diameter collimator, through a 120 cm thick paraffin wall. The neutron flux emitted from the D-T source was counted by an NE-213 scintillator, simultaneously with the irradiation of the copper foil. Thus, the determination of the normalization factor of the D-T source is used for an absolute flux calibration of the NE-213 scintillator. The major contributions to the uncertainty in the determination of the normalization factor, and their origins, are discussed.

Otolith and Vertical Canal Contributions to Dynamic Postural Control

NASA Technical Reports Server (NTRS)

Black, F. Owen

1999-01-01

The objective of this project is to determine: 1) how do normal subjects adjust postural movements in response to changing or altered otolith input, for example, due to aging? and 2) how do patients adapt postural control after altered unilateral or bilateral vestibular sensory inputs such as ablative inner ear surgery or ototoxicity, respectively? The following hypotheses are under investigation: 1) selective alteration of otolith input or abnormalities of otolith receptor function will result in distinctive spatial, frequency, and temporal patterns of head movements and body postural sway dynamics. 2) subjects with reduced, altered, or absent vertical semicircular canal receptor sensitivity but normal otolith receptor function or vice versa, should show predictable alterations of body and head movement strategies essential for the control of postural sway and movement. The effect of altered postural movement control upon compensation and/or adaptation will be determined. These experiments provide data for the development of computational models of postural control in normals, vestibular deficient subjects and normal humans exposed to unusual force environments, including orbital space flight.

Using the manual ability classification system in young adults with cerebral palsy and normal intelligence.

PubMed

van Meeteren, Jetty; Nieuwenhuijsen, Channah; de Grund, Arthur; Stam, Henk J; Roebroeck, Marij E

2010-01-01

The study aimed to establish whether the manual ability classification system (MACS), a valid classification system for manual ability in children with cerebral palsy (CP), is applicable in young adults with CP and normal intelligence. The participants (n = 83) were young adults with CP and normal intelligence and had a mean age of 19.9 years. In this study, inter observer reliability of the MACS was determined. We investigated relationships between the MACS level and patient characteristics (such as the gross motor function classification system (GMFCS) level, limb distribution of the spastic paresis and educational level) and with functional activities of the upper extremity (assessed with the Melbourne assessment, the Abilhand questionnaire and the domain self-care of the functional independence measure (FIM)). Furthermore, with a linear regression analysis it was determined whether the MACS is a significant determinant of activity limitations and participation restrictions. The reliability was good (intraclass correlation coefficient 0.83). The Spearman correlation coefficients with GMFCS level, limb distribution of the spastic paresis and educational level were 0.53, 0.46, and 0.26, respectively. MACS level correlated moderately with outcome measures of functional activities (correlations ranging from -0.38 to -0.55). MACS level is, in addition to the GMFCS level, an important determinant for limitations in activities and restrictions in participation. We conclude that the MACS is a feasible method to classify manual ability in young adults with CP and normal intelligence with a good manual ability.

Normal IQ is possible in Smith-Lemli-Opitz syndrome.

PubMed

Eroglu, Yasemen; Nguyen-Driver, Mina; Steiner, Robert D; Merkens, Louise; Merkens, Mark; Roullet, Jean-Baptiste; Elias, Ellen; Sarphare, Geeta; Porter, Forbes D; Li, Chumei; Tierney, Elaine; Nowaczyk, Małgorzata J; Freeman, Kurt A

2017-08-01

Children with Smith-Lemli-Opitz syndrome (SLOS) are typically reported to have moderate to severe intellectual disability. This study aims to determine whether normal cognitive function is possible in this population and to describe clinical, biochemical and molecular characteristics of children with SLOS and normal intelligent quotient (IQ). The study included children with SLOS who underwent cognitive testing in four centers. All children with at least one IQ composite score above 80 were included in the study. Six girls, three boys with SLOS were found to have normal or low-normal IQ in a cohort of 145 children with SLOS. Major/multiple organ anomalies and low serum cholesterol levels were uncommon. No correlation with IQ and genotype was evident and no specific developmental profile were observed. Thus, normal or low-normal cognitive function is possible in SLOS. Further studies are needed to elucidate factors contributing to normal or low-normal cognitive function in children with SLOS. © 2017 Wiley Periodicals, Inc.

System for determining the angle of impact of an object on a structure

NASA Technical Reports Server (NTRS)

Prosser, William H. (Inventor); Gorman, Michael R. (Inventor)

1993-01-01

A method for determining the angle of impact of an object on a thin-walled structure which determines the angle of impact through analysis of the acoustic waves which result when an object impacts a structure is presented. Transducers are placed on and in the surface of the structure which sense the wave caused in the structure by impact. The waves are recorded and saved for analysis. For source motion normal to the surface, the antisymmetric mode has a large amplitude while that of the symmetric mode is very small. As the source angle increases with respect to the surface normal, the symmetric mode amplitude increases while the antisymmetric mode amplitude decreases. Thus, the angle of impact is determined by measuring the relative amplitudes of these two lowest order modes.

Gene Expression Profile Analysis as a Prognostic Indicator of Normal Tissue Response to Simulated Space Radiations

NASA Technical Reports Server (NTRS)

Story, Michael; Stivers, David N.

2004-01-01

This project was funded as a pilot project to determine the feasibility of using gene expression profiles to characterize the response of human cells to exposure to particulate radiations such as those encountered in the spaceflight environment. We proposed to use microarray technology to examine the gene expression patterns of a bank of well-characterized human fibroblast cell cultures. These fibroblast cultures were derived from breast or head and neck cancer patients who exhibited normal, minimal, or severe normal tissue reactions following low LET radiation exposure via radiotherapy. Furthermore, determination of SF2 values from fibroblasts cultured from these individuals were predictive of risk for severe late reactions. We hypothesized that by determining the expression of thousands of genes we could identify gene expression patterns that reflect how normal tissues respond to high Z and energy (HZE) particles, that is, that there are molecular signatures for HZE exposures. We also hypothesized that individuals who are intrinsically radiosensitive may elicit a unique response. Because this was funded as a pilot project we focused our initial studies on logistics and appropriate experimental design, and then to test our hypothesis that there is a unique molecular response to specific particles, in this case C and Fe, for primary human skin fibroblasts.

Quaternion normalization in spacecraft attitude determination

NASA Technical Reports Server (NTRS)

Deutschmann, J.; Markley, F. L.; Bar-Itzhack, Itzhack Y.

1993-01-01

Attitude determination of spacecraft usually utilizes vector measurements such as Sun, center of Earth, star, and magnetic field direction to update the quaternion which determines the spacecraft orientation with respect to some reference coordinates in the three dimensional space. These measurements are usually processed by an extended Kalman filter (EKF) which yields an estimate of the attitude quaternion. Two EKF versions for quaternion estimation were presented in the literature; namely, the multiplicative EKF (MEKF) and the additive EKF (AEKF). In the multiplicative EKF, it is assumed that the error between the correct quaternion and its a-priori estimate is, by itself, a quaternion that represents the rotation necessary to bring the attitude which corresponds to the a-priori estimate of the quaternion into coincidence with the correct attitude. The EKF basically estimates this quotient quaternion and then the updated quaternion estimate is obtained by the product of the a-priori quaternion estimate and the estimate of the difference quaternion. In the additive EKF, it is assumed that the error between the a-priori quaternion estimate and the correct one is an algebraic difference between two four-tuple elements and thus the EKF is set to estimate this difference. The updated quaternion is then computed by adding the estimate of the difference to the a-priori quaternion estimate. If the quaternion estimate converges to the correct quaternion, then, naturally, the quaternion estimate has unity norm. This fact was utilized in the past to obtain superior filter performance by applying normalization to the filter measurement update of the quaternion. It was observed for the AEKF that when the attitude changed very slowly between measurements, normalization merely resulted in a faster convergence; however, when the attitude changed considerably between measurements, without filter tuning or normalization, the quaternion estimate diverged. However, when the quaternion estimate was normalized, the estimate converged faster and to a lower error than with tuning only. In last years, symposium we presented three new AEKF normalization techniques and we compared them to the brute force method presented in the literature. The present paper presents the issue of normalization of the MEKF and examines several MEKF normalization techniques.

Does osteoporosis reduce the primary tilting stability of cementless acetabular cups?

PubMed

von Schulze Pellengahr, Christoph; von Engelhardt, Lars V; Wegener, Bernd; Müller, Peter E; Fottner, Andreas; Weber, Patrick; Ackermann, Ole; Lahner, Matthias; Teske, Wolfram

2015-04-21

Cementless hip cups need sufficient primary tilting stability to achieve osseointegration. The aim of the study was to assess differences of the primary implant stability in osteoporotic bone and in bone with normal bone density. To assess the influence of different cup designs, two types of threaded and two types of press-fit cups were tested. The maximum tilting moment for two different cementless threaded cups and two different cementless press-fit cups was determined in macerated human hip acetabuli with reduced (n=20) and normal bone density (n=20), determined using Q-CT. The tilting moments for each cup were determined five times in the group with reduced bone density and five times in the group with normal bone density, and the respective average values were calculated. The mean maximum extrusion force of the threaded cup Zintra was 5670.5 N (max. tilting moment 141.8 Nm) in bone with normal density and.5748.3 N (max. tilting moment 143.7 Nm) in osteoporotic bone. For the Hofer Imhof (HI) threaded cup it was 7681.5 N (192.0 Nm) in bone with normal density and 6828.9 N (max. tilting moment 170.7 Nm) in the group with osteoporotic bone. The mean maximum extrusion force of the macro-textured press-fit cup Metallsockel CL was 3824.6 N (max. tilting moment 95.6 Nm) in bone with normal and 2246.2 N (max. tilting moment 56.2 Nm) in osteoporotic bone. For the Monoblock it was 1303.8 N (max. tilting moment 32.6 Nm) in normal and 1317 N (max. tilting moment 32.9 Nm) in osteoporotic bone. There was no significance. A reduction of the maximum tilting moment in osteoporotic bone of the ESKA press-fit cup Metallsockel CL was noticed. Results on macerated bone specimens showed no statistically significant reduction of the maximum tilting moment in specimens with osteoporotic bone density compared to normal bone, neither for threaded nor for the press-fit cups. With the limitation that the results were obtained using macerated bone, we could not detect any restrictions for the clinical indication of the examined cementless cups in osteoporotic bone.

Effects of water immersion on plasma catecholamines in normal humans

NASA Technical Reports Server (NTRS)

Epstein, M.; Johnson, G.; Denunzio, A. G.

1983-01-01

An investigation was conducted in order to determine whether water immersion to the neck (NI) alters plasma catecholamines in normal humans. Eight normal subjects were studied during a seated control study (C) and during 4 hr of NI, and the levels of norepinephrine (NE) and epinephrine (E) as determined by radioenzymatic assay were measured hourly. Results show that despite the induction of a marked natriuresis and diuresis indicating significant central hypervolemia, NI failed to alter plasma NE or E levels compared with those of either C or the corresponding prestudy 1.5 hr. In addition, the diuresis and natriuresis was found to vary independently of NE. These results indicate that the response of the sympathetic nervous system to acute volume alteration may differ from the reported response to chronic volume expansion.

Edge technique for measurement of laser frequency shifts including the Doppler shift

NASA Technical Reports Server (NTRS)

Korb, Larry (Inventor)

1991-01-01

A method is disclosed for determining the frequency shift in a laser system by transmitting an outgoing laser beam. An incoming laser beam having a frequency shift is received. A first signal is acquired by transmitting a portion of the incoming laser beam to an energy monitor detector. A second signal is acquired by transmitting a portion of the incoming laser beam through an edge filter to an edge detector, which derives a first normalized signal which is proportional to the transmission of the edge filter at the frequency of the incoming laser beam. A second normalized signal is acquired which is proportional to the transmission of the edge filter at the frequency of the outgoing laser beam. The frequency shift is determined by processing the first and second normalized signals.

Telomere length in normal and neoplastic canine tissues.

PubMed

Cadile, Casey D; Kitchell, Barbara E; Newman, Rebecca G; Biller, Barbara J; Hetler, Elizabeth R

2007-12-01

To determine the mean telomere restriction fragment (TRF) length in normal and neoplastic canine tissues. 57 solid-tissue tumor specimens collected from client-owned dogs, 40 samples of normal tissue collected from 12 clinically normal dogs, and blood samples collected from 4 healthy blood donor dogs. Tumor specimens were collected from client-owned dogs during diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital, whereas 40 normal tissue samples were collected from 12 control dogs. Telomere restriction fragment length was determined by use of an assay kit. A histologic diagnosis was provided for each tumor by personnel at the Veterinary Diagnostic Laboratory at the University of Illinois. Mean of the mean TRF length for 44 normal samples was 19.0 kilobases (kb; range, 15.4 to 21.4 kb), and the mean of the mean TRF length for 57 malignant tumors was 19.0 kb (range, 12.9 to 23.5 kb). Although the mean of the mean TRF length for tumors and normal tissues was identical, tumor samples had more variability in TRF length. Telomerase, which represents the main mechanism by which cancer cells achieve immortality, is an attractive therapeutic target. The ability to measure telomere length is crucial to monitoring the efficacy of telomerase inhibition. In contrast to many other mammalian species, the length of canine telomeres and the rate of telomeric DNA loss are similar to those reported in humans, making dogs a compelling choice for use in the study of human anti-telomerase strategies.

Reduced β-Cell Secretory Capacity in Pancreatic-Insufficient, but Not Pancreatic-Sufficient, Cystic Fibrosis Despite Normal Glucose Tolerance.

PubMed

Sheikh, Saba; Gudipaty, Lalitha; De Leon, Diva D; Hadjiliadis, Denis; Kubrak, Christina; Rosenfeld, Nora K; Nyirjesy, Sarah C; Peleckis, Amy J; Malik, Saloni; Stefanovski, Darko; Cuchel, Marina; Rubenstein, Ronald C; Kelly, Andrea; Rickels, Michael R

2017-01-01

Patients with pancreatic-insufficient cystic fibrosis (PI-CF) are at increased risk for developing diabetes. We determined β-cell secretory capacity and insulin secretory rates from glucose-potentiated arginine and mixed-meal tolerance tests (MMTTs), respectively, in pancreatic-sufficient cystic fibrosis (PS-CF), PI-CF, and normal control subjects, all with normal glucose tolerance, in order to identify early pathophysiologic defects. Acute islet cell secretory responses were determined under fasting, 230 mg/dL, and 340 mg/dL hyperglycemia clamp conditions. PI-CF subjects had lower acute insulin, C-peptide, and glucagon responses compared with PS-CF and normal control subjects, indicating reduced β-cell secretory capacity and α-cell function. Fasting proinsulin-to-C-peptide and proinsulin secretory ratios during glucose potentiation were higher in PI-CF, suggesting impaired proinsulin processing. In the first 30 min of the MMTT, insulin secretion was lower in PI-CF compared with PS-CF and normal control subjects, and glucagon-like peptide 1 and gastric inhibitory polypeptide were lower compared with PS-CF, and after 180 min, glucose was higher in PI-CF compared with normal control subjects. These findings indicate that despite "normal" glucose tolerance, adolescents and adults with PI-CF have impairments in functional islet mass and associated early-phase insulin secretion, which with decreased incretin responses likely leads to the early development of postprandial hyperglycemia in CF. © 2017 by the American Diabetes Association.

Discordances between follicle stimulating hormone (FSH) and anti-Müllerian hormone (AMH) in female infertility

PubMed Central

2010-01-01

Background Follicle stimulating hormone (FSH) and anti-Müllerian hormone (AMH) represent the two most frequently utilized laboratory tests in determining ovarian reserve (OR). This study determined the clinical significance of their concordance and discordance in female infertility patients. Methods We investigated 366 consecutive infertility patients (350 reached IVF), excluding women with polycystic ovarian syndrome (PCOS). They were considered to have normal FSH and AMH if values fell within age-specific (as-) 95% confidence intervals (CI), and to suffer from diminished ovarian reserve (DOR) if FSH exceeded and/or AMH fell below those. The two hormones, thus, could be concordant (Group I), both normal (IA) or abnormal (IB), show normal AMH/abnormal FSH (Group II) or normal FSH/abnormal AMH (Group III). Oocyte yields, stratified for age categories, were then studied in each group as reflection of OR. Results Oocyte yields significantly decreased from groups IA to II to III and IB. Predictive values of as-FSH/AMH patterns changed, however, at different ages. Except at very young and very old ages, normal as-AMH better predicted higher oocytes yields than normal as-FSH, though above age 42 years normal as-FSH predicts good oocyte yields even with abnormally low AMH. Under age 42 discrepancies between as- FSH and as-AMH remain similarly predictive of oocyte yields at all ages. Discussion Concordances and discordances between as-FSH and as-AMH improve OR assessments and predictability of oocyte yields in IVF. PMID:20565808

Multivariate normality

NASA Technical Reports Server (NTRS)

Crutcher, H. L.; Falls, L. W.

1976-01-01

Sets of experimentally determined or routinely observed data provide information about the past, present and, hopefully, future sets of similarly produced data. An infinite set of statistical models exists which may be used to describe the data sets. The normal distribution is one model. If it serves at all, it serves well. If a data set, or a transformation of the set, representative of a larger population can be described by the normal distribution, then valid statistical inferences can be drawn. There are several tests which may be applied to a data set to determine whether the univariate normal model adequately describes the set. The chi-square test based on Pearson's work in the late nineteenth and early twentieth centuries is often used. Like all tests, it has some weaknesses which are discussed in elementary texts. Extension of the chi-square test to the multivariate normal model is provided. Tables and graphs permit easier application of the test in the higher dimensions. Several examples, using recorded data, illustrate the procedures. Tests of maximum absolute differences, mean sum of squares of residuals, runs and changes of sign are included in these tests. Dimensions one through five with selected sample sizes 11 to 101 are used to illustrate the statistical tests developed.

A novel automated instrument designed to determine photosensitivity thresholds (Conference Presentation)

NASA Astrophysics Data System (ADS)

Aguilar, Mariela C.; Gonzalez, Alex; Rowaan, Cornelis; De Freitas, Carolina; Rosa, Potyra R.; Alawa, Karam; Lam, Byron L.; Parel, Jean-Marie A.

2016-03-01

As there is no clinically available instrument to systematically and reliably determine the photosensitivity thresholds of patients with dry eyes, blepharospasms, migraines, traumatic brain injuries, and genetic disorders such as Achromatopsia, retinitis pigmentosa and other retinal dysfunctions, a computer-controlled optoelectronics system was designed. The BPEI Photosensitivity System provides a light stimuli emitted from a bi-cupola concave, 210 white LED array with varying intensity ranging from 1 to 32,000 lux. The system can either utilize a normal or an enhanced testing mode for subjects with low light tolerance. The automated instrument adjusts the intensity of each light stimulus. The subject is instructed to indicate discomfort by pressing a hand-held button. Reliability of the responses is tracked during the test. The photosensitivity threshold is then calculated after 10 response reversals. In a preliminary study, we demonstrated that subjects suffering from Achromatopsia experienced lower photosensitivity thresholds than normal subjects. Hence, the system can safely and reliably determine the photosensitivity thresholds of healthy and light sensitive subjects by detecting and quantifying the individual differences. Future studies will be performed with this system to determine the photosensitivity threshold differences between normal subjects and subjects suffering from other conditions that affect light sensitivity.

28 CFR 42.712 - Exception; normal operation or statutory objective.

Code of Federal Regulations, 2014 CFR

2014-07-01

... objective. 42.712 Section 42.712 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION; EQUAL... Programs or Activities; Implementation of the Age Discrimination Act of 1975 Standards for Determining Age Discrimination § 42.712 Exception; normal operation or statutory objective. (a) A recipient may take an action...

Analysis of the Astronomy Diagnostic Test

ERIC Educational Resources Information Center

Brogt, Erik; Sabers, Darrell; Prather, Edward E.; Deming, Grace L.; Hufnagel, Beth; Slater, Timothy F.

2007-01-01

Seventy undergraduate class sections were examined from the database of Astronomy Diagnostic Test (ADT) results of Deming and Hufnagel to determine if course format correlated with ADT normalized gain scores. Normalized gains were calculated for four different classroom scenarios: lecture, lecture with discussion, lecture with lab, and lecture…

Endothelial function in youth: A biomarker modulated by adiposity-related insulin resistance

USDA-ARS?s Scientific Manuscript database

To investigate the physical and metabolic determinants of endothelial dysfunction, an early marker of subclinical atherosclerosis, in normal weight and overweight adolescents with and without type 2 diabetes mellitus. A cross-sectional study of 81 adolescents: 21 normal weight, 25 overweight with no...

Antecedents of cell aging research.

PubMed

Hayflick, L

1989-01-01

Our observation that normal human and animal cells have a limited capacity to divide and function in vitro overturned a dogma held since the turn of the century. The dogma held that cultured normal cells are immortal and gerontologists interpreted this to mean that aging, therefore, could not be the result of intracellular events. We concluded that longevity and aging do result from intracellular events, and, in the subsequent 30 years, the validity of our finding has been widely confirmed. Other major findings have been made: (a) The number of population doublings and functional events that a cultured normal cell can undergo is inversely proportional to donor age and, probably, directly proportional to species longevity; (b) the limit on cell division and function also occurs in vivo when normal cells are transplanted seriatim; (c) as cell doublings or functional events reach their limit, changes occur in hundreds of variables from the molecular to the whole cell. Most importantly, many of these changes are identical to those seen in intact humans and animals as they age; (d) WI-38, the first widely distributed normal human cell strain has retained its memory of population doubling level during 27 years of cryogenic storage. This is the longest time that any normal human cell has ever been preserved. Evidence that longevity is determined by genetic events is overwhelming but evidence that age changes are the result of gene expression is not. Normal age changes must be distinguished from disease. Because few feral animals ever become old, natural selection could not have favored the development of a genetically programmed aging process. In the 2 or 3 million years of human existence, too few old humans existed to have provided a selective advantage favoring the development of a genetic program that would determine age changes. The selective advantage of maintaining physiological vigor for as long as possible in order to insure maximum reproductive success may be the essential indirect determinant of longevity. Natural selection has provided sexually mature animals with extraordinary reserve capacities in virtually all organs. After sexual maturation, animals continue to function by utilizing the reserve capacity that evolved to insure that they would attain reproductive success. The magnitude of reserve capacity is the essential element in determining postdevelopmental longevity. Thus "Why do we age?" may be the wrong question. The right question may be "Why do we live as long as we do?"

Histologic Normalization Occurs in Ulcerative Colitis and Is Associated With Improved Clinical Outcomes.

PubMed

Christensen, Britt; Hanauer, Stephen B; Erlich, Jonathan; Kassim, Olufemi; Gibson, Peter R; Turner, Jerrold R; Hart, John; Rubin, David T

2017-10-01

Mucosal healing, determined by histologic analysis, is a potential therapeutic target for patients with ulcerative colitis (UC). However, the histologic features of tissue normalization, as an outcome of treatment, have not been well described. We examined the prevalence and predictive values of normalization of the colonic mucosa, based on histologic analysis (histologic normalization) in patients with UC, and determined its association with risk of clinical relapse, compared with histologic disease quiescence and endoscopic mucosal healing. We performed a retrospective study of 646 patients with confirmed UC who underwent colonoscopy at a tertiary medical center from August 2005 through October 2013. We reviewed reports from pathology analyses of random mucosal biopsies from each colon segment, and categorized them into 3 groups based on histology findings: (1) normalization (completely normal mucosa with no features of chronicity present), (2) quiescence (crypt atrophy or branching without signs of active inflammation including erosions, abscesses, or focal neutrophil infiltration), or (3) active disease (epithelial infiltration by neutrophils, crypt abscesses, erosions, or ulceration). Histology findings were compared with clinical and endoscopic findings. We assessed variables associated with histology findings and, in patients in clinical remission (Simple Clinical Colitis Activity Index score ≤2 and subscore of ≤1 for stool frequency or rectal bleeding), predictive values for clinical relapse at follow-up evaluations 6 months later or more were calculated. Of the 646 patients included in the study, 60% had endoscopic mucosal healing, 40% had histologic quiescence, and 10% had histologic normalization. The level of agreement between mucosal and histologic activity was moderate (agreement for 68% of samples; κ = 0.50; P < .001). On multivariate analysis, only proctitis associated with histologic normalization (P = .002). Of 310 patients in clinical remission at initial review, 25% had a clinical relapse, after a median time of 16 months (interquartile range, 10-23 months). Histologic normalization was independently associated with increased odds of relapse-free survival compared with histologic quiescence (hazard ratio, 4.31; 95% confidence interval, 1.48-12.46; P = .007) and histologic activity (hazard ratio, 6.69; 95% confidence interval, 2.16-20.62; P = .001); mucosal healing was not associated with increased odds of relapse-free survival compared with no mucosal healing (hazard ratio, 1.02; 95% confidence interval, 0.56-1.85; P = .954). Histologic normalization of colonic mucosa can be used as a clinical endpoint for patients with UC. We associated histologic normalization with increased odds of relapse-free survival compared with endoscopic healing or histologic quiescence. Further studies are needed to determine whether histologic normalization should be a goal of treatment for patients with UC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

System for determining position of normal shock in supersonic flow

NASA Technical Reports Server (NTRS)

Iverson, Jr., Donald G. (Inventor); Daiber, Troy D. (Inventor)

1991-01-01

Light from a plurality of light emitting diodes is transmitted through optical cables (12) to a lens system. The lenses (56, 58) expand and collimate the light and project it in a sheet (16) across the supersonic inlet of an aircraft power plant perpendicular to incoming airflow. A normal shock bends a portion of the sheet of light (16). A linear array of a multiplicity of optical fiber ends collects discrete samples of light. The samples are processed and compared to a predetermined profile to determine the shock location.

Method of measuring heat influx of a cryogenic transfer system

DOEpatents

Niemann, Ralph C.; Zelipsky, Steven A.; Rezmer, Ronald R.; Smelser, Peter

1981-01-01

A method is provided for measuring the heat influx of a cryogenic transfer system. A gaseous phase of the cryogen used during normal operation of the system is passed through the system. The gaseous cryogen at the inlet to the system is tempered to duplicate the normal operating temperature of the system inlet. The temperature and mass flow rate of the gaseous cryogen is measured at the outlet of the system, and the heat capacity of the cryogen is determined. The heat influx of the system is then determined from known thermodynamic relationships.

Experimental philosophy.

PubMed

Knobe, Joshua; Buckwalter, Wesley; Nichols, Shaun; Robbins, Philip; Sarkissian, Hagop; Sommers, Tamler

2012-01-01

Experimental philosophy is a new interdisciplinary field that uses methods normally associated with psychology to investigate questions normally associated with philosophy. The present review focuses on research in experimental philosophy on four central questions. First, why is it that people's moral judgments appear to influence their intuitions about seemingly nonmoral questions? Second, do people think that moral questions have objective answers, or do they see morality as fundamentally relative? Third, do people believe in free will, and do they see free will as compatible with determinism? Fourth, how do people determine whether an entity is conscious?

Local bifurcations in differential equations with state-dependent delay.

PubMed

Sieber, Jan

2017-11-01

A common task when analysing dynamical systems is the determination of normal forms near local bifurcations of equilibria. As most of these normal forms have been classified and analysed, finding which particular class of normal form one encounters in a numerical bifurcation study guides follow-up computations. This paper builds on normal form algorithms for equilibria of delay differential equations with constant delay that were developed and implemented in DDE-Biftool recently. We show how one can extend these methods to delay-differential equations with state-dependent delay (sd-DDEs). Since higher degrees of regularity of local center manifolds are still open for sd-DDEs, we give an independent (still only partial) argument which phenomena from the truncated normal must persist in the full sd-DDE. In particular, we show that all invariant manifolds with a sufficient degree of normal hyperbolicity predicted by the normal form exist also in the full sd-DDE.

Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?

PubMed Central

Rusek, Jakub; Thiel, Michaela; Wolinska, Justyna; Laforsch, Christian

2017-01-01

Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological (body length, width and tail spine length) or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits (body length, width and tail spine length). For adult Daphnia, alterations in expression of genes related to stress responses (i.e. HSP60, HSP70 & GST) as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment. PMID:29145427

Effect of Ca2+ Efflux Pathway Distribution and Exogenous Ca2+ Buffers on Intracellular Ca2+ Dynamics in the Rat Ventricular Myocyte: A Simulation Study

PubMed Central

Šimurda, Jiří; Orchard, Clive H.

2014-01-01

We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca2+ efflux pathways (SERCA, Na+/Ca2+ exchange, and sarcolemmal Ca2+ ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca2+ buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca2+ in the dyad and bulk cytoplasm, on cellular Ca2+ cycling. Increasing the dyadic fraction of a particular Ca2+ efflux pathway increases the amount of Ca2+ removed by that pathway, with corresponding changes in Ca2+ efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca2+ removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca2+-dependent inactivation of the L-type Ca2+ current, resulted from the buffers acting as slow and fast “shuttles,” respectively, removing Ca2+ from the dyadic space. The data suggest that complex changes in dyadic Ca2+ and cellular Ca2+ cycling occur as a result of changes in the location of Ca2+ removal pathways or the presence of exogenous Ca2+ buffers, although changing the distribution of Ca2+ efflux pathways has relatively small effects on the systolic Ca2+ transient. PMID:24971358

Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma).

PubMed

Plattner, H; Sehring, I M; Mohamed, I K; Miranda, K; De Souza, W; Billington, R; Genazzani, A; Ladenburger, E-M

2012-05-01

The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effects of antioxidants on apoptosis induced by dasatinib and nilotinib in K562 cells.

PubMed

Damiano, Sara; Montagnaro, Serena; Puzio, Maria V; Severino, Lorella; Pagnini, Ugo; Barbarino, Marcella; Cesari, Daniele; Giordano, Antonio; Florio, Salvatore; Ciarcia, Roberto

2018-06-01

In clinical practice for the treatment of chronic myeloid leukemia, second generation of tyrosine kinase inhibitors such as Nilotinib (NIL) specific and potent inhibitor of the BCR/ABL kinase and Dasatinib (DAS) a inhibitor of BCR/ABL and Src family kinase were developed to clinically overcome imatinib resistance. In this study, we wanted to test the ability of some antioxidants such Resveratrol (RES) or a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) or δ-tocotrienol (δ-TOCO) to interact with DAS and NIL on viability, reactive oxygen species (ROS) production, lipid peroxidation, and apoptosis. To test the possible mechanisms of action of such antioxidants, we utilized N-acetyl-L-cysteine (NAC) a specific inhibitor ROS production or PP1 a specific Src tyrosine kinase inhibitor or BAPTA a specific chelator of intracellular calcium. Our data demonstrated: 1) RES, rMnSOD, δ-TOCO, and NAC, at dose used, significantly reduced the intracellular levels of MDA induced by DAS or NIL; 2) RES, rMnSOD, and δ-TOCO increased the intracellular ROS levels; 3) The increase ROS levels is related to higher levels of oligonucleosomesi induced by DAS and NIL and that NAC significantly reduced this activity. Interestingly, our data showed that apoptotic activity of DAS and NIL have significantly increased the production of oligonucleosomes by triggering excessive ROS generation as well as functionality of SERCA receptors. © 2018 Wiley Periodicals, Inc.

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

PubMed

Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

2017-08-01

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

The anti-diabetic drug repaglinide induces vasorelaxation via activation of PKA and PKG in aortic smooth muscle.

PubMed

Kim, Hye Won; Li, Hongliang; Kim, Han Sol; Shin, Sung Eun; Jung, Won-Kyo; Ha, Kwon-Soo; Han, Eun-Taek; Hong, Seok-Ho; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Park, Won Sun

2016-09-01

We investigated the vasorelaxant effect of repaglinide and its related signaling pathways using phenylephrine (Phe)-induced pre-contracted aortic rings. Repaglinide induced vasorelaxation in a concentration-dependent manner. The repaglinide-induced vasorelaxation was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor (L-NAME) and a small-conductance Ca(2+)-activated K(+) (SKCa) channel inhibitor (apamin) did not alter the vasorelaxant effect of repaglinide on endothelium-intact arteries. Pretreatment with an adenylyl cyclase inhibitor (SQ 22536) or a PKA inhibitor (KT 5720) effectively reduced repaglinide-induced vasorelaxation. Also, pretreatment with a guanylyl cyclase inhibitor (ODQ) or a PKG inhibitor (KT 5823) inhibited repaglinide-induced vasorelaxation. However, pretreatment with a voltage-dependent K(+) (Kv) channel inhibitor (4-AP), ATP-sensitive K(+) (KATP) channel inhibitor (glibenclamide), large-conductance Ca(2+)-activated K(+) (BKCa) channel inhibitor (paxilline), or the inwardly rectifying K(+) (Kir) channel inhibitor (Ba(2+)) did not affect the vasorelaxant effect of repaglinide. Furthermore, pretreatment with a Ca(2+) inhibitor (nifedipine) and a sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor (thapsigargin) did not affect the vasorelaxant effect of repaglinide. The vasorelaxant effect of repaglinide was not affected by elevated glucose (50mM). Based on these results, we conclude that repaglinide induces vasorelaxation via activation of adenylyl cyclase/PKA and guanylyl cyclase/PKG signaling pathways independently of the endothelium, K(+) channels, Ca(2+) channels, and intracellular Ca(2+) ([Ca(2+)]i). Copyright © 2016 Elsevier Inc. All rights reserved.

Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?

PubMed

Imhof, Hannes K; Rusek, Jakub; Thiel, Michaela; Wolinska, Justyna; Laforsch, Christian

2017-01-01

Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological (body length, width and tail spine length) or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits (body length, width and tail spine length). For adult Daphnia, alterations in expression of genes related to stress responses (i.e. HSP60, HSP70 & GST) as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment.

17β-Estradiol and/or estrogen receptor alpha signaling blocks protein phosphatase 1 mediated ISO induced cardiac hypertrophy.

PubMed

Fang, Hsin-Yuan; Hung, Meng-Yu; Lin, Yueh-Min; Pandey, Sudhir; Chang, Chia-Chien; Lin, Kuan-Ho; Shen, Chia-Yao; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-01-01

Earlier studies have shown that estrogen possess protective function against the development of pathological cardiac hypertrophy. However, the molecular mechanisms of estrogens (E2) protective effect are poorly understood. Additionally, abnormal activation of β-adrenergic signaling have been implicated in the development of pathological cardiac remodeling. However, the role of serine/threonine protein phosphatase 1 (PP1) in pathological cardiac remodeling under the influence of β-adrenergic signaling have been sparsely investigated. In this study, we assessed the downstream effects of abnormal activation of PP1 upon isoproterenol (ISO) induced pathological cardiac changes. We found that pre-treatment of 17β-estradiol (E2), tet-on estrogen receptor-α, or both significantly inhibited ISO-induced increase in cell size, hypertrophy marker gene expression and cytosolic calcium accumulation in H9c2 cells. Additionally, treatment with estrogen receptor inhibitor (ICI) reversed those effects, implicating role of E2 in inhibiting pathological cardiac remodeling. However, specific inhibition of ERα using melatonin, reduced ISO-induced PP1c expression and enhanced the level of ser-16 phosphorylated phospholamban (PLB), responsible for regulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Furthermore, hypertrophic effect caused by overexpression of PP1cα was reduced by treatment with specific inhibitor of ERα. Collectively, we found that estrogen and estrogen receptor-α have protective effect against pathological cardiac changes by suppressing PP1 expression and its downstream signaling pathway, which further needs to be elucidated.

Greenhouse gas emissions and N turnover along an altitudinal gradient at Mt. Kilimanjaro, Tanzania.

NASA Astrophysics Data System (ADS)

Gütlein, Adrian; Gerschlauer, Friederike; Zistl-Schlingmann, Marcus; Dannenmann, Michael; Meier, Rudolf; Kolar, Alison; Butterbach-Bahl, Klaus; Kiese, Ralf

2016-04-01

Worldwide climate and land-use change force alterations in various ecosystem properties and functions such as diversity and activity of soil microbial communities which are responsible for biogeochemical processes like soil nitrogen (N) turnover and associated greenhouse gas (GHG) exchange. Tropical deforestation is highest in Africa and despite the importance of those ecosystems to global climate and biogeochemical cycles, data for greenhouse gas exchange is still rare (Serca et al., 1994, Werner et al., 2007) and no study regarding N turnover processes has been published yet. For that reason, we focused on seven different land-use types extending along an altitudinal gradient (950 -- 3880m) at Mt. Kilimanjaro, East Africa, covering (semi-) natural savanna, two montane forests and one afro alpine ecosystem, an extensive agroforest (homegarden) and an intensively managed coffee plantation. On all ecosystems we measured CO_2, CH4 and N_2O fluxes and gross rates of ammonification, nitrification, N immobilization, and dissimilatory nitrate reduction to ammonium (DNRA). GHG results reveal pronounced N_2O fluxes depending mainly on soil moisture and to a lesser extent on soil temperature. Emissions are highest during the rainy seasons while lowest at dry season conditions. The largest N_2O emissions are recognizable at Ocotea forest, most likely due to the generally higher SOC/ totN and wetter conditions favoring formation and emission of N_2O via denitrification. Soils of the studied ecosystems were a sink of atmospheric CH

An iterative procedure for obtaining maximum-likelihood estimates of the parameters for a mixture of normal distributions

NASA Technical Reports Server (NTRS)

Peters, B. C., Jr.; Walker, H. F.

1975-01-01

A general iterative procedure is given for determining the consistent maximum likelihood estimates of normal distributions. In addition, a local maximum of the log-likelihood function, Newtons's method, a method of scoring, and modifications of these procedures are discussed.

The Theoretical Distribution of Evoked Brainstem Activity in Preterm, High-Risk, and Healthy Infants.

ERIC Educational Resources Information Center

Salamy, A.

1981-01-01

Determines the frequency distribution of Brainstem Auditory Evoked Potential variables (BAEP) for premature babies at different stages of development--normal newborns, infants, young children, and adults. The author concludes that the assumption of normality underlying most "standard" statistical analyses can be met for many BAEP…

Frog egg growth, experiment S003

NASA Technical Reports Server (NTRS)

Young, R. S.; Tremor, J. W.

1971-01-01

The objective of experiment was to determine the effect of weightlessness on the ability of a fertilized frog egg to divide normally and to differentiate and form a normal embryo. This experiment was first attempted on the Gemini 8 mission and was completed only partially because of the early termination of that mission.

Systems of Generators for the Normalizers of Certain Elements of the Braid Group

NASA Astrophysics Data System (ADS)

Gurzo, G. G.

1985-06-01

Systems of generators of normalizers are determined for certain elements of the braid group {\\mathfrak{B}}_{n+1}. These systems of generators consist of fewer than 2n explicitly written words in the positive alphabet of {\\mathfrak{B}}_{n+1}. Bibliography: 10 titles.

36 CFR 907.8 - Actions that normally require an EIS.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 36 Parks, Forests, and Public Property 3 2012-07-01 2012-07-01 false Actions that normally require an EIS. 907.8 Section 907.8 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT... prepare or have prepared the environmental impact statement. To assist in determining if a proposal or...

Seasonal changes in carbohydrate levels in roots of sugar maple

Treesearch

Philip M. Wargo; Philip M. Wargo

1971-01-01

This study was done to determine the normal complement of individual carbohydrates present in roots of sugar maples duringthe year and to obtain, as a basis for future comparison, an estimate of the normal variation and range of concentrations of individual carbohydrates in the roots during the year.

Electrocardiogram reference intervals for clinically normal wild-born chimpanzees (Pan troglodytes).

PubMed

Atencia, Rebeca; Revuelta, Luis; Somauroo, John D; Shave, Robert E

2015-08-01

To generate reference intervals for ECG variables in clinically normal chimpanzees (Pan troglodytes). 100 clinically normal (51 young [< 10 years old] and 49 adult [≥ 10 years old]) wild-born chimpanzees. Electrocardiograms collected between 2009 and 2013 at the Tchimpounga Chimpanzee Rehabilitation Centre were assessed to determine heart rate, PR interval, QRS duration, QT interval, QRS axis, P axis, and T axis. Electrocardiographic characteristics for left ventricular hypertrophy (LVH) and morphology of the ST segment, T wave, and QRS complex were identified. Reference intervals for young and old animals were calculated as mean ± 1.96•SD for normally distributed data and as 5th to 95th percentiles for data not normally distributed. Differences between age groups were assessed by use of unpaired Student t tests. RESULTS Reference intervals were generated for young and adult wild-born chimpanzees. Most animals had sinus rhythm with small or normal P wave morphology; 24 of 51 (47%) young chimpanzees and 30 of 49 (61%) adult chimpanzees had evidence of LVH as determined on the basis of criteria for humans. Cardiac disease has been implicated as the major cause of death in captive chimpanzees. Species-specific ECG reference intervals for chimpanzees may aid in the diagnosis and treatment of animals with, or at risk of developing, heart disease. Chimpanzees with ECG characteristics outside of these intervals should be considered for follow-up assessment and regular cardiac monitoring.

Prevalence and etiology of false normal aEEG recordings in neonatal hypoxic-ischaemic encephalopathy

PubMed Central

2013-01-01

Background Amplitude-integrated electroencephalography (aEEG) is a useful tool to determine the severity of neonatal hypoxic-ischemic encephalopathy (HIE). Our aim was to assess the prevalence and study the origin of false normal aEEG recordings based on 85 aEEG recordings registered before six hours of age. Methods Raw EEG recordings were reevaluated retrospectively with Fourier analysis to identify and describe the frequency patterns of the raw EEG signal, in cases with inconsistent aEEG recordings and clinical symptoms. Power spectral density curves, power (P) and median frequency (MF) were determined using the raw EEG. In 7 patients non-depolarizing muscle relaxant (NDMR) exposure was found. The EEG sections were analyzed and compared before and after NDMR administration. Results The reevaluation found that the aEEG was truly normal in 4 neonates. In 3 neonates, high voltage electrocardiographic (ECG) artifacts were found with flat trace on raw EEG. High frequency component (HFC) was found as a cause of normal appearing aEEG in 10 neonates. HFC disappeared while P and MF decreased significantly upon NDMR administration in each observed case. Conclusion Occurrence of false normal aEEG background pattern is relatively high in neonates with HIE and hypothermia. High frequency EEG artifacts suggestive of shivering were found to be the most common cause of false normal aEEG in hypothermic neonates while high voltage ECG artifacts are less common. PMID:24268061

Investigation of phosphoserine and cytidine 5'-phosphate by heteronuclear two-dimensional spectroscopy: samples with strong proton coupling

NASA Astrophysics Data System (ADS)

Bolton, Philip H.

Heteronuclear two-dimensional magnetic resonance is a novel method for investigating the conformations of cellular phosphates. The two-dimensional proton spectra are detected indirectly via the phosphorus-31 nucleus and thus allow determination of proton chemical shifts and coupling constants in situations in which the normal proton spectrum is obscured. Previous investigations of cellular phosphates with relatively simple spin systems have shown that the two-dimensional proton spectrum can be readily related to the normal proton spectrum by subspectral analysis. The normal proton spectrum can be decomposed into two subspectra, one for each polarization of the phosphorus-31 nucleus. The two-dimensional spectrum arises from the difference between the subspectra, and the normal proton spectrum is the sum. This allows simulation of the two-dimensional spectra and hence determination of the proton chemical shifts and coupling constants. Many cellular phosphates of interest, such as 5'-nucleotides and phosphoserine, contain three protons coupled to the phosphorus which are strongly coupled to one another. These samples are amenable to the two-dimensional method and the straightforward subspectral analysis is preserved when a 90° pulse is applied to the protons in the magnetization transfer step. The two-dimensional proton spectra of the samples investigated here have higher resolution than the normal proton spectra, revealing spectral features not readily apparent in the normal proton spectra.

Assessment of 1H NMR-based metabolomics analysis for normalization of urinary metals against creatinine.

PubMed

Cassiède, Marc; Nair, Sindhu; Dueck, Meghan; Mino, James; McKay, Ryan; Mercier, Pascal; Quémerais, Bernadette; Lacy, Paige

2017-01-01

Proton nuclear magnetic resonance ( 1 H NMR, or NMR) spectroscopy and inductively coupled plasma-mass spectrometry (ICP-MS) are commonly used for metabolomics and metal analysis in urine samples. However, creatinine quantification by NMR for the purpose of normalization of urinary metals has not been validated. We assessed the validity of using NMR analysis for creatinine quantification in human urine samples in order to allow normalization of urinary metal concentrations. NMR and ICP-MS techniques were used to measure metabolite and metal concentrations in urine samples from 10 healthy subjects. For metabolite analysis, two magnetic field strengths (600 and 700MHz) were utilized. In addition, creatinine concentrations were determined by using the Jaffe method. Creatinine levels were strongly correlated (R 2 =0.99) between NMR and Jaffe methods. The NMR spectra were deconvoluted with a target database containing 151 metabolites that are present in urine. A total of 50 metabolites showed good correlation (R 2 =0.7-1.0) at 600 and 700MHz. Metal concentrations determined after NMR-measured creatinine normalization were comparable to previous reports. NMR analysis provided robust urinary creatinine quantification, and was sufficient for normalization of urinary metal concentrations. We found that NMR-measured creatinine-normalized urinary metal concentrations in our control subjects were similar to general population levels in Canada and the United Kingdom. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, A.L.; Kohrs, M.B.; Horwitz, D.L.

To determine the effect of glucose loading on serum zinc concentrations, 34 elderly subjects aged 60-86 y were studied. Anthropometric data, medical and dietary histories were obtained. Serum zinc and glucose concentrations were obtained fasting and 1/2, 1, 1 1/2, 2 and 3 h after 75 g oral glucose load; glycohemoglobin and fasting serum lipids were also determined. For comparison, the subjects were categorized as: normal or low serum zinc concentrations; normal or high body mass index BMI; normal or high sum of skinfolds and normal or high serum cholesterol. Results showed that low serum zinc concentrations increased significantly overmore » baseline values after the glucose load and did not return to fasting levels. On the other hand, mean serum zinc concentrations significantly declined without recovery for those with normal zinc values. For the total group, no significant differences were noted between fasting values and subsequent time periods. No correlations were noted between fasting serum zinc and area under the curve for zinc except in the high BMI group (positive correlation observed). For the high BMI group, fasting serum zinc differed significantly from the succeeding measurements except for 30 min. For the group as a whole, mean serum zinc concentration was within normal limits (76.9 +/- 2.8 mcg/ml): mean zinc intake was less than 2/3rds the RDA. They conclude that glucose ingestion may alter serum zinc and should be considered in interpreting these levels.« less

DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors.

PubMed

Rechache, Nesrin S; Wang, Yonghong; Stevenson, Holly S; Killian, J Keith; Edelman, Daniel C; Merino, Maria; Zhang, Lisa; Nilubol, Naris; Stratakis, Constantine A; Meltzer, Paul S; Kebebew, Electron

2012-06-01

It is not known whether there are any DNA methylation alterations in adrenocortical tumors. The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

Data-driven identification of intensity normalization region based on longitudinal coherency of 18F-FDG metabolism in the healthy brain.

PubMed

Zhang, Huiwei; Wu, Ping; Ziegler, Sibylle I; Guan, Yihui; Wang, Yuetao; Ge, Jingjie; Schwaiger, Markus; Huang, Sung-Cheng; Zuo, Chuantao; Förster, Stefan; Shi, Kuangyu

2017-02-01

In brain 18 F-FDG PET data intensity normalization is usually applied to control for unwanted factors confounding brain metabolism. However, it can be difficult to determine a proper intensity normalization region as a reference for the identification of abnormal metabolism in diseased brains. In neurodegenerative disorders, differentiating disease-related changes in brain metabolism from age-associated natural changes remains challenging. This study proposes a new data-driven method to identify proper intensity normalization regions in order to improve separation of age-associated natural changes from disease related changes in brain metabolism. 127 female and 128 male healthy subjects (age: 20 to 79) with brain 18 F-FDG PET/CT in the course of a whole body cancer screening were included. Brain PET images were processed using SPM8 and were parcellated into 116 anatomical regions according to the AAL template. It is assumed that normal brain 18 F-FDG metabolism has longitudinal coherency and this coherency leads to better model fitting. The coefficient of determination R 2 was proposed as the coherence coefficient, and the total coherence coefficient (overall fitting quality) was employed as an index to assess proper intensity normalization strategies on single subjects and age-cohort averaged data. Age-associated longitudinal changes of normal subjects were derived using the identified intensity normalization method correspondingly. In addition, 15 subjects with clinically diagnosed Parkinson's disease were assessed to evaluate the clinical potential of the proposed new method. Intensity normalizations by paracentral lobule and cerebellar tonsil, both regions derived from the new data-driven coherency method, showed significantly better coherence coefficients than other intensity normalization regions, and especially better than the most widely used global mean normalization. Intensity normalization by paracentral lobule was the most consistent method within both analysis strategies (subject-based and age-cohort averaging). In addition, the proposed new intensity normalization method using the paracentral lobule generates significantly higher differentiation from the age-associated changes than other intensity normalization methods. Proper intensity normalization can enhance the longitudinal coherency of normal brain glucose metabolism. The paracentral lobule followed by the cerebellar tonsil are shown to be the two most stable intensity normalization regions concerning age-dependent brain metabolism. This may provide the potential to better differentiate disease-related changes from age-related changes in brain metabolism, which is of relevance in the diagnosis of neurodegenerative disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Blockade of renal medullary bradykinin B2 receptors increases tubular sodium reabsorption in rats fed a normal-salt diet

PubMed Central

Sivritas, Sema-Hayriye; Ploth, David W.; Fitzgibbon, Wayne R.

2008-01-01

The present study was performed to test the hypothesis that under normal physiological conditions and/or during augmentation of kinin levels, intrarenal kinins act on medullary bradykinin B2 (BKB2) receptors to acutely increase papillary blood flow (PBF) and therefore Na+ excretion. We determined the effect of acute inner medullary interstitial (IMI) BKB2 receptor blockade on renal hemodynamics and excretory function in rats fed either a normal (0.23%)- or a low (0.08%)-NaCl diet. For each NaCl diet, two groups of rats were studied. Baseline renal hemodynamic and excretory function were determined during IMI infusion of 0.9% NaCl into the left kidney. The infusion was then either changed to HOE-140 (100 μg·kg−1·h−1, treated group) or maintained with 0.9% NaCl (time control group), and the parameters were again determined. In rats fed a normal-salt diet, HOE-140 infusion decreased left kidney Na+ excretion (urinary Na+ extraction rate) and fractional Na+ excretion by 40 ± 5% and 40 ± 4%, respectively (P < 0.01), but did not alter glomerular filtration rate, inner medullary blood flow (PBF), or cortical blood flow. In rats fed a low-salt diet, HOE-140 infusion did not alter renal regional hemodynamics or excretory function. We conclude that in rats fed a normal-salt diet, kinins act tonically via medullary BKB2 receptors to increase Na+ excretion independent of changes in inner medullary blood flow. PMID:18632797

Low-intensity laser irradiation at 660 nm stimulates cytochrome c oxidase in stressed fibroblast cells.

PubMed

Houreld, Nicolette N; Masha, Roland T; Abrahamse, Heidi

2012-07-01

Low-intensity laser irradiation (LILI) has been used to modulate a variety of biological processes, including diabetic wound healing. The mechanism of action is thought to exist primarily with the mitochondria. This study aimed to determine the effect of irradiation on normal, diabetic, and ischemic mitochondrial electron transport chain (ETC) complexes. Normal, diabetic and ischemic human skin fibroblast mitochondria were irradiated in vitro at a wavelength of 660 nm and a fluence of either 5 or 15 J/cm(2). Non-irradiated mitochondria served as controls. Enzyme activities of mitochondrial complexes I, II, III, and IV were determined immediately post-irradiation. Normal, diabetic, and ischemic cells were irradiated and adenosine triphosphate (ATP) and active mitochondria were determined by luminescence and fluorescent microscopy, respectively. Irradiated diabetic mitochondria at a fluence of 15 J/cm(2) showed a significant decrease in complex III activity (P < 0.05). Normal (P < 0.01) and diabetic (P < 0.05) mitochondria irradiated at either 5 or 15 J/cm(2) showed a significant increase in complex IV activity. ATP results showed a significant increase in irradiated normal cells (5 J/cm(2); P < 0.05) and diabetic cells (15 J/cm(2); P < 0.01). There was a higher accumulation of active mitochondria in irradiated cells than non-irradiated cells. Irradiation at 660 nm has the ability to influence mitochondrial enzyme activity, in particular cytochrome c oxidase. This leads to increased mitochondrial activity and ATP synthesis. Copyright © 2012 Wiley Periodicals, Inc.

A replacement for islet equivalents with improved reliability and validity.

PubMed

Huang, Han-Hung; Ramachandran, Karthik; Stehno-Bittel, Lisa

2013-10-01

Islet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R2 value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter≤100 μm) and large (diameter≥200 μm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

Ischemia-reperfusion injury in rat fatty liver: role of nutritional status.

PubMed

Caraceni, P; Nardo, B; Domenicali, M; Turi, P; Vici, M; Simoncini, M; De Maria, N; Trevisani, F; Van Thiel, D H; Derenzini, M; Cavallari, A; Bernardi, M

1999-04-01

Fatty livers are more sensitive to the deleterious effects of ischemia-reperfusion than normal livers. Nutritional status greatly modulates this injury in normal livers, but its role in the specific setting of fatty liver is unknown. This study aimed to determine the effect of nutritional status on warm ischemia-reperfusion injury in rat fatty livers. Fed and fasted rats with normal or fatty liver induced by a choline deficient diet underwent 1 hour of lobar ischemia and reperfusion. Rat survival was determined for 7 days. Serum transaminases, liver histology and cell ultrastructure were assessed before and after ischemia, and at 30 minutes, 2 hours, 8 hours, and 24 hours after reperfusion. Survival was also determined in fatty fasted rats supplemented with glucose before surgery. The preischemic hepatic glycogen was measured in all groups. Whereas survival was similar in fasted and fed rats with normal liver (90% vs. 100%), fasting dramatically reduced survival in rats with fatty liver (14% vs. 64%, P <.01). Accordingly, fasting and fatty degeneration had a synergistic effect in exacerbating liver injury. Mitochondrial damage was a predominant feature of ultrastructural hepatocyte injury in fasted fatty livers. Glucose supplementation partially prevented the fasting-induced depletion of glycogen and improved the 7-day rat survival to 45%. These data indicate that rat fatty livers exposed to normothermic ischemia-reperfusion injury are much more sensitive to fasting than histologically normal livers. Because glucose supplementation improves both the hepatic glycogen stores and the rat survival, a nutritional repletion procedure may be part of a treatment strategy aimed to prevent ischemia-reperfusion injury in fatty livers.

A Preliminary Quantitative Comparison of Vibratory Amplitude Using Rigid and Flexible Stroboscopic Assessment.

PubMed

Hosbach-Cannon, Carly J; Lowell, Soren Y; Kelley, Richard T; Colton, Raymond H

2016-07-01

The purpose of this study was to establish preliminary, quantitative data on amplitude of vibration during stroboscopic assessment in healthy speakers with normal voice characteristics. Amplitude of vocal fold vibration is a core physiological parameter used in diagnosing voice disorders, yet quantitative data are lacking to guide the determination of what constitutes normal vibratory amplitude. Eleven participants were assessed during sustained vowel production using rigid and flexible endoscopy with stroboscopy. Still images were extracted from digital recordings of a sustained /i/ produced at a comfortable pitch and loudness, with F0 controlled so that levels were within ±15% of each participant's comfortable mean level as determined from connected speech. Glottal width (GW), true vocal fold (TVF) length, and TVF width were measured from still frames representing the maximum open phase of the vibratory cycle. To control for anatomic and magnification differences across participants, GW was normalized to TVF length. GW as a ratio of TVF width was also computed for comparison with prior studies. Mean values and standard deviations were computed for the normalized measures. Paired t tests showed no significant differences between rigid and flexible endoscopy methods. Interrater and intrarater reliability values for raw measurements were found to be high (0.89-0.99). These preliminary quantitative data may be helpful in determining normality or abnormality of vocal fold vibration. Results indicate that quantified amplitude of vibration is similar between endoscopic methods, a clinically relevant finding for individuals performing and interpreting stroboscopic assessments. Copyright © 2016 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Ambiguous genitalia

MedlinePlus

... chromosome. These X and Y chromosomes determine the child's genetic sex. Normally, an infant inherits 1 pair of sex ... from the father. The father "determines" the genetic sex of the child. A baby who inherits the X chromosome from ...

Attitude determination of planetary exploration rovers using solar panels characteristics and accelerometer

NASA Astrophysics Data System (ADS)

Ishida, Takayuki; Takahashi, Masaki

2014-12-01

In this study, we propose a new attitude determination system, which we call Irradiance-based Attitude Determination (IRAD). IRAD employs the characteristics and geometry of solar panels. First, the sun vector is estimated using data from solar panels including current, voltage, temperature, and the normal vectors of each solar panel. Because these values are obtained using internal sensors, it is easy for rovers to provide redundancy for IRAD. The normal vectors are used to apply to various shapes of rovers. Second, using the gravity vector obtained from an accelerometer, the attitude of a rover is estimated using a three-axis attitude determination method. The effectiveness of IRAD is verified through numerical simulations and experiments that show IRAD can estimate all the attitude angles (roll, pitch, and yaw) within a few degrees of accuracy, which is adequate for planetary explorations.

Influences of Normalization Method on Biomarker Discovery in Gas Chromatography-Mass Spectrometry-Based Untargeted Metabolomics: What Should Be Considered?

PubMed

Chen, Jiaqing; Zhang, Pei; Lv, Mengying; Guo, Huimin; Huang, Yin; Zhang, Zunjian; Xu, Fengguo

2017-05-16

Data reduction techniques in gas chromatography-mass spectrometry-based untargeted metabolomics has made the following workflow of data analysis more lucid. However, the normalization process still perplexes researchers, and its effects are always ignored. In order to reveal the influences of normalization method, five representative normalization methods (mass spectrometry total useful signal, median, probabilistic quotient normalization, remove unwanted variation-random, and systematic ratio normalization) were compared in three real data sets with different types. First, data reduction techniques were used to refine the original data. Then, quality control samples and relative log abundance plots were utilized to evaluate the unwanted variations and the efficiencies of normalization process. Furthermore, the potential biomarkers which were screened out by the Mann-Whitney U test, receiver operating characteristic curve analysis, random forest, and feature selection algorithm Boruta in different normalized data sets were compared. The results indicated the determination of the normalization method was difficult because the commonly accepted rules were easy to fulfill but different normalization methods had unforeseen influences on both the kind and number of potential biomarkers. Lastly, an integrated strategy for normalization method selection was recommended.

Design of air-gapped magnetic-core inductors for superimposed direct and alternating currents

NASA Technical Reports Server (NTRS)

Ohri, A. K.; Wilson, T. G.; Owen, H. A., Jr.

1976-01-01

Using data on standard magnetic-material properties and standard core sizes for air-gap-type cores, an algorithm designed for a computer solution is developed which optimally determines the air-gap length and locates the quiescent point on the normal magnetization curve so as to yield an inductor design with the minimum number of turns for a given ac voltage and frequency and with a given dc bias current superimposed in the same winding. Magnetic-material data used in the design are the normal magnetization curve and a family of incremental permeability curves. A second procedure, which requires a simpler set of calculations, starts from an assigned quiescent point on the normal magnetization curve and first screens candidate core sizes for suitability, then determines the required turns and air-gap length.

Effects of genetic captive-breeding protocols on sperm quality and fertility in the white-footed mouse.

PubMed

Malo, Aurelio F; Martinez-Pastor, Felipe; Alaks, Glen; Dubach, Jean; Lacy, Robert C

2010-10-01

Mice (Peromyscus leucopus noveboracensis) from a captive-breeding program were used to test the effects of three genetic breeding protocols (minimizing mean kinship [MK], random breeding, and selection for docility [DOC]) and inbreeding levels on sperm traits and fertility. Earlier, in generation 8, one DOC replicate went extinct because of poor reproductive success. By generation 10, spermatozoa from DOC mice had more acrosome and midpiece abnormalities, which were shown to be strong determinants of fertility, as well as lower sperm production and resistance to osmotic stress. In addition, determinants of fertility, including male and female components, were assessed in a comprehensive manner. Results showed that the probability (P) of siring litters is determined by sperm number, sperm viability, and midpiece and acrosome abnormalities; that the P of siring one versus two litters is determined by tail abnormalities; and that the total number of offspring is influenced by female size and proportion of normal sperm, showing the relative importance of different sperm traits on fertility. On average, males with 20% normal sperm sired one pup per litter, and males with 70% normal sperm sired eight pups per litter. Interestingly, the proportion of normal sperm was affected by docility but not by relatively low inbreeding. However, inbreeding depression in sperm motility was detected. In the MK group, inbreeding depression not only affected sperm motility but also fertility: An increase in the coefficient of inbreeding (f) of 0.03 reduced sperm motility by 30% and translated into an offspring reduction of three pups in second litters. A genetic load of 48 fecundity equivalents was calculated.

On the possibility of spectroscopic cancer diagnostics

NASA Astrophysics Data System (ADS)

Khairullina, Alphiya Y.; Oleinik, Tatiana V.; Korolevich, Alexander N.; Sevkovsky, Yacob I.

1993-07-01

The diffuse reflection and transmission coefficients, other optical parameters of normal and cancer tissues have been investigated in visible and infrared spectra. The optimal spectral range for distinguishing the cancer is found. The spectral absorption coefficients and size of cells parameter determined using our approach are analyzed to be different for normal and pathological tissues. The method is proposed for calculating the diffuse reflectance and transmittance of multiple tissue layers. The investigations have shown that cancer may be distinguished under the layers of skin and normal tissue.

A Whole Word and Number Reading Machine Based on Two Dimensional Low Frequency Fourier Transforms

DTIC Science & Technology

1990-12-01

they are energy normalized. The normalization process accounts for brightness variations and is equivalent to graphing each 2DFT onto the surface of an n...determined empirically (trial and error). Each set is energy normalized based on the number of coefficients within the set. Therefore, the actual...using the 6 font group case with the top 1000 words, where the energy has been renormalized based on the particular number of coefficients being used

Liquid jet impingement normal to a disk in zero gravity. Ph.D. Thesis - Toledo Univ.

NASA Technical Reports Server (NTRS)

Labus, T. L.

1976-01-01

An experimental and analytical investigation was conducted to determine the free surface shapes of circular liquid jets impinging normal to sharp-edged disks under both normal and zero gravity conditions. An order of magnitude analysis was conducted indicating regions where viscous forces were not significant when computing free surface shapes. The demarcation between the viscous and inviscid region was found to depend upon the flow Reynolds number and the ratio between the jet and disk radius.

Normal and abnormal human vestibular ocular function

NASA Technical Reports Server (NTRS)

Peterka, R. J.; Black, F. O.

1986-01-01

The major motivation of this research is to understand the role the vestibular system plays in sensorimotor interactions which result in spatial disorientation and motion sickness. A second goal was to explore the range of abnormality as it is reflected in quantitative measures of vestibular reflex responses. The results of a study of vestibular reflex measurements in normal subjects and preliminary results in abnormal subjects are presented in this report. Statistical methods were used to define the range of normal responses, and determine age related changes in function.

Critical dynamics of Hopf bifurcations in the corticothalamic system: Transitions from normal arousal states to epileptic seizures.

PubMed

Yang, Dong-Ping; Robinson, P A

2017-04-01

A physiologically based corticothalamic model of large-scale brain activity is used to analyze critical dynamics of transitions from normal arousal states to epileptic seizures, which correspond to Hopf bifurcations. This relates an abstract normal form quantitatively to underlying physiology that includes neural dynamics, axonal propagation, and time delays. Thus, a bridge is constructed that enables normal forms to be used to interpret quantitative data. The normal form of the Hopf bifurcations with delays is derived using Hale's theory, the center manifold theorem, and normal form analysis, and it is found to be explicitly expressed in terms of transfer functions and the sensitivity matrix of a reduced open-loop system. It can be applied to understand the effect of each physiological parameter on the critical dynamics and determine whether the Hopf bifurcation is supercritical or subcritical in instabilities that lead to absence and tonic-clonic seizures. Furthermore, the effects of thalamic and cortical nonlinearities on the bifurcation type are investigated, with implications for the roles of underlying physiology. The theoretical predictions about the bifurcation type and the onset dynamics are confirmed by numerical simulations and provide physiologically based criteria for determining bifurcation types from first principles. The results are consistent with experimental data from previous studies, imply that new regimes of seizure transitions may exist in clinical settings, and provide a simplified basis for control-systems interventions. Using the normal form, and the full equations from which it is derived, more complex dynamics, such as quasiperiodic cycles and saddle cycles, are discovered near the critical points of the subcritical Hopf bifurcations.

Significant effect of topographic normalization of airborne LiDAR data on the retrieval of plant area index profile in mountainous forests

NASA Astrophysics Data System (ADS)

Liu, Jing; Skidmore, Andrew K.; Heurich, Marco; Wang, Tiejun

2017-10-01

As an important metric for describing vertical forest structure, the plant area index (PAI) profile is used for many applications including biomass estimation and wildlife habitat assessment. PAI profiles can be estimated with the vertically resolved gap fraction from airborne LiDAR data. Most research utilizes a height normalization algorithm to retrieve local or relative height by assuming the terrain to be flat. However, for many forests this assumption is not valid. In this research, the effect of topographic normalization of airborne LiDAR data on the retrieval of PAI profile was studied in a mountainous forest area in Germany. Results show that, although individual tree height may be retained after topographic normalization, the spatial arrangement of trees is changed. Specifically, topographic normalization vertically condenses and distorts the PAI profile, which consequently alters the distribution pattern of plant area density in space. This effect becomes more evident as the slope increases. Furthermore, topographic normalization may also undermine the complexity (i.e., canopy layer number and entropy) of the PAI profile. The decrease in PAI profile complexity is not solely determined by local topography, but is determined by the interaction between local topography and the spatial distribution of each tree. This research demonstrates that when calculating the PAI profile from airborne LiDAR data, local topography needs to be taken into account. We therefore suggest that for ecological applications, such as vertical forest structure analysis and modeling of biodiversity, topographic normalization should not be applied in non-flat areas when using LiDAR data.

Critical dynamics of Hopf bifurcations in the corticothalamic system: Transitions from normal arousal states to epileptic seizures

NASA Astrophysics Data System (ADS)

Yang, Dong-Ping; Robinson, P. A.

2017-04-01

A physiologically based corticothalamic model of large-scale brain activity is used to analyze critical dynamics of transitions from normal arousal states to epileptic seizures, which correspond to Hopf bifurcations. This relates an abstract normal form quantitatively to underlying physiology that includes neural dynamics, axonal propagation, and time delays. Thus, a bridge is constructed that enables normal forms to be used to interpret quantitative data. The normal form of the Hopf bifurcations with delays is derived using Hale's theory, the center manifold theorem, and normal form analysis, and it is found to be explicitly expressed in terms of transfer functions and the sensitivity matrix of a reduced open-loop system. It can be applied to understand the effect of each physiological parameter on the critical dynamics and determine whether the Hopf bifurcation is supercritical or subcritical in instabilities that lead to absence and tonic-clonic seizures. Furthermore, the effects of thalamic and cortical nonlinearities on the bifurcation type are investigated, with implications for the roles of underlying physiology. The theoretical predictions about the bifurcation type and the onset dynamics are confirmed by numerical simulations and provide physiologically based criteria for determining bifurcation types from first principles. The results are consistent with experimental data from previous studies, imply that new regimes of seizure transitions may exist in clinical settings, and provide a simplified basis for control-systems interventions. Using the normal form, and the full equations from which it is derived, more complex dynamics, such as quasiperiodic cycles and saddle cycles, are discovered near the critical points of the subcritical Hopf bifurcations.

Neither Hematocrit Normalization nor Exercise Training Restores Oxygen Consumption to Normal Levels in Hemodialysis Patients

PubMed Central

Stray-Gundersen, James; Parsons, Dora Beth; Thompson, Jeffrey R.

2016-01-01

Patients treated with hemodialysis develop severely reduced functional capacity, which can be partially ameliorated by correcting anemia and through exercise training. In this study, we determined perturbations of an erythroid-stimulating agent and exercise training to examine if and where limitation to oxygen transport exists in patients on hemodialysis. Twenty-seven patients on hemodialysis completed a crossover study consisting of two exercise training phases at two hematocrit (Hct) values: 30% (anemic) and 42% (physiologic; normalized by treatment with erythroid-stimulating agent). To determine primary outcome measures of peak power and oxygen consumption (VO2) and secondary measures related to components of oxygen transport and utilization, all patients underwent numerous tests at five time points: baseline, untrained at Hct of 30%, after training at Hct of 30%, untrained at Hct of 42%, and after training at Hct of 42%. Hct normalization, exercise training, or the combination thereof significantly improved peak power and VO2 relative to values in the untrained anemic phase. Hct normalization increased peak arterial oxygen and arteriovenous oxygen difference, whereas exercise training improved cardiac output, citrate synthase activity, and peak tissue diffusing capacity. However, although the increase in arterial oxygen observed in the combination phase reached a value similar to that in healthy sedentary controls, the increase in peak arteriovenous oxygen difference did not. Muscle biopsy specimens showed markedly thickened endothelium and electron–dense interstitial deposits. In conclusion, exercise and Hct normalization had positive effects but failed to normalize exercise capacity in patients on hemodialysis. This effect may be caused by abnormalities identified within skeletal muscle. PMID:27153927

Changes in neurocranium thickness in early childhood

NASA Astrophysics Data System (ADS)

Gajawelli, Niharika; Deoni, Sean; Shi, Jie; Xu, Liang; Dirks, Holly; Dean, Douglas; O'Muircheartaigh, Jonathan; Sawardekar, Siddhant; Ezis, Andrea; Nelson, Marvin D.; Wang, Yalin; Lepore, Natasha

2015-12-01

Several developmental disorders involve shape abnormalities of the neurocranium, the most common one being craniosynostosis, that affects about 1 in 2000 infants. A key step in determining how these disorders affect neurodevelopment is to establish how the brain and neurocranium co-evolve in the normally developing child. However, due to the scarcity of normally developing infant and pediatric imaging data, there have been a lack of imaging studies pertaining to normal neurocranial development. Here, taking advantage of a large data bank of high quality brain MRI from healthy children ages 0-4 years old, and of a novel conformal geometry-based analysis pipeline, we have been determining a set of statistical atlases of the neurocranium, divided into age groups. In this first part of the study, we focus more specifically on a comparison of 1 and 2 year old infants. Characterizing neurocranium shape changes will enable us to understand how the cranial bones develop in relation to brain development. This in turn will allow a better determination of the effects of neurocranial disorders, which will help inform treatment strategies.

Normal and abnormal tissue identification system and method for medical images such as digital mammograms

NASA Technical Reports Server (NTRS)

Heine, John J. (Inventor); Clarke, Laurence P. (Inventor); Deans, Stanley R. (Inventor); Stauduhar, Richard Paul (Inventor); Cullers, David Kent (Inventor)

2001-01-01

A system and method for analyzing a medical image to determine whether an abnormality is present, for example, in digital mammograms, includes the application of a wavelet expansion to a raw image to obtain subspace images of varying resolution. At least one subspace image is selected that has a resolution commensurate with a desired predetermined detection resolution range. A functional form of a probability distribution function is determined for each selected subspace image, and an optimal statistical normal image region test is determined for each selected subspace image. A threshold level for the probability distribution function is established from the optimal statistical normal image region test for each selected subspace image. A region size comprising at least one sector is defined, and an output image is created that includes a combination of all regions for each selected subspace image. Each region has a first value when the region intensity level is above the threshold and a second value when the region intensity level is below the threshold. This permits the localization of a potential abnormality within the image.

7 CFR 58.133 - Methods for quality and wholesomeness determination.

Code of Federal Regulations, 2010 CFR

2010-01-01

... and Grading Service 1 Quality Specifications for Raw Milk § 58.133 Methods for quality and wholesomeness determination. (a) Appearance and odor. The appearance of acceptable raw milk shall be normal and...

7 CFR 58.133 - Methods for quality and wholesomeness determination.

Code of Federal Regulations, 2011 CFR

2011-01-01

... and Grading Service 1 Quality Specifications for Raw Milk § 58.133 Methods for quality and wholesomeness determination. (a) Appearance and odor. The appearance of acceptable raw milk shall be normal and...

Normal-incidence reflectance of optimized W/B4C x-ray multilayers in the range 1.4 nm < λ < 2.4 nm

NASA Astrophysics Data System (ADS)

Windt, David L.; Gullikson, Eric M.; Walton, Christopher C.

2002-12-01

We have fabricated W/B4C multilayers having periods in the range d = 0.8-1.2 nm and measured their soft-x-ray performance near normal incidence in the wavelength range 1.4 < λ < 2.4 nm. By adjusting the fractional layer thickness of W we have produced structures having interface widths σ ~ 0.29 nm (i.e., as determined from normal-incidence reflectometry), thus having optimal soft-x-ray performance. We describe our results and discuss their implications, particularly with regard to the development of short-wavelength normal-incidence x-ray optics.

In vivo electrical conductivity of hepatic tumours.

PubMed

Haemmerich, Dieter; Staelin, S T; Tsai, J Z; Tungjitkusolmun, S; Mahvi, D M; Webster, J G

2003-05-01

Knowledge of electrical tissue conductivity is necessary to determine deposition of electromagnetic energy and can further be used to diagnostically differentiate between normal and neoplastic tissue. We measured 17 rats with a total of 24 tumours of the K12/TRb rat colon cancer cell line. In each animal we measured in vivo hepatic tumour and normal tissue conductivity at seven frequencies from 10 Hz to 1 MHz, at different tumour stages between 6 and 12 weeks after induction. Conductivity of normal liver tissue was 1.26 +/- 0.15 mS cm(-1) at 10 Hz, and 4.61 +/- 0.42 mS cm(-1) at 1 MHz. Conductivity of tumour was 2.69 +/- 0.91 mS cm(-1) at 10 Hz, and 5.23 +/- 0.82 mS cm(-1) at 1 MHz. Conductivity was significantly different between normal and tumour tissue (p < 0.05). We determined the percentage of necrosis and fibrosis at the measurement site. We fitted the conductivity data to the Cole-Cole model. For the tumour data we determined Spearman's correlation coefficients between the Cole-Cole parameters and age, necrosis, fibrosis and tumour volume and found significant correlation between necrosis and the Cole-Cole parameters (p < 0.05). We conclude that necrosis within the tumour and the associated membrane breakdown is likely responsible for the observed change in conductivity.

Confidence Intervals for True Scores Using the Skew-Normal Distribution

ERIC Educational Resources Information Center

Garcia-Perez, Miguel A.

2010-01-01

A recent comparative analysis of alternative interval estimation approaches and procedures has shown that confidence intervals (CIs) for true raw scores determined with the Score method--which uses the normal approximation to the binomial distribution--have actual coverage probabilities that are closest to their nominal level. It has also recently…

Levels of Conformity to Leader in Normal and Critical Situations

ERIC Educational Resources Information Center

Gündüz, Yüksel

2017-01-01

The aim of this study is to determine primary school, middle school, high school and university students' levels of conformity to leader in normal and critical situations. Experimental model was used in the research. Study group is comprised of 80 students chosen randomly from Karadeniz Bakir Primary School, Gazi Middle School, Kazim Karabekir…

APPLICATION OF ORGANIC IODINE SPECIES ANALYTICS: DETERMINING THYROID HORMONE STATUS IN ADULT DANIO RERIO AND DEVELOPING XENOPUS LAEVIS USING LC/ICP-MS

EPA Science Inventory

Disruption of normal thyroid function by xenobiotic chemicals is an important ecological issue. Theoretically, normal thyroid hormone (TH) homeostasis and action can be disrupted at several sites in the synthetic and elimination pathways. Indeed, xenobiotic chemicals, which are k...

Problem Child or Quirky Kid? A Commonsense Guide.

ERIC Educational Resources Information Center

Sommers-Flanagan, Rita; Sommers-Flanagan, John

Determining whether one's child is just "going through a phase," is just a bit quirky but developing normally, or is in need of professional help can be a complex and confusing process for parents. This book provides parents and caregivers with practical information to differentiate normal and problematic child development, offers tips…

Comprehensive Experiment--Clinical Biochemistry: Determination of Blood Glucose and Triglycerides in Normal and Diabetic Rats

ERIC Educational Resources Information Center

Jiao, Li; Xiujuan, Shi; Juan, Wang; Song, Jia; Lei, Xu; Guotong, Xu; Lixia, Lu

2015-01-01

For second year medical students, we redesigned an original laboratory experiment and developed a combined research-teaching clinical biochemistry experiment. Using an established diabetic rat model to detect blood glucose and triglycerides, the students participate in the entire experimental process, which is not normally experienced during a…

Maximal isometric muscle strength values obtained By hand-held dynamometry in children between 6 and 15 years of age.

PubMed

Escobar, Raul G; Munoz, Karin T; Dominguez, Angelica; Banados, Pamela; Bravo, Maria J

2017-01-01

In this study we aimed to determine the maximal isometric muscle strength of a healthy, normal-weight, pediatric population between 6 and 15 years of age using hand-held dynamometry to establish strength reference values. The secondary objective was determining the relationship between strength and anthropometric parameters. Four hundred normal-weight Chilean children, split into 10 age groups, separated by 1-year intervals, were evaluated. Each age group included between 35 and 55 children. The strength values increased with increasing age and weight, with a correlation of 0.83 for age and 0.82 for weight. The results were similar to those reported in previous studies regarding the relationships among strength, age, and anthropometric parameters, but the reported strength differed. These results provide normal strength parameters for healthy and normal-weight Chilean children between 6 and 15 years of age and highlight the relevance of ethnicity in defining reference values for muscle strength in a pediatric population. Muscle Nerve 55: 16-22, 2017. © 2016 Wiley Periodicals, Inc.

Economics of Future Growth in Photovoltaics Manufacturing; NREL (National Renewable Energy Laboratory)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basore, Paul; Chung, Donald; Buonassisi, Tonio

2015-06-14

The past decade’s record of growth in the photovoltaic manufacturing industry indicates that global investment in manufacturing capacity for photovoltaic modules tends to increase in proportion to the size of the industry. The slope of this proportionality determines how fast the industry will grow in the future. Two key parameters determine this slope. One is the annual global investment in manufacturing capacity normalized to the manufacturing capacity for the previous year (capacity-normalized capital investment rate, CapIR, units $/W). The other is how much capital investment is required for each watt of annual manufacturing capacity, normalized to the service life ofmore » the assets (capacity-normalized capital demand rate, CapDR, units $/W). If these two parameters remain unchanged from the values they have held for the past few years, global manufacturing capacity will peak in the next few years and then decline. However, it only takes a small improvement in CapIR to ensure future growth in photovoltaics. Any accompanying improvement in CapDR will accelerate that growth.« less

Measurement of the normalized 238U(n ,f )/235U(n ,f ) cross section ratio from threshold to 30 MeV with the NIFFTE fission Time Projection Chamber

NASA Astrophysics Data System (ADS)

Casperson, R. J.; Asner, D. M.; Baker, J.; Baker, R. G.; Barrett, J. S.; Bowden, N. S.; Brune, C.; Bundgaard, J.; Burgett, E.; Cebra, D. A.; Classen, T.; Cunningham, M.; Deaven, J.; Duke, D. L.; Ferguson, I.; Gearhart, J.; Geppert-Kleinrath, V.; Greife, U.; Grimes, S.; Guardincerri, E.; Hager, U.; Hagmann, C.; Heffner, M.; Hensle, D.; Hertel, N.; Higgins, D.; Hill, T.; Isenhower, L. D.; King, J.; Klay, J. L.; Kornilov, N.; Kudo, R.; Laptev, A. B.; Loveland, W.; Lynch, M.; Lynn, W. S.; Magee, J. A.; Manning, B.; Massey, T. N.; McGrath, C.; Meharchand, R.; Mendenhall, M. P.; Montoya, L.; Pickle, N. T.; Qu, H.; Ruz, J.; Sangiorgio, S.; Schmitt, K. T.; Seilhan, B.; Sharma, S.; Snyder, L.; Stave, S.; Tate, A. C.; Tatishvili, G.; Thornton, R. T.; Tovesson, F.; Towell, D. E.; Towell, R. S.; Walsh, N.; Watson, S.; Wendt, B.; Wood, L.; Yao, L.; Younes, W.; Niffte Collaboration

2018-03-01

The normalized 238U(n ,f )/235U(n ,f ) cross section ratio has been measured using the NIFFTE fission Time Projection Chamber (fissionTPC) from the reaction threshold to 30 MeV . The fissionTPC is a two-volume MICROMEGAS time projection chamber that allows for full three-dimensional reconstruction of fission-fragment ionization profiles from neutron-induced fission. The measurement was performed at the Los Alamos Neutron Science Center, where the neutron energy is determined from neutron time of-flight. The 238U(n ,f )/235U(n ,f ) ratio reported here is the first cross section measurement made with the fissionTPC, and will provide new experimental data for evaluation of the 238U(n ,f ) cross section, an important standard used in neutron-flux measurements. Use of a development target in this work prevented the determination of an absolute normalization, to be addressed in future measurements. Instead, the measured cross section ratio has been normalized to ENDF/B-VIII.β 5 at 14.5 MeV.

Atomistic modeling of the low-frequency mechanical modes and Raman spectra of icosahedral virus capsids

NASA Astrophysics Data System (ADS)

Dykeman, Eric C.; Sankey, Otto F.

2010-02-01

We describe a technique for calculating the low-frequency mechanical modes and frequencies of a large symmetric biological molecule where the eigenvectors of the Hessian matrix are determined with full atomic detail. The method, which follows order N methods used in electronic structure theory, determines the subset of lowest-frequency modes while using group theory to reduce the complexity of the problem. We apply the method to three icosahedral viruses of various T numbers and sizes; the human viruses polio and hepatitis B, and the cowpea chlorotic mottle virus, a plant virus. From the normal-mode eigenvectors, we use a bond polarizability model to predict a low-frequency Raman scattering profile for the viruses. The full atomic detail in the displacement patterns combined with an empirical potential-energy model allows a comparison of the fully atomic normal modes with elastic network models and normal-mode analysis with only dihedral degrees of freedom. We find that coarse-graining normal-mode analysis (particularly the elastic network model) can predict the displacement patterns for the first few (˜10) low-frequency modes that are global and cooperative.

Estimating the concentration of urea and creatinine in the human serum of normal and dialysis patients through Raman spectroscopy.

PubMed

de Almeida, Maurício Liberal; Saatkamp, Cassiano Junior; Fernandes, Adriana Barrinha; Pinheiro, Antonio Luiz Barbosa; Silveira, Landulfo

2016-09-01

Urea and creatinine are commonly used as biomarkers of renal function. Abnormal concentrations of these biomarkers are indicative of pathological processes such as renal failure. This study aimed to develop a model based on Raman spectroscopy to estimate the concentration values of urea and creatinine in human serum. Blood sera from 55 clinically normal subjects and 47 patients with chronic kidney disease undergoing dialysis were collected, and concentrations of urea and creatinine were determined by spectrophotometric methods. A Raman spectrum was obtained with a high-resolution dispersive Raman spectrometer (830 nm). A spectral model was developed based on partial least squares (PLS), where the concentrations of urea and creatinine were correlated with the Raman features. Principal components analysis (PCA) was used to discriminate dialysis patients from normal subjects. The PLS model showed r = 0.97 and r = 0.93 for urea and creatinine, respectively. The root mean square errors of cross-validation (RMSECV) for the model were 17.6 and 1.94 mg/dL, respectively. PCA showed high discrimination between dialysis and normality (95 % accuracy). The Raman technique was able to determine the concentrations with low error and to discriminate dialysis from normal subjects, consistent with a rapid and low-cost test.

Experimental investigation of internal tides generated by finite-height topography

NASA Astrophysics Data System (ADS)

Wang, Shuya; Chen, Xu; Wang, Jinhu; Meng, Jing

2018-06-01

Internal tides generated by finite-height topography are investigated in the laboratory, and the particle image velocimetry (PIV) technique is applied to measure the velocity fields. The energy, energy flux, and vertical mode structure of the internal tides are calculated and analyzed. The experimental results indicate that the strength of the wave field is mainly affected by the normalized topography height. The rays radiated from the taller topography are wider than those radiated from the lower topography. Both the experimental and theoretical results indicate that the normalized energy and energy flux of the internal tides are mainly determined by the normalized topography height, and the increase of the two quantities follows a quadratic function, and they almost remain unchanged with different normalized frequencies except for higher frequency. The percentage of energy for mode-1 and mode-2 internal tides is determined not only by frequency but also by topography height. In addition, an "inherent normalized frequency" is observed in the experiment, at which the percentage of energy for mode 1 and mode 2 does not vary with topography height. The decay rate of internal tide energy in the near field and far field is also estimated, with average values of 36.5 and 7.5%, respectively.

Gaussian fitting for carotid and radial artery pressure waveforms: comparison between normal subjects and heart failure patients.

PubMed

Liu, Chengyu; Zheng, Dingchang; Zhao, Lina; Liu, Changchun

2014-01-01

It has been reported that Gaussian functions could accurately and reliably model both carotid and radial artery pressure waveforms (CAPW and RAPW). However, the physiological relevance of the characteristic features from the modeled Gaussian functions has been little investigated. This study thus aimed to determine characteristic features from the Gaussian functions and to make comparisons of them between normal subjects and heart failure patients. Fifty-six normal subjects and 51 patients with heart failure were studied with the CAPW and RAPW signals recorded simultaneously. The two signals were normalized first and then modeled by three positive Gaussian functions, with their peak amplitude, peak time, and half-width determined. Comparisons of these features were finally made between the two groups. Results indicated that the peak amplitude of the first Gaussian curve was significantly decreased in heart failure patients compared with normal subjects (P<0.001). Significantly increased peak amplitude of the second Gaussian curves (P<0.001) and significantly shortened peak times of the second and third Gaussian curves (both P<0.001) were also presented in heart failure patients. These results were true for both CAPW and RAPW signals, indicating the clinical significance of the Gaussian modeling, which should provide essential tools for further understanding the underlying physiological mechanisms of the artery pressure waveform.

30 CFR 27.41 - Test to determine resistance to moisture.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Test to determine resistance to moisture. 27.41... determine resistance to moisture. Components, subassemblies, or assemblies, the normal functioning of which might be affected by moisture, shall be tested in atmospheres of high relative humidity (80 percent or...

30 CFR 27.41 - Test to determine resistance to moisture.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Test to determine resistance to moisture. 27.41... determine resistance to moisture. Components, subassemblies, or assemblies, the normal functioning of which might be affected by moisture, shall be tested in atmospheres of high relative humidity (80 percent or...

30 CFR 27.41 - Test to determine resistance to moisture.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Test to determine resistance to moisture. 27.41... determine resistance to moisture. Components, subassemblies, or assemblies, the normal functioning of which might be affected by moisture, shall be tested in atmospheres of high relative humidity (80 percent or...

30 CFR 27.41 - Test to determine resistance to moisture.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Test to determine resistance to moisture. 27.41... determine resistance to moisture. Components, subassemblies, or assemblies, the normal functioning of which might be affected by moisture, shall be tested in atmospheres of high relative humidity (80 percent or...

Sample Size Determination for One- and Two-Sample Trimmed Mean Tests

ERIC Educational Resources Information Center

Luh, Wei-Ming; Olejnik, Stephen; Guo, Jiin-Huarng

2008-01-01

Formulas to determine the necessary sample sizes for parametric tests of group comparisons are available from several sources and appropriate when population distributions are normal. However, in the context of nonnormal population distributions, researchers recommend Yuen's trimmed mean test, but formulas to determine sample sizes have not been…

William Wales and the 1769 transit of Venus: puzzle solving and the determination of the astronomical unit

NASA Astrophysics Data System (ADS)

Metz, Don

2009-05-01

According to Thomas Kuhn, a significant part of “normal science” is the fact gathering, empirical work which is intended to illustrate an existing paradigm. Some of this effort focuses on the determination of physical constants such as the astronomical unit (AU). For Kuhn, normal science is also what prepares students for membership in a particular scientific community and is embodied in some form in our science textbooks. However, neither Kuhn nor the textbook says much about the individuals who practice normal science, especially those who had been relegated to the “hack” duties of long and arduous measurement and calculation. In this paper, to provide a context for students of astronomy, I will outline the story of the determination of the AU and in particular the contribution of William Wales, an obscure British astronomer. Wales, toiling in the shadow of Halley (of Halley’s comet fame), Mason and Dixon (of Mason and Dixon line fame) and the infamous Captain Cook endured a brutal winter in northern Canada for a brief glimpse of the 1769 transit of Venus. In the end, Wales supplied one small piece of the puzzle in the determination of the AU and he exemplified the human spirit and persistence of a Kuhnian “puzzle solver”.

Increased determinism in brain electrical activity occurs in association with multiple sclerosis.

PubMed

Carrubba, Simona; Minagar, Alireza; Chesson, Andrew L; Frilot, Clifton; Marino, Andrew A

2012-04-01

Increased determinism (decreased complexity) of brain electrical activity has been associated with some brain diseases. Our objective was to determine whether a similar association occurred for multiple sclerosis (MS). Ten subjects with a relapsing-remitting course of MS who were in remission were studied; the controls were age- and gender-matched clinically normal subjects. Recurrence plots were calculated using representative electroencephalogram (EEG) epochs (1-7 seconds) from six derivations; the plots were quantified using the nonlinear variables percent recurrence (%R) and percent determinism (%D). The results were averaged over all derivations for each participant, and the means were compared between the groups. As a linear control procedure the groups were also compared using spectral analysis. The mean±SD of %R for the MS subjects was 6·6±1·3%, compared with 5·1±1·3% in the normal group (P = 0·017), indicating that brain activity in the subjects with MS was less complex, as hypothesized. The groups were not distinguishable using %D or spectral analysis. Taken together with our earlier report that %R could be used to discriminate between MS and normal subjects based on the ability to exhibit evoked potentials, the evidence suggests that complexity analysis of the EEG has potential for development as a diagnostic test for MS.

Impact of heat release on strain rate field in turbulent premixed Bunsen flames

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coriton, Bruno Rene Leon; Frank, Jonathan H.

2016-08-10

The effects of combustion on the strain rate field are investigated in turbulent premixed CH 4/air Bunsen flames using simultaneous tomographic PIV and OH LIF measurements. Tomographic PIV provides three-dimensional velocity measurements, from which the complete strain rate tensor is determined. The OH LIF measurements are used to determine the position of the flame surface and the flame-normal orientation within the imaging plane. This combination of diagnostic techniques enables quantification of divergence as well as flame-normal and tangential strain rates, which are otherwise biased using only planar measurements. Measurements are compared in three lean-to-stoichiometric flames that have different amounts ofmore » heat release and Damköhler numbers greater than unity. The effects of heat release on the principal strain rates and their alignment relative to the local flame normal are analyzed. The extensive strain rate preferentially aligns with the flame normal in the reaction zone, which has been indicated by previous studies. The strength of this alignment increases with increasing heat release and, as a result, the flame-normal strain rate becomes highly extensive. These effects are associated with the gas expansion normal to the flame surface, which is largest for the stoichiometric flame. In the preheat zone, the compressive strain rate has a tendency to align with the flame normal. Away from the flame front, the flame – strain rate alignment is arbitrary in both the reactants and products. The flame-tangential strain rate is on average positive across the flame front, and therefore the turbulent strain rate field contributes to the enhancement of scalar gradients as in passive scalar turbulence. As a result, increases in heat release result in larger positive values of the divergence as well as flame-normal and tangential strain rates, the tangential strain rate has a weaker dependence on heat release than the flame-normal strain rate and the divergence.« less

Protein Degradation in Normal and Beige (Chediak-Higashi) Mice

PubMed Central

Lyons, Robert T.; Pitot, Henry C.

1978-01-01

The beige mouse, C57BL/6 (bg/bg), is an animal model for the Chediak-Higashi syndrome in man, a disease characterized morphologically by giant lysosomes in most cell types. Half-lives for the turnover of [14C]bicarbonate-labeled total soluble liver protein were determined in normal and beige mice. No significant differences were observed between the normal and mutant strain for both rapidly and slowly turning-over classes of proteins. Glucagon treatment during the time-course of protein degradation had similar effects on both normal and mutant strains and led to the conclusion that the rate of turnover of endogenous intracellular protein in the beige mouse liver does not differ from normal. The rates of uptake and degradation of an exogenous protein were determined in normal and beige mice by intravenously injecting 125I-bovine serum albumin and following, in peripheral blood, the loss with time of phosphotungstic acid-insoluble bovine serum albumin and the parallel appearance of phosphotungstic acid-soluble (degraded) material. No significant differences were observed between beige and normal mice in the uptake by liver lysosomes of 125I-bovine serum albumin (t½ = 3.9 and 2.8 h, respectively). However, it was found that lysosomes from livers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slower than normal (t½ = 6.8 and 3.1 h, respectively). This defect in beige mice could be corrected by chronic administration of carbamyl choline (t½ = 3.5 h), a cholinergic agonist which raises intracellular cyclic GMP levels. However, no significant differences between normal and beige mice were observed either in the ability of soluble extracts of liver and kidney to bind [3H]cyclic GMP in vitro or in the basal levels of cyclic AMP in both tissues. The relevance of these observations to the presumed biochemical defect underlying the Chediak-Higashi syndrome is discussed. PMID:202611

Sonographic Assessment of the Normal Dimensions of Liver, Spleen, and Kidney in Healthy Children at Tertiary Care Hospital.

PubMed

Thapa, N B; Shah, S; Pradhan, A; Rijal, K; Pradhan, A; Basnet, S

2015-01-01

Background Ultrasonography is one of the most common imaging modality to measure dimensions of visceral organs in children. However, the normal limit of size of visceral organs according to age and body habitus has not been specified in the standard textbooks. This might result in under detection of organomegaly in pediatrics population. Objective The objective of this study was to determine the normal range of dimensions for the liver, spleen, and kidney in healthy children. Method This is prospective cross-sectional, hospital-based study done at Tertiary-care teaching hospital. Participants included 272 pediatric subjects (152 male and 120 female) with normal physical or sonographic findings who were examined because of problems unrelated to the measured organs. The subjects were one month to 15 year (180 months) old. All measured organs were sonographically normal. Relationships of the dimensions of these organs with sex, age, body weight and height were investigated. Limits of normal dimensions of these organs were defined. Result Normal length of liver, kidneys and spleen were obtained sonographically for 272 children (152 male [55.9%] and 120 female [44.1%]) in the age group from 1 months to 15 (180 months) years. The mean age was 45.78 months (SD, 44.73). The measured dimensions of all these organs showed highest correlation with height and age so the descriptive analysis of the organ dimensions (mean, minimum, and maximum values, SD and 5th and 95th percentiles) were expressed in 10 age groups along with height range of the included children. The mean length of right kidney was shorter than the left kidney length, and the difference was statistically significant (p = 0.001). Conclusion This study provides practical and comprehensive guide to the normal visceral organ dimension in pediatric population. The normal range limit of the liver, spleen, and kidney determined in this study could be used as a reference in daily practice in local radiology clinics.

Group normalization for genomic data.

PubMed

Ghandi, Mahmoud; Beer, Michael A

2012-01-01

Data normalization is a crucial preliminary step in analyzing genomic datasets. The goal of normalization is to remove global variation to make readings across different experiments comparable. In addition, most genomic loci have non-uniform sensitivity to any given assay because of variation in local sequence properties. In microarray experiments, this non-uniform sensitivity is due to different DNA hybridization and cross-hybridization efficiencies, known as the probe effect. In this paper we introduce a new scheme, called Group Normalization (GN), to remove both global and local biases in one integrated step, whereby we determine the normalized probe signal by finding a set of reference probes with similar responses. Compared to conventional normalization methods such as Quantile normalization and physically motivated probe effect models, our proposed method is general in the sense that it does not require the assumption that the underlying signal distribution be identical for the treatment and control, and is flexible enough to correct for nonlinear and higher order probe effects. The Group Normalization algorithm is computationally efficient and easy to implement. We also describe a variant of the Group Normalization algorithm, called Cross Normalization, which efficiently amplifies biologically relevant differences between any two genomic datasets.

Group Normalization for Genomic Data

PubMed Central

Ghandi, Mahmoud; Beer, Michael A.

2012-01-01

Data normalization is a crucial preliminary step in analyzing genomic datasets. The goal of normalization is to remove global variation to make readings across different experiments comparable. In addition, most genomic loci have non-uniform sensitivity to any given assay because of variation in local sequence properties. In microarray experiments, this non-uniform sensitivity is due to different DNA hybridization and cross-hybridization efficiencies, known as the probe effect. In this paper we introduce a new scheme, called Group Normalization (GN), to remove both global and local biases in one integrated step, whereby we determine the normalized probe signal by finding a set of reference probes with similar responses. Compared to conventional normalization methods such as Quantile normalization and physically motivated probe effect models, our proposed method is general in the sense that it does not require the assumption that the underlying signal distribution be identical for the treatment and control, and is flexible enough to correct for nonlinear and higher order probe effects. The Group Normalization algorithm is computationally efficient and easy to implement. We also describe a variant of the Group Normalization algorithm, called Cross Normalization, which efficiently amplifies biologically relevant differences between any two genomic datasets. PMID:22912661

Industrial process surveillance system

DOEpatents

Gross, Kenneth C.; Wegerich, Stephan W.; Singer, Ralph M.; Mott, Jack E.

1998-01-01

A system and method for monitoring an industrial process and/or industrial data source. The system includes generating time varying data from industrial data sources, processing the data to obtain time correlation of the data, determining the range of data, determining learned states of normal operation and using these states to generate expected values, comparing the expected values to current actual values to identify a current state of the process closest to a learned, normal state; generating a set of modeled data, and processing the modeled data to identify a data pattern and generating an alarm upon detecting a deviation from normalcy.

Industrial process surveillance system

DOEpatents

Gross, K.C.; Wegerich, S.W.; Singer, R.M.; Mott, J.E.

1998-06-09

A system and method are disclosed for monitoring an industrial process and/or industrial data source. The system includes generating time varying data from industrial data sources, processing the data to obtain time correlation of the data, determining the range of data, determining learned states of normal operation and using these states to generate expected values, comparing the expected values to current actual values to identify a current state of the process closest to a learned, normal state; generating a set of modeled data, and processing the modeled data to identify a data pattern and generating an alarm upon detecting a deviation from normalcy. 96 figs.