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Sample records for sarcoplasmic reticulum suggesting

  1. Decavanadate interactions with sarcoplasmic reticulum calcium pump

    OpenAIRE

    2007-01-01

    Although not stable, once formed, decameric vanadate (V10) disintegration is in general slow enough to allow the study of its effects even in the micromolar range. Besides, it may become inaccessible to decomposition due to their specific interaction upon target proteins such as the Ca2+-ATPase from sarcoplasmic reticulum (SR). Characterization of the vanadate solutions and interactions with compounds containing phosphate as well as with the SR Ca2+-ATPase was analysed by 51V NMR spectr...

  2. The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

    Science.gov (United States)

    Papp, S; Kracke, G; Joshi, N; Martonosi, A

    1986-01-01

    The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence. PMID:2937461

  3. Dynamic Changes in Sarcoplasmic Reticulum Structure in Ventricular Myocytes

    Directory of Open Access Journals (Sweden)

    Amanda L. Vega

    2011-01-01

    sarcoplasmic reticulum (SR and the sarcolemma where Ca2+ release is activated. Here, we tested the hypothesis that the SR is a structurally inert organelle in ventricular myocytes. Our data suggest that rather than being static, the SR undergoes frequent dynamic structural changes. SR boutons expressing functional ryanodine receptors moved throughout the cell, approaching or moving away from the sarcolemma of ventricular myocytes. These changes in SR structure occurred in the absence of changes in [Ca2+] during EC coupling. Microtubules and the molecular motors dynein and kinesin 1(Kif5b were important regulators of SR motility. These findings support a model in which the SR is a motile organelle capable of molecular motor protein-driven structural changes.

  4. Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

    Science.gov (United States)

    Kang, J J; Cheng, Y W

    1998-02-01

    The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

  5. Discontinuity of sarcoplasmic reticulum in the mid-sarcomere region in flight muscle of dragonflies.

    Science.gov (United States)

    de Eguileor, M; Valvassori, R; Lanzavecchia, G

    1980-01-01

    The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.

  6. Affi-gel blue treatment simplifies the protein composition of sarcoplasmic reticulum vesicles.

    Science.gov (United States)

    Papp, S; Dux, L; Martonosi, A

    1986-04-01

    Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

  7. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  8. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  9. Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation.

    Science.gov (United States)

    Heilmann, C; Pette, D

    1979-02-01

    Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+-dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+-dependent and Ca2+, Mg2+-dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.

  10. Interactions of vanadate oligomers with sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Aureliano, M; Mdeira, V M

    1994-04-28

    Upon addition of sarcoplasmic reticulum (SR), the line width of tetrameric vanadate signal of 51V-NMR spectra narrowed in the presence of ATP and Ca2+, whereas monomeric vanadate line widths were broadened. Thus, ATP decreases the affinity of the enzyme for tetravanadate whereas it induces the interaction with monomeric vanadate. In the presence of Ca2+ it was observed that tetrameric and decameric vanadate bind to SR ATPase whereas monomeric vanadate only binds to SR when ATP is present. However, decameric vanadate clearly differs from vanadate oligomers present in monovanadate solutions in preventing the accumulation of Ca2+ by sarcoplasmic reticulum (SR) vesicles coupled to ATP hydrolysis. Mg2+ increased the inhibitory effect promoted by decavanadate whereas a slight enhancement of Ca2+ uptake was observed in the presence of monovanadate. For 5 mM Mg2+, a nominal 2 mM vanadium 'decavanadate' solution containing about 190 to 200 microM decameric and less than 100 microM monomeric species depressed the rate of Ca2+ uptake by 50% whereas a nominal 2 mM monovanadate solution containing about 662 microM monomeric, 143 microM dimeric and 252 microM tetrameric species had no effect on the rate of Ca2+ accumulation. However, 2 mM 'decavanadate' inhibits by 75% the SR Ca(2+)-ATPase activity whereas the presence of 2 mM 'monovanadate' produces an inhibitory effect below 50%. Therefore, the Ca:ATP stoichiometry of Ca2+ transport is enhanced by monovanadate. In the presence of oxalate, inhibition of SR Ca(2+)-ATPase activity by these solutions is enhanced to 97% and 86% whereas in the presence of the ionophore lasalocid, the inhibitory values were 87% and 19% for 2 mM decavanadate and 2 mM monovanadate solutions, respectively. Apparently, the increase of vesicular Ca2+ concentration counteracts monovanadate inhibition of SR Ca(2+)-ATPase activity but it does not significantly affect decavanadate inhibition.

  11. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  12. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  13. Ion pathways in the sarcoplasmic reticulum Ca2+-ATPase.

    Science.gov (United States)

    Bublitz, Maike; Musgaard, Maria; Poulsen, Hanne; Thøgersen, Lea; Olesen, Claus; Schiøtt, Birgit; Morth, J Preben; Møller, Jesper Vuust; Nissen, Poul

    2013-04-12

    The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.

  14. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  15. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  16. Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

    DEFF Research Database (Denmark)

    Frías, J A; Cadefau, J A; Prats, C;

    2005-01-01

    -migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural...... damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may......Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit...

  17. Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.

    Science.gov (United States)

    Paran, Christopher W; Zou, Kai; Ferrara, Patrick J; Song, Haowei; Turk, John; Funai, Katsuhiko

    2015-12-01

    Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.

  18. Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Suárez-Isla, B. A.; Irribarra, V.; Oberhauser, A.; Larralde, L.; Bull, R.; Hidalgo, C.; Jaimovich, E.

    1988-01-01

    Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process. PMID:2852037

  19. Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

    Science.gov (United States)

    Restall, C J; Coke, M; Murray, E K; Chapman, D

    1985-02-28

    The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

  20. Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

    DEFF Research Database (Denmark)

    Nielsen, J S; Sahlin, K; Ørtenblad, N

    2007-01-01

    AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity...

  1. Sub-sarcolemmal swelling of sarcoplasmic reticulum after isometric contractions in rat semimembranosus lateralis muscle

    NARCIS (Netherlands)

    Willems, M.E.T.; Huijing, P.A.J.B.M.; Friden, J.

    1999-01-01

    The decline in isometric force, swelling of sarcoplasmic reticulum and loss of desmin was measured in semimembranosus lateralis muscle of male Wistar rats immediately after a short series of brief (500 ms) maximal isometric contractions. For the active muscle, the series ended below (protocol A) and

  2. Effect of losartan on sarcoplasmic reticulum Ca2+ handing proteins in heart failure rabbit

    Institute of Scientific and Technical Information of China (English)

    姚艳

    2006-01-01

    Objective To investigate the effects of losartan on mRNA expression of myocardial sarcoplasmic reticulum calcium handling proteins (SERCA2, RyR2 and PLB) and the role of which in prevention of chronic heart failure in rabbit. Methods After chronic heart failure was

  3. Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

    Directory of Open Access Journals (Sweden)

    V.A. Vizotto

    2007-01-01

    Full Text Available We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2, phospholamban (PLB, and ryanodine channel (RYR2 mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01. The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively. Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

  4. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, J. [Lodz Univ. (Poland)

    1995-12-31

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 {mu}M and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca{sup 2+} accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca{sup 2+}, the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs.

  5. Cylindrical Spirals in Skeletal Muscles Originate From the Longitudinal Sarcoplasmic Reticulum.

    Science.gov (United States)

    Xu, Jing-Wen; Liu, Fu-Chen; Li, Wei; Zhao, Yu-Ying; Zhao, Dan-Dan; Luo, Yue-Bei; Lu, Jian-Qiang; Yan, Chuan-Zhu

    2016-02-01

    Cylindrical spirals (CSs) are rare but distinct subsarcolemmal accumulations in skeletal muscle fibers. To date, CSs have been reported in only 16 patients with a variety of neuromuscular conditions. The origin and composition of CSs are unknown, although there are some morphologic similarities between CSs and tubular aggregates (TAs). To clarify the nature of CSs, we characterized the sarcoplasmic reticulum (SR) and other intracellular membrane system proteins in CSs of muscle biopsies from 2 adult Chinese siblings. Immunohistochemical studies revealed subsarcolemmal immunoreactivity for sarco/endoplasmic reticulum Ca2þ-ATPase 1 (SERCA 1) in the longitudinal SR, but no immunoreactivity for calsequestrin in the terminal cisternae or type 1 ryanodine receptor (RYR1) in the junctional SR. Muscles biopsied from 2 patients with TAs showed immunoreactivity not only for SERCA1 but also for other SR proteins, including calsequestrin and RYR1. CSs exhibited no immunoreactivity for the Golgi apparatus marker GM130, the nuclear membrane emerin, desmin, the autophagosome marker LC3, the lysosomal membrane marker LAMP2, dystrophin, or myosin. Our results suggest CSs may originate only from the longitudinal SR, whereas TAs are composed of both the junctional and longitudinal SR. Immunochemical staining with antibodies against calsequestrin and RYR1 help to distinguish these 2 pathological alterations.

  6. Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle.

    Science.gov (United States)

    Sumida, M; Wang, T; Mandel, F; Froehlich, J P; Schwartz, A

    1978-12-25

    Current evidence supports similar functions and mechanisms for cardiac sarcoplasmic reticulum (CSR) as for skeletal sarcoplasmic reticulum (SSR). It is thought that the slower relaxation rate of cardiac muscle compared to fast skeletal muscle reflects the lower ATPase activity and calcium transport of CSR. Possible quantitative differences is phosphorylation, dephosphorylation, and calcium transport of the isolated preparations are studied using a quench-flow apparatus. The results show that both CSR and SSR bind calcium tightly in the absence of ATP, and coupling of E approximately P formation and calcium transport occurs in the transient phase of ATP hydrolysis. The rate of phosphorylation (t-1/2 - 10 ms) of sarcoplasmic reticulum (SR) preloaded with calcium is the same for cardiac and skeletal preparations. However, the rates of dissociation of extra vesicular calcium (10 s-1 versus 15 s-1), phosphorylation of calcium-free SR, and dephosphorylation of E approximately P (8 s-1 versus 12 s-1) are lower for CSR than for SSR. By computer simulation, the apparent rate constants associated with the reduced rates of phosphorylation of calcium-free SR were: 12 s-1 for CSR and 63 s-1 for SSR in the presence of high Mg2+. The difference in the rates may be partly responsible for the lower levels of ATPase and calcium transport activity with characterize cardiac muscle preparations.

  7. Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles

    Science.gov (United States)

    Treves, Susan; Vukcevic, Mirko; Maj, Marcin; Thurnheer, Raphael; Mosca, Barbara; Zorzato, Francesco

    2009-01-01

    In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca2+ stored in the sarcoplasmic reticulum by a process called excitation–contraction coupling. Excitation–contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation–contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca2+ release channel, and Ca2+ storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation–contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation–contraction coupling is important in order to understand how mutations in genes involved in Ca2+ signalling cause neuromuscular disorders. PMID:19403606

  8. Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles.

    Science.gov (United States)

    Kaasik, A; Minajeva, A; Paju, K; Eimre, M; Seppet, E K

    1997-11-01

    The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca(2+)-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca(2+)-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca(2+)-pump in atria compared to ventricles.

  9. Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

    Science.gov (United States)

    Szentesi, P; Zaremba, R; Stienen, G J

    1998-08-01

    Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

  10. Vanadate oligoanions interact with the proton ejection by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M; Madeira, V M

    1994-11-30

    Decameric vanadate differs from other oligomeric vanadate species in inhibiting Ca2+ uptake and H+ ejection promoted by sarcoplasmic reticulum ATPase. A decavanadate solution, 2 mM in total vanadium, containing about 200 microM decameric species, inhibits by about 50% the uptake of Ca2+ and by 75% the H+ ejection, whereas 2 mM nominal monovanadate slightly increases the uptake of Ca2+ and inhibits the ejection of H+ by 25%. Moreover, decavanadate linearly increases the Ca2+/H+ ratio, whereas monovanadate mimicks decavanadate behavior only at concentrations up to 1.2 mM. For higher concentrations of monovanadate, this effect is reversed probably due to the formation of metavanadates, namely tetravandate. It is concluded that Ca2+ uptake is tightly coupled to proton ejection through molecular events that are sensitive to the interaction of vanadate species. Apparently, the stoichiometry is variable and modulated by molecular events involved in vanadate interaction suggesting alterations in the energetic coupling associated with Ca2+ translocation.

  11. Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

    Science.gov (United States)

    Chen, L Y; Chen, X; Tian, X L; Yu, X H

    2000-10-01

    To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

  12. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    OpenAIRE

    1993-01-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular...

  13. Alterations in mitochondria and sarcoplasmic reticulum from heart and skeletal muscle of horizontally casted primates

    Science.gov (United States)

    Sordahl, L. A.; Stone, H. L.

    1982-01-01

    Horizontally body-casted rhesus monkeys are used as an animal model in order to study the physiological changes known as cardiovascular deconditioning which occur during weightless conditions. No difference was found between the experimental and control animals in heart mitochondrial oxidative phosphorylation which indicates that no apparent changes occurred in the primary energy-producing system of the heart. A marked increase in cytochrome oxidase activity was observed in the casted primate heart mitochondria compared to controls, while a 25% decrease in respiratory substrate-supported calcium uptake was found in casted primate heart mitochondria compared to controls. Sacroplasmic reticulum isolated from the primate hearts revealed marked changes in calcium transport activities. It is concluded that the marked depression in cardiac sarcoplasmic reticulum functions indicates altered calcium homeostasis in the casted-primate heart which could be a factor in cardiovascular deconditioning.

  14. Vanadate oligomer inhibition of passive and active Ca2+ translocation by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M

    2000-05-30

    'Monovanadate' containing mainly monomeric, dimeric and tetrameric vanadate species or 'decavanadate', containing mainly decameric vanadate species inhibits the passive and the active efflux of Ca2+ through the sarcoplasmic reticulum calcium pump. When the efflux of Ca2+ by sarcoplasmic reticulum vesicles is not associated with ATP synthesis both vanadate solutions inhibit the passive efflux of Ca2+. However, only 'decavanadate' exerts noticeable effects when the efflux of Ca2+ is associated with ATP synthesis being the active efflux of Ca2+ almost completely inhibited by decameric species concentration as low as 40 microM.

  15. Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding.

    Science.gov (United States)

    Feher, J J; Davis, M D

    1991-03-01

    The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.

  16. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  17. H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

    Science.gov (United States)

    Yu, X; Carroll, S; Rigaud, J L; Inesi, G

    1993-04-01

    The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

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

    Science.gov (United States)

    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

  19. Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate.

    Science.gov (United States)

    Fraqueza, Gil; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-02-01

    Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca(2+)-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca(2+)-ATPase, with the following IC(50) values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb(10)), is the strongest P-type enzyme inhibitor, after decavanadate (V(10)). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca(2+)-ATPase stoichiometry. Furthermore, V(10) binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H(2)VO(4)(2-); V(1)) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca(2+)-ATPase, with a dissociation constant, k(d) of 1 μM(-1). The interaction of decavanadate V(10)O(28)(6-) (V(10)) with Ca(2+)-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb(10)O(28)(6-) (Nb(10)), whereas no significant effects were

  20. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    OpenAIRE

    Pierce, D H; Scarpa, A.; Trentham, D R; Topp, M. R.; Blasie, J K

    1983-01-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane...

  1. Quantitative Measurement of Ca2+ in the Sarcoplasmic Reticulum Lumen of Mammalian Skeletal Muscle

    Science.gov (United States)

    Ziman, Andrew P.; Ward, Christopher W.; Rodney, George G.; Lederer, W. Jonathan; Bloch, Robert J.

    2010-01-01

    Skeletal muscle stores Ca2+ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca2+ in the SR ([Ca2+]SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca2+]SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca2+]SR, free is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca2+]SR, free to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca2+ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca2+ and for future studies of the role of SR Ca2+ in healthy and diseased mammalian muscle. PMID:20959112

  2. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid.

    Science.gov (United States)

    Taffet, G E; Pham, T T; Bick, D L; Entman, M L; Pownall, H J; Bick, R J

    1993-01-01

    Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

  3. Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs

    2000-01-01

    to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which......The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate...... during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P

  4. Alteration of Sarcoplasmic Reticulum Ca2+ Release in Skeletal Muscle from Calpain 3-Deficient Mice

    Directory of Open Access Journals (Sweden)

    Govindan Dayanithi

    2009-01-01

    Full Text Available Mutations of Ca2+-activated proteases (calpains cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

  5. Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

    Science.gov (United States)

    Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

    2015-11-01

    The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

  6. Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

    Science.gov (United States)

    Chen, L; Sumbilla, C; Lewis, D; Zhong, L; Strock, C; Kirtley, M E; Inesi, G

    1996-05-01

    Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

  7. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Science.gov (United States)

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

  8. Cellular mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin-induced rat ventricular hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Lai-jing SONG; Guan-lei WANG; Jie LIU; Qin-ying QIU; Jing-hua OU; Yong-yuan GUAN

    2008-01-01

    Aim:To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods:Echocardiography was used to confirm the establishment of the cardiac hypertro-phy model. The confocal microscopy and fluorescent indicator Fluo-3 was ap-plied to examine the intracellular Ca2+ concentration ([Ca2+]I), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results:L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]I increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion:The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

  9. Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.

    Science.gov (United States)

    Picard, Martin; Dahmane, Tassadite; Garrigos, Manuel; Gauron, Carole; Giusti, Fabrice; le Maire, Marc; Popot, Jean-Luc; Champeil, Philippe

    2006-02-14

    Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.

  10. Matching of sarcoplasmic reticulum and contractile properties in rat fast- and slow-twitch muscle fibres.

    Science.gov (United States)

    Trinh, Huong H; Lamb, Graham D

    2006-07-01

    1. The twitch characteristics (fast-twitch or slow-twitch) of skeletal muscle fibres are determined not only by the contractile apparatus properties of the fibre, but also by the time-course of Ca2+ release and re-uptake by the sarcoplasmic reticulum (SR). The present study examined, in individual fibres from non-transforming muscle of the rat, whether particular SR properties are matched to the contractile apparatus properties of the fibre, in particular in the case of fibres with fast-twitch contractile apparatus located in a slow-twitch muscle, namely the soleus. 2. Force was recorded in single, mechanically skinned fibres from extensor digitorum longus (EDL), gastrocnemius, peroneus longus and soleus muscles. Using repeated cycles in which the SR was emptied of all releasable Ca2+ and then reloaded, it was possible to determine the relative amount of Ca2+ present in the SR endogenously, the maximum SR capacity and the rate of Ca2+ loading. The sensitivity of the contractile apparatus to Ca2+ and Sr2+ was used to classify the fibres as fast-twitch (FT), slow-twitch (ST) or mixed (< 3% of the fibres examined) and thereby identify the likely troponin C and myosin heavy chain types present. 3. There was no significant difference in SR properties between the groups of FT fibres obtained from the four different muscles, including soleus. Despite some overlap in the SR properties of individual fibres between the FT and ST groups, the properties of the FT fibres in all four muscles studied were significantly different from those of the ST and mixed fibres. 4. In general, in FT fibres the SR had a larger capacity and the endogenous Ca2+ content was a relatively lower percentage of maximum compared with ST fibres. Importantly, in terms of their SR properties, FT fibres from soleus muscle more closely resembled FT fibres from other muscles than they did ST fibres from soleus muscle.

  11. Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

    Directory of Open Access Journals (Sweden)

    Yael Yaniv

    Full Text Available BACKGROUND: Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP. OBJECTIVE: To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity. RESULTS: Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84. CONCLUSION: A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.

  12. CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency dependent acceleration of relaxation and Ca2+ current facilitation

    OpenAIRE

    Picht, Eckard; DeSantiago, Jaime; Huke, Sabine; Kaetzel, Marcia A.; Dedman, John R.; Bers, Donald M.

    2006-01-01

    Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca2+ current (ICa) facilitation, enhanced sarcoplasmic reticulum (SR) Ca2+ release and frequency dependent acceleration of relaxation (FDAR) via enhanced SR Ca2+ uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaM-KII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide...

  13. Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump.

    Science.gov (United States)

    Aureliano, Manuel; Tiago, Teresa; Gândara, Ricardo M C; Sousa, Andrea; Moderno, A; Kaliva, M; Salifoglou, A; Duarte, Rui O; Moura, José J G

    2005-12-01

    Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.

  14. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  15. Effects of prior exercise and a low-carbohydrate diet on muscle sarcoplasmic reticulum function during cycling in women.

    Science.gov (United States)

    Duhamel, T A; Green, H J; Perco, J G; Ouyang, J

    2006-09-01

    The effects of exercise and diet on sarcoplasmic reticulum Ca(2+)-cycling properties in female vastus lateralis muscle were investigated in two groups of women following four different conditions. The conditions were 4 days of a low-carbohydrate (Lo CHO) and glycogen-depleting exercise plus a Lo CHO diet (Ex + Lo CHO) (experiment 2) and 4 days of normal CHO (Norm CHO) and glycogen-depleting exercise plus Norm CHO (Ex + Norm CHO) (experiment 1). Peak aerobic power (Vo2peak)) was 38.1 +/- 1.4 (SE); n = 9 and 35.6 +/- 1.4 ml.kg(-1).min(-1); n = 9, respectively. Sarcoplasmic reticulum properties measured in vitro in homogenates (micromol.g protein(-1).min(-1)) indicated exercise-induced reductions (P 30, 60 min > fatigue), Ca(2+) uptake (0 > 30 > 60 min, fatigue), and Ca(2+) release, both phase 1 (0, 30 > 60 min, fatigue) and phase 2 (0 > 30, 60 min, fatigue; 30 min > fatigue) in Norm CHO. Exercise was without effect in altering the Hill slope (n(H)), defined as the slope of relationship between Ca(2+)-ATPase activity and Ca(2+) concentration. No differences were observed between Norm CHO and Ex+Norm CHO. Compared with Norm CHO, Lo CHO resulted in a lower (P cycling and that, with the exception of Ca(2+) release, a glycogen-depleting session of exercise before Lo CHO can reverse the effects.

  16. Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

    Science.gov (United States)

    Salazar-Cantú, Ayleen; Pérez-Treviño, Perla; Montalvo-Parra, Dolores; Balderas-Villalobos, Jaime; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

    2016-08-15

    In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

  17. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    Science.gov (United States)

    McGarry, S J; Williams, A J

    1993-04-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or

  18. Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.

    Science.gov (United States)

    Baylor, S M; Chandler, W K; Marshall, M W

    1983-01-01

    Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7-3.9 microns. In fibres heavily injected with dye (1.5-2.2 mM-dye) at least 0.1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2-0.3 mM-Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2

  19. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    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.

  20. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology

    Institute of Scientific and Technical Information of China (English)

    范平

    2012-01-01

    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  1. Sarcoplasmic reticulum calcium mobilization in right ventricular pressure-overload hypertrophy in the ferret: relationships to diastolic dysfunction and a negative treppe.

    Science.gov (United States)

    Gwathmey, J K; Morgan, J P

    1993-03-01

    In a model of right-ventricular pressure-overload hypertrophy (POH) in the ferret, action potential duration (to 90% repolarization) was found to be significantly longer (228 +/- 11 vs 314 +/- 12 ms) with no change in amplitude (85 +/- 3 vs 85 +/- 2 mV) or resting membrane potential (-79 +/- 1.5 vs -79 +/- 1 mV) for control and POH, respectively. Peak sarcoplasmic reticulum Ca2+ release (expressed as the logarithm of the fractional luminescence, -4.2 +/- 0.1 vs -4.4 +/- 0.3) and resting calcium concentrations (-5.5 +/- 0.1 vs -5.7 +/- 0.1) were not different between the two groups (control vs POH respectively). Muscles from control and POH animals demonstrated a positive force/interval relationship in the presence of physiological extracellular [Ca2+]. However, unlike muscles from control animals, muscles from animals with POH subjected to increasing frequencies of contraction in the presence of increased extracellular [Ca2+] demonstrated further impairment of diastolic relaxation and a negative treppe. Exposure of muscles from POH animals to isoproterenol returned the slowed Ca2+ uptake by the sarcoplasmic reticulum as detected with aequorin to control values, although the relaxation phase of the isometric twitch remained prolonged compared to non-hypertrophied muscles. Exposure to milrinone also abbreviated the time course of the intracellular Ca2+ transient, but did not return it to that seen in normal myocardium. The exposure of non-hypertrophied isolated muscles to caffeine resulted in similar prolongation of the isometric twitch duration to that seen in hypertrophied myocardium. Results of these experiments suggest that impaired muscle relaxation in POH reflects changes at the level of the myofilaments. Thus, although slowed intracellular calcium mobilization contributes to diastolic relaxation abnormalities, it can not be the sole factor responsible for the slowed relaxation as has been suggested.

  2. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2.......5) between 60 and 250 muM. Results indicated that interaction between SR Ca2+-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated. (C) 2002 Elsevier Science Inc. All rights reserved....

  3. [Calcium transport in sarcoplasmic reticulum in the presence of AR-L 115 BS].

    Science.gov (United States)

    Hasselbach, W

    1981-01-01

    2-[(2-Methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS) is a substance with positive inotropic activity which does not influence the activity of the sarcoplasmic calcium pump. It can, therefore, be expected that AR-L 115 BS does not interfere with the distribution and movement of calcium in the resting and active muscle.

  4. Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

    Science.gov (United States)

    Picher, M; Decrouy, A; Rousseau, E

    1996-02-21

    Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

  5. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

    Science.gov (United States)

    Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G

    1998-02-01

    The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase

  6. Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

    Science.gov (United States)

    Launikonis, B S; Stephenson, D G

    1997-10-15

    1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 micrograms ml-1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min-1. Saponin concentrations up to 150 micrograms ml-1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 micrograms ml-1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 micrograms ml-1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min-1 in the presence of 150 micrograms ml-1 saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 microM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 microns) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 micrograms ml-1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species

  7. Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

    OpenAIRE

    1988-01-01

    We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneo...

  8. Luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum Ca2+-ATPase 2a

    Science.gov (United States)

    Hu, Wenjing; Xu, Tongda; Wu, Pei; Pan, Defeng; Chen, Junhong; Chen, Jing; Zhang, Buchun; Zhu, Hong; Li, Dongye

    2017-01-01

    We previously found that luteolin (Lut) appeared to improve the contractility of cardiomyocytes during ischemia/reperfusion in rats. The enhancement was associated with the alteration in sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). This finding prompted us to consider if the mechanism worked in heart failure (HF). We studied the regulation of SERCA2a by Lut in failing cardiomyocytes and intact heart of rats. Improvement of contractility and the mechanisms centered on SERCA2a were studied in isolated cardiomyocytes and intact heart. We found that Lut significantly improved contractility and Ca2+ transients, ameliorated expression, activity and stability of SERCA2a and upregulated expression of small ubiquitin-related modifier (SUMO) 1, which is a newfound SERCA2a regulator. Lut also increased phosphorylation of protein kinase B (Akt), phospholaban (PLB) and sumoylation of SERCA2a, specificity protein 1 (Sp1). Transcriptions of SUMO1 and SERCA2a were concurrently increased. Inhibition of posphatidylinositol 3 kinase/Akt (PI3K/Akt) pathway and SERCA2a activity both markedly abolished Lut-induced benefits in vitro and in vivo. Lut upregulated the expression ratio of Bcl-2/Bax, caspase-3/cleaved-Caspase3. Meanwhile, Lut ameliorated the myocardium fibrosis of HF. These discoveries provide an important potential therapeutic strategy that Lut targeted SERCA2a SUMOylation related to PI3K/Akt-mediated regulations on rescuing the dysfunction of HF.

  9. Effect of Zn2+ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione

    Institute of Scientific and Technical Information of China (English)

    Hong XIE; Ke-ying CHEN; Pei-hong ZHU

    2004-01-01

    AIM: To explore whether the differential effects of Zn2+ on ryanodine binding to the sarcoplasmic reticulum (SR)of skeletal and cardiac muscles resulted from different permeability of the SR to Zn2+. METHODS: [3H]ryanodine binding assays were performed to examine the effect of Zn2+ on ryanodine binding to the SR in the presence of pyrithione sodium (PyNa), a specific Zn2+ ionophore. RESULTS: As a control, PyNa up to 50 μmol/L did not induce any effect on ryanodine binding to the SR of cardiac muscle. But PyNa 1-100 μmol/L increased ryanodine binding in skeletal muscle with maximum binding (222.2 %+20.9 % of the control) and inhibited ryanodine binding to 50 % of the control at about 500 μrnol/L. In the presence of PyNa 10 and 50 μmol/L the dose-dependence of the effect of Zn2+ in cardiac muscle was still monophasic and not changed by PyNa, while the biphasic effect of Zn2+in skeletal muscle became monophasic. CONCLUSION: Different permeability of the SR to Zn2+ may account for the differential effects of Zn2+on ryanodine binding in skeletal and cardiac muscles. PyNa is not a strictly specific Zn2+ ionophore.

  10. Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres.

    Science.gov (United States)

    Coonan, J R; Lamb, G D

    1998-04-01

    The effect of intracellular Cl- on Ca2+ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ loading. Both in rat and toad fibres, the presence of 20 mM Cl- in the myoplasm increased Ca2+ leakage from the SR at pCa (i.e. -log10 [Ca2+]) 6.7, but not at pCa 8. Ca2+ uptake was not significantly affected by the presence of Cl-. This Ca2+-dependent effect of Cl- on Ca2+ leakage was most likely due to a direct action on the ryanodine receptor/Ca2+ release channel, and could influence channel sensitivity and the resting [Ca2+] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl- to the myoplasm caused a 40-50% reduction in Ca2+ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl- induces a potential across the SR (lumen negative) which might reduce Ca2+ release via several different mechanisms.

  11. Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle.

    Science.gov (United States)

    Du, G G; Ashley, C C; Lea, T J

    1994-12-01

    Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.

  12. Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

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    Changsheng Nai

    2015-01-01

    Full Text Available The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications.

  13. Altered calcium pump and secondary deficiency of gamma-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from delta-sarcoglycan knockout mice.

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    Solares-Pérez, Alhondra; Alvarez, Rocío; Crosbie, Rachelle H; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J; Ortega, Alicia; Coral-Vazquez, Ramón

    2010-07-01

    Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

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    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

    2015-12-15

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

  15. Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

    Science.gov (United States)

    Cantilina, T; Sagara, Y; Inesi, G; Jones, L R

    1993-08-15

    Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

  16. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

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    Lu Fang-hao

    2010-06-01

    Full Text Available Abstract Communication between the SR (sarcoplasmic reticulum, SR and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM. Although it has been demonstrated that CaR (calcium sensing receptor activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re, the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  17. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation.

    Science.gov (United States)

    Lu, Fang-hao; Tian, Zhiliang; Zhang, Wei-hua; Zhao, Ya-jun; Li, Hu-lun; Ren, Huan; Zheng, Hui-shuang; Liu, Chong; Hu, Guang-xia; Tian, Ye; Yang, Bao-feng; Wang, Rui; Xu, Chang-qing

    2010-06-17

    Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  18. Decrease in sarcoplasmic reticulum calcium content, not myofilament function, contributes to muscle twitch force decline in isolated cardiac trabeculae

    Science.gov (United States)

    Milani-Nejad, Nima; Brunello, Lucia; Gyorke, Sándor; Janssen, Paul M.L.

    2014-01-01

    We set out to determine the factors responsible for twitch force decline in isolated intact rat cardiac trabeculae. The contractile force of trabeculae declined over extended periods of isometric twitch contractions. The force-frequency relationship within the frequency range of 4–8 Hz, at 37 °C, became more positive and the frequency optimum shifted to higher rates with this decline in baseline twitch tensions. The post-rest potentiation (37 °C), a phenomenon highly dependent on calcium handling mechanisms, became more pronounced with decrease in twitch tensions. We show that the main abnormality during muscle run-down was not due to a deficit in the myofilaments; maximal tension achieved using a K+ contracture protocol was either unaffected or only slightly decreased. Conversely, the sarcoplasmic reticulum (SR) calcium content, as assessed by rapid cooling contractures (from 27 °C to 0 °C), decreased, and had a close association with the declining twitch tensions (R2 ~ 0.76). SR Ca2+-ATPase, relative to Na+/Ca2+ exchanger activity, was not altered as there was no significant change in paired rapid cooling contracture ratios. Furthermore, confocal microscopy detected no abnormalities in the overall structure of the cardiomyocytes and t-tubules in the cardiac trabeculae (~23 °C). Overall, the data indicates that the primary mechanism responsible for force run-down in multi-cellular cardiac preparations is a decline in the SR calcium content and not the maximal tension generation capability of the myofilaments. PMID:25056841

  19. Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice

    Science.gov (United States)

    Wang, Wei; Landstrom, Andrew P.; Wang, Qiongling; Munro, Michelle L.; Beavers, David; Ackerman, Michael J.; Soeller, Christian

    2014-01-01

    Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development. PMID:25193470

  20. Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres.

    Science.gov (United States)

    Kabbara, A A; Stephenson, D G

    1994-10-01

    The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Topp, M R; Blasie, J K

    1983-11-08

    The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

  2. Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction.

    Science.gov (United States)

    Dolnikov, Katya; Shilkrut, Mark; Zeevi-Levin, Naama; Gerecht-Nir, Sharon; Amit, Michal; Danon, Asaf; Itskovitz-Eldor, Joseph; Binah, Ofer

    2006-02-01

    Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.

  3. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle.

    Science.gov (United States)

    Baylor, S M; Hollingworth, S

    2003-08-15

    Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 degrees C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Delta[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 +/- 1.0 microM (mean +/- S.E.M.); half-duration, 7.7 +/- 0.6 ms; fast-twitch: peak amplitude 18.5 +/- 0.5 microM; half-duration, 4.9 +/- 0.3 ms). SR Ca2+ release was estimated from Delta[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Delta[CaT]) and the peak rate of Ca2+ release ((d/dt)Delta[CaT]) differed about 3-fold between the fibre types (slow-twitch: Delta[CaT], 127 +/- 7 microM; (d/dt)Delta[CaT], 70 +/- 6 microM ms-1; fast-twitch: Delta[CaT], 346 +/- 6 microM; (d/dt)Delta[CaT], 212 +/- 4 microM ms-1). In contrast, the half-duration of (d/dt)Delta[CaT] was very similar in the two fibre types (slow-twitch, 1.8 +/- 0.1 ms; fast-twitch, 1.6 +/- 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Delta[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2-0.3; fast-twitch, 0.1-0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.

  4. Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle.

    Science.gov (United States)

    Damiani, E; Sacchetto, R; Margreth, A

    2000-12-09

    Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.

  5. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Trentham, D R; Topp, M R; Blasie, J K

    1983-12-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants

  6. Biphasic contractions induced by milrinone at low temperature in ferret ventricular muscle: role of the sarcoplasmic reticulum and transmembrane calcium influx.

    Science.gov (United States)

    Malecot, C O; Bers, D M; Katzung, B G

    1986-08-01

    The effects of milrinone were studied in ferret papillary muscle stimulated at various rates and temperatures from 23 degrees to 36 degrees C. In voltage-clamp experiments, 50 micrograms/ml (0.237 mM) milrinone induced a 2.1-fold increase in calcium current at 28 degrees or 36 degrees C. At 50 micrograms/ml, milrinone transiently increased contractility in all muscles at 28 degrees C, but its steady-state effect was either increased (+50%) or decreased (-24.7%) steady-state twitch amplitude. A negative inotropic effect always occurred below 27 degrees C. Milrinone decreased the total twitch duration and split the twitch into two components (P1 and P2) in the absence of any evidence of aberrant conduction. Increasing milrinone concentration from 50 to 300 micrograms/ml decreased P1 and increased P2. Ryanodine (100 mM) or caffeine (10 mM) suppressed P1. Contractions elicited after 30 seconds of rest were also biphasic in the presence of milrinone, but not in its absence. P2 of post-rest contraction was increased by caffeine or calcium (10 mM) and decreased by cobalt (2 mM) when drugs were applied at the beginning of the rest. Ryanodine and caffeine also suppressed P1 of post-rest contraction. The evidence suggests that P1 may be caused by Ca release from the sarcoplasmic reticulum and P2 by increased Ca influx during the action potential via the calcium channel. It is also suggested that P2 may be present under control conditions, but to a lesser extent, and masked by a large P1.

  7. Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue.

    Science.gov (United States)

    Aureliano, Manuel; Henao, Fernando; Tiago, Teresa; Duarte, Rui O; Moura, J J G; Baruah, Bharat; Crans, Debbie C

    2008-07-07

    The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

  8. A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions.

    Directory of Open Access Journals (Sweden)

    Michael A Colman

    2017-08-01

    Full Text Available Intracellular calcium cycling is a vital component of cardiac excitation-contraction coupling. The key structures responsible for controlling calcium dynamics are the cell membrane (comprising the surface sarcolemma and transverse-tubules, the intracellular calcium store (the sarcoplasmic reticulum, and the co-localisation of these two structures to form dyads within which calcium-induced-calcium-release occurs. The organisation of these structures tightly controls intracellular calcium dynamics. In this study, we present a computational model of intracellular calcium cycling in three-dimensions (3-D, which incorporates high resolution reconstructions of these key regulatory structures, attained through imaging of tissue taken from the sheep left ventricle using serial block face scanning electron microscopy. An approach was developed to model the sarcoplasmic reticulum structure at the whole-cell scale, by reducing its full 3-D structure to a 3-D network of one-dimensional strands. The model reproduces intracellular calcium dynamics during control pacing and reveals the high-resolution 3-D spatial structure of calcium gradients and intracellular fluxes in both the cytoplasm and sarcoplasmic reticulum. We also demonstrated the capability of the model to reproduce potentially pro-arrhythmic dynamics under perturbed conditions, pertaining to calcium-transient alternans and spontaneous release events. Comparison with idealised cell models emphasised the importance of structure in determining calcium gradients and controlling the spatial dynamics associated with calcium-transient alternans, wherein the probabilistic nature of dyad activation and recruitment was constrained. The model was further used to highlight the criticality in calcium spark propagation in relation to inter-dyad distances. The model presented provides a powerful tool for future investigation of structure-function relationships underlying physiological and pathophysiological

  9. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca(2+)-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition.

    Science.gov (United States)

    Fraqueza, Gil; Batista de Carvalho, Luís A E; Marques, M Paula M; Maia, Luisa; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-11-07

    Recently we demonstrated that the decavanadate (V(10)) ion is a stronger Ca(2+)-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V(10) interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb(10) = Nb(10)O(28)](6-), is a useful tool in deducing the interaction and the non-competitive Ca(2+)-ATPase inhibition by the decavanadate ion [V(10) = V(10)O(28)](6-). Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca(2+)-ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V(10), Nb(10) and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V(10) and Nb(10) decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V(10) to inhibit the Ca(2+)-ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These

  10. Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

    Institute of Scientific and Technical Information of China (English)

    Zhi-Qing Fu; Xiao-Ying Li; Xiao-Chun Lu; Ya-Fei Mi; Tao Liu; Wei-Hua Ye

    2012-01-01

    Background Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin Ⅱ) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory

  11. Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

    Science.gov (United States)

    Delmotte, Philippe; Sieck, Gary C

    2015-02-01

    Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

  12. The Ca(2+)-ATPase of the sarcoplasmic reticulum in skeletal and cardiac muscle. An overview from the very beginning to more recent prospects.

    Science.gov (United States)

    Hasselbach, W

    1998-09-16

    The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar (World War II) years and would not be possible without the generous support by the American scientific community. It was this community that in pre- and postwar years gave shelter to many European scientists, which in return stimulated scientific development in the United States. These pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells. The pump and its counterpart, the calcium release channel, proved to be controlled by various intrinsic mechanisms. Rising hydrogen concentrations as occurring in ischemic muscles switch off pump activity and counteract allosterically caffeine-induced calcium release (CICR). Rising phosphate or the presence of other calcium-precipitating anions, on the other hand, prevents pump inhibition by intraluminal calcium precipitation, which, simultaneously, can increase the quantity of releasable calcium. The inactivation of CICR by removing medium chloride must be considered as a hint of additional mechanisms by which calcium-dependent activity regulation can be modified.

  13. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    Science.gov (United States)

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  14. Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate.

    Science.gov (United States)

    Hasselbach, W

    1988-01-01

    The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport in sarcoplasmic reticulum

    Science.gov (United States)

    Vostrikov, Vitaly V.; Mote, Kaustubh R.; Verardi, Raffaello; Veglia, Gianluigi

    2013-01-01

    Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window. PMID:24207128

  16. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine;

    2009-01-01

    Abstract: We have determined the structure of the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) in an E2.P-i-like form stabilized as a complex with MgF42-, an ATP analog, adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.......5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  17. The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad.

    Science.gov (United States)

    Kabbara, A A; Allen, D G

    2001-07-01

    1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degrees C in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K(Ca) 90 microM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the SR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 microM 2,5-di(tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca(2+) from the SR; this procedure reduced the fluorescence to 46 +/- 4 % of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 microg ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degrees C) the component of signal from the SR declined by 33 +/- 4 %. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca(2+) pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50 %. The SR signal during the periods between tetani declined steadily and the SR Ca(2+) signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca(2+) signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the

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

    Science.gov (United States)

    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.

  19. Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

    Directory of Open Access Journals (Sweden)

    Bassani R.A.

    2003-01-01

    Full Text Available Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A, Na+-Ca2+ exchanger (NCX and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake. To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW. Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM, whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM. The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

  20. NO donors-relaxation is impaired in aorta from hypertensive rats due to a reduced involvement of K(+) channels and sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Bonaventura, Daniella; de Lima, Renata Galvão; da Silva, Roberto Santana; Bendhack, Lusiane Maria

    2011-10-24

    To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO](3+) (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100nM). We have analyzed the maximal relaxation (Emax) and potency (pD(2)) of NO donors. Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K(+) channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. Taken together, our results provide evidences that in this model of hypertension, impaired K(+) channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1

    Science.gov (United States)

    Glukhov, Alexey V.; Kalyanasundaram, Anuradha; Lou, Qing; Hage, Lori T.; Hansen, Brian J.; Belevych, Andriy E.; Mohler, Peter J.; Knollmann, Björn C.; Periasamy, Muthu; Györke, Sandor; Fedorov, Vadim V.

    2015-01-01

    Aims Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. Methods and results In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca2+ cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2−/−) mice. Casq2−/− mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca2+ cycling, including abnormal Ca2+ release, periods of significantly elevated diastolic Ca2+ levels leading to pauses and unstable pacemaker rate. Importantly, Ca2+ cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca2+ transient upstrokes throughout the atrial pacemaker complex. Conclusions Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca2+ release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients. PMID:24216388

  2. Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

    Directory of Open Access Journals (Sweden)

    Wei XIN

    2011-04-01

    Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

  3. Improvement in cardiac function after sarcoplasmic reticulum Ca2+-ATPase gene transfer in a beagle heart failure model

    Institute of Scientific and Technical Information of China (English)

    MI Ya-fei; LI Xiao-ying; TANG Li-jiang; LU Xiao-chun; FU Zhi-qing; YE Wei-hua

    2009-01-01

    Background Heart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.Methods HF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n=4); (b) HF group (n=4); (c) enhanced green fluorescent protein group (n=4); and (d) SERCA2.a group (n=4). rAAVl-EGFP (lx1012 μg) and rAAVl-SERCA2a (lx1012 μg) were delivered intramyocardially. SERCA2.a expression was assessed by Western blotting and immunohistochemistry.Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P <0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dtmax), and the maximal rate of decline of left ventricular pressure (-dp/dtmax) (P <0.05). Left ventricular end-diastole pressure significantly decreased (P <0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P<0.05). Conclusions Intramyocardial injection of rAAVl-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a's long-term safety, efficacy, dosage

  4. Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

    Directory of Open Access Journals (Sweden)

    Fang-Hao Lu

    2013-05-01

    Full Text Available Aims: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. Methods and Results: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso. Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours

  5. A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

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    Tobias Gröβl

    Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

  6. Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Lu, Fang-Hao; Fu, Song-Bin; Leng, Xiaoning; Zhang, Xinying; Dong, Shiyun; Zhao, Ya-Jun; Ren, Huan; Li, Hulun; Zhong, Xin; Xu, Chang-Qing; Zhang, Wei-Hua

    2013-01-01

    Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR

  7. Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

    Directory of Open Access Journals (Sweden)

    Jerry Curran

    Full Text Available Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+ leak through ryanodine receptors. Beta-adrenergic (β-AR tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis

  8. Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

    Science.gov (United States)

    Fénelon, Karine; Lamboley, Cédric R H; Carrier, Nicole; Pape, Paul C

    2012-10-01

    Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits

  9. Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling [v1; ref status: indexed, http://f1000r.es/32q

    Directory of Open Access Journals (Sweden)

    Nicola Fameli

    2014-04-01

    Full Text Available Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca2+ waves. In pulmonary artery smooth muscle cells (PASMCs it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR via ryanodine receptor (RyR subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L

  10. A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

    Science.gov (United States)

    Größl, Tobias; Hammer, Elke; Bien-Möller, Sandra; Geisler, Anja; Pinkert, Sandra; Röger, Carsten; Poller, Wolfgang; Kurreck, Jens; Völker, Uwe

    2014-01-01

    In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile. PMID:24670775

  11. 姜黄素对心力衰竭兔肌浆网钙泵表达的影响%Effects of curcumin on sarcoplasmic reticulum Ca~(2+) -ATPase in rabbits with heart failure

    Institute of Scientific and Technical Information of China (English)

    张艳; 林国生; 包明威; 武欣迎; 王澈; 杨波

    2010-01-01

    目的 探讨姜黄素对心力衰竭(心衰)兔肌浆网钙泵表达的影响.方法 采用主动脉瓣反流联合腹主动脉缩窄制作慢性心衰家兔模型.随机分为心衰姜黄素组、心衰安慰剂组、对照姜黄素组、对照安慰剂组.8周后计算心脏重量与体重比值,观察超微结构,检测肌浆网钙泵mRNA和蛋白的表达水平及活性.结果 心衰姜黄素组和心衰安慰剂组心脏重量与体重比值均大于对照组(P<0.05);且心衰姜黄素组比值小于心衰安慰剂组(P<0.05).电子显微镜显示心衰姜黄素组的心脏超微结构有所改善.心衰姜黄素组和心衰安慰剂组肌浆网钙泵mRNA、蛋白表达及活性均小于对照组(P<0.05),但心衰姜黄素组均显著高于心衰安慰剂组(P<0.05).结论 姜黄素能在mRNA水平和蛋白水平提高心衰家兔肌浆网钙泵的表达,提高肌浆网钙泵的活性,这可能是姜黄素改善心衰的机制之一.%Objective To investigate the effects of curcumin on sarcoplasmic reticulum Ca~(2+)-ATPase in heart failure rabbits.Methods Rabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including:(1) heart failure treated with curcumin;(2) heart failure treated with placebo;(3) healthy control treated with curcumin and (4) healthy control treated with placebo.All rabbits were administrated with curcumin capsules or placebo capsules 100 mg·kg~(-1)·d~(-1),respectively.All groups were sacrificed after eight weeks.Myocardial ultrastructural organization was detected by transmission electron microscope.RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca~(2+)-ATPase in mRNA and protein levels,respectively.Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca~(2+) -ATPase.Results All detected parameters were similar between control curcumin group and control placebo

  12. The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase.

    Science.gov (United States)

    Medda, P; Fassold, E; Hasselbach, W

    1987-06-01

    sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].

  13. Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum.

    Science.gov (United States)

    Loy, Ryan E; Orynbayev, Murat; Xu, Le; Andronache, Zoita; Apostol, Simona; Zvaritch, Elena; MacLennan, David H; Meissner, Gerhard; Melzer, Werner; Dirksen, Robert T

    2011-01-01

    The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.

  14. Effect of downhill exercise on sarcoplasmic reticulum function in rat skeletal muscle%下坡运动对大鼠骨骼肌肌浆网功能的影响

    Institute of Scientific and Technical Information of China (English)

    陈万; Ruell PA; Thompson MW

    2007-01-01

    To investigate the effect of downhill(eccentric)exercise(ECE)on sarcoplasmic reticulum(SR) Ca2+-ATPase activity,Ca2+ uptake and release in rat skeletal muscle,in terms of both magnitude and time course.In addition,ionophore stimulation was determined to assess vesicle integrity by measuring the ratio of calciumdependent ATPase activities in the presence and absence of A23187.Method:Adult male SD rats were randomly assigned to control and ECE groups.The ECE rats were sacrificed at the Oth,4th,24th,48th,72nd and 144th h following ECE(n=7).The ECE protocol consisted of 90min continuous downhill exercise(-16 deg;15m·min-1).Red vastus muscles were sampled separately for each group and muscle homogenates were prepared.The rates of SR Ca2+-ATPase activity.Ca2+uptake and release were measured in vitro.Result:SR Ca2+uptake was significantly lower(P<0.05)compared with control values[19.25+1.38 nmol·min-1·(mg protein)-1],by 29% and 36% immediately and 4h after ECE,respectively,and remained depressed(P<0.05)24h following ECE.SR Ca2+ release was also significantly lower(P<0.05)compared with control values[31.06±2.36 nmol·min-1·(mg protein)-1],by 37% and 39% immediately and 4h after ECE,respectively,and remained depressed(P<0.05)24h following ECE.SR Ca2+-ATPaseactivity measured with ionophore was 31% lower(P<0.05)4h after ECE,and remained lower(P<0.05)24h following ECE.The ratio of Ca2+-ATPase activities in the presence and absence of A23187 was not significantly changed following ECE,indicating that membrane integrity was not altered by the exercise.Conclusion:The present remits suggest that a bout of low-intensity,prolonged downhill exereise results in a long-lasting depression of SR function that is not fully restored after two days of recovery,which may underlie some muscle functional impairments induced by ECE.These changes could be the results of stress from sarcomere length inhomogeneities during eccentric contractions.%目的:观测研究下坡(离心)

  15. Sarcoplasmic Reticulum Ca2+ Cycling Protein Phosphorylation in a Physiologic Ca2+ Milieu Unleashes a High-Power, Rhythmic Ca2+ Clock in Ventricular Myocytes: Relevance to Arrhythmias and Bio-Pacemaker Design

    Science.gov (United States)

    Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A; Yang, Dongmei; Lukyanenko, Yevgeniya; Vinogradova, Tatiana; Jones, Larry; Lakatta, Edward G.

    2014-01-01

    Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature

  16. Single-channel properties of the sarcoplasmic reticulum calcium-release channel in slow- and fast-twitch muscles of Rhesus monkeys.

    Science.gov (United States)

    Bastide, B; Mounier, Y

    1998-08-01

    RyR1 is the main isoform of ryanodine receptor expressed in fast- and slow-twitch mammalian skeletal muscles although differences in Ca2+-release kinetics and properties have been reported. Single-channel measurements reveal that a large proportion (82%) of Ca2+-release channels measured in slow-twitch muscle preparations have properties similar to those of the Ca2+-release channels of fast-twitch preparations, i.e. the same conductance, an identical sensitivity to caffeine and a bell-shaped Ca2+ activation curve for pCa (-log10[Ca2+]) 7 to 3. A low proportion (18%) of Ca2+-release channels observed in preparations from slow-twitch muscles were characterized by a very high activity level. These channels were not inhibited at a millimolar concentration of Ca2+. Our data suggest that the different properties of Ca2+ release in slow- and fast-twitch muscles might not be related to intrinsic properties of the Ca2+-release channels of each type of muscle but rather to the co-expression of two isoforms of ryanodine receptor and the lower amount of Ca2+-release channels expressed in slow- than in fast-twitch muscles.

  17. Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

    Science.gov (United States)

    Medraño-Fernandez, Iria; Fagioli, Claudio; Mezghrani, Alexandre; Otsu, Mieko; Sitia, Roberto

    2014-04-01

    To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

  18. 精-甘-天冬-丝氨酸对脓毒性休克大鼠心肌肌浆网钙转运的影响%Effects of Arg-Gly-Asp-Ser on Ca2 + transport of myocardial sarcoplasmic reticulum in rat septic shock

    Institute of Scientific and Technical Information of China (English)

    吉勇; 赵明; 齐鹰; 董林旺; 吴立玲; 彭师奇; 苏静怡

    1996-01-01

    To study the effects of Arg-Gly-Asp-Ser (RGDS), a synthetic short peptide of fibrinogen degradation, on the Ca2 + transport function of cardiac sarcoplasmic reticulum in rat septic shock.4 h and 14 h after cecal ligation and puncture (CLP) operation on rats. Highly purified membrane of sarcoplasmic reticulum (SR) was prepared from rat hearts. Assays were made of ATP-dependent Ca2 + uptake by cardiac SR and [3H] ryanodine binding to SR. RESULTS: The initial rate and the capacity of SR Ca2 + uptake were increased by 104 % (P<0.01) and 12 % (P<0.05), respectively, paralleled by an increase in Ca2 +-ATPase activity and a decrease in calcium accumulation of myo- cardium of septic rats, whereas the Bmax and Kd values of Ca2+ activated [3H]ryanodine binding to SR were unaffected after RGDS administration. CONCLUTIONS: The results indicated that RGDS have cardioprotective effects of maintaining Ca2+ homeostasis of cardiac myocytes by enhancing SR Ca2 + uptake in rat septic shock.%探讨一种人工合成的纤维蛋白原降解肽片段RGDS对脓毒性休克大鼠心肌肌浆网钙转运功能的影响.方法:大鼠盲肠结扎穿孔术后4 h和14 h分两次尾静脉注射RGDS 5 μmol·kg-1.制备大鼠心肌肌浆网(SR)膜;测定SR Ca2+摄取和[3H]ryanodine受体结合功能.结果:RGDS组大鼠心肌SR摄Ca2+率及摄Ca2+量分别较休克组提高104%(P<0.01)和12%(P<0.05),而心肌SR钙释放通道-[3H]ryanodine受体结合Bmax和Ka值没有明显变化.同时RGDS还可以减轻休克大鼠心肌组织钙聚积.结论:RGDS提高休克大鼠心肌SR Ca2+摄取功能,维持心肌细胞钙稳态,具有心肌保护作用.

  19. Taurine protects cardiac contractility in killifish, Fundulus heteroclitus, by enhancing sarcoplasmic reticular Ca(2+) cycling.

    Science.gov (United States)

    Henry, Elenor F; MacCormack, Tyson J

    2017-05-23

    Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L(-1)) protected peak tension development (F max) at high stimulation frequencies, an effect abolished by treatment with ryanodine, a blocker of sarcoplasmic reticulum Ca(2+) release. In the presence of ryanodine, taurine-treated preparations were also better able to maintain F max at supraphysiological extracellular Ca(2+) levels, but a prior anoxia exposure abolished this effect. Taurine had no impact on basal F max during or after anoxia, but it provided additive protection to high-frequency contractility post-anoxia. Tissue oxygen consumption and extracellular glucose utilization were unaffected by taurine in non-contracting preparations, indicating that it does not impact energy metabolism. Overall, the results suggest that cardiac taurine levels are well maintained on acute time scales in this highly stress-tolerant species. Supplemental taurine has no effect on aerobic metabolism in vitro, but it significantly improved cardiac contractility in a manner dependent upon sarcoplasmic reticulum Ca(2+) cycling. The data indicate that taurine likely plays an important role in the regulation of cardiac performance in teleosts.

  20. 心肺复苏后心功能障碍与心肌内质网Ca2+调控蛋白表达关系的研究%The relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction

    Institute of Scientific and Technical Information of China (English)

    黄煜; 何庆

    2014-01-01

    Objective To investigate the relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction. Methods Thirty-eight SPF male Sprague-Dawley (SD) rats were randomly divided into control group(n=12)and cardiac arrest(CA)group(n=26). CA was induced by intravenous bolus of potassium chloride(40μg/g),and cardiopulmonary resuscitation(CPR)was conducted 8 minutes later. No CA was induced in control group except catheter placement for monitoring cardiopulmonary parameters after anesthesia. Invasive hemodynamic parameters were monitored for 1 hour after CPR. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after restoration of spontaneous circulation (ROSC),and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),phosphorylated phospholamban (p-PLB) and rynodine receptor(RyR)were determined by Western Blot. Results ROSC rate of CA group was 92.3%(24/26),and mean recovery time was (68 ±39)seconds. Cardiac function was significantly impaired in CA group at 1 hour after resuscitation, and ejection fraction, fraction shortening (FS), the maximal rate of left ventricular pressure increase/decline (±dp/dt max)were significantly decreased compared with those in control group 〔ejection fraction:0.548±0.060 vs. 0.809±0.043,F=71.692,P=0.000;FS:(34.4±4.4)%vs. (46.0±3.5)%,F=55.443,P=0.000;+dp/dt max(mmHg/s):4 718±743 vs. 7 098±394,P0.05). Conclusions The impairment of the p-PLB is closely related to postresuscitation myocardial dysfunction.%目的探讨心肌内质网Ca2+调控蛋白表达与心肺复苏(CPR)后心功能障碍的关系。方法38只SPF级雄性SD大鼠按随机数字表法分为对照组(12只)和心搏骤停组(26只)。静脉弹丸式注射氯化钾40μg/g诱导心搏骤停,8 min后进行CPR;对照组大鼠仅麻醉后置管并监测指标,不诱导心搏骤停。在复苏后进行有创血流动力学监测1 h,采用超声心动

  1. LPCES对慢性低压缺氧兔颏舌肌肌球蛋白重链和SR Ca2+摄取-释放动力学的影响%Electrical stimulation at lower physiological frequency induces myosin heavy chain isoform transformation and improves sarcoplasmic reticulum Ca2+ uptake/release in genioglossus of rabbits exposed to chronic hypoxia

    Institute of Scientific and Technical Information of China (English)

    刘熙; 刘刚; 张妮; 欧娜; 张鹏

    2011-01-01

    Objective To identify the effect of chronic electrical stimulation at a lower physiological frequency on the expressions of myosin heavy chain (MHC) isoforms and kinetics of sarcoplasmic reticulum (SR) Ca2 + uptake/release in the genioglossus of rabbits exposed to chronic hypoxia. Methods Twenty-four adult rabbits were randomized into control group ( A), chronic hypoxia group ( B ), 2.5 Hz electrical stimulation group (C) and (2.5 + 40) Hz electrical stimulation group (low frequency plus physical frequency, D).After the rabbits from group B, C and D had been fed with free access to food and water in a hypoxia cabin ( simulating 5 000 m altitude) in 10 h a day for 4 weeks, the rabbits in group C and D received electrical stimulation in their genioglossus at a frequency of 2.5 Hz and (2.5 +40) Hz respectively in 10 h per day for 14 d,while those in group B received no electrical stimulation. Expressions of MHC isoforms in the genioglossus of rabbits in 4 groups were detected by Western blotting, and Fura-2 fluorophotometry was used to assay the kinetics changes of SR Ca2 + uptake-release. Restlts The expression level of MHC l a was significantly higher while that of MHC I was significantly lower in group B than that in group A (P < 0.05 ). Meanwhile,the genioglossus SR Ca2+ uptake/release velocity in group B was significantly decreased compared with that in group A ( P < 0. 05 ). The expression levels of MHC Ⅱ a and MHC I in group C and D after electrical stimulation were significantly higher, while those of MHC Ⅱ b, especially in group D, were significantly lower than those in group B (P < 0.05 ). The genioglossus SR Ca2+ uptake/release velocity in group C and D, especially in group D, was significantly increased compared with that in group B ( P < 0.05 ). No significant difference was found in expression levels of MHC Ⅱ a and MHC I between group C and D after electrical stimulation ( P > 0.05). Conclusion MHC Ⅱb in the genioglossus of rabbits with

  2. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates.

    Science.gov (United States)

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2014-03-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.

  3. Evidence against inhibition of sarcoplasmic reticulum Ca2 + -pump as mechanism of H202-induced contraction of rat aorta%肌浆网钙泵的抑制不参与过氧化氢诱导的大鼠主动脉收缩

    Institute of Scientific and Technical Information of China (English)

    沈建中; 郑秀凤; 魏尔清; 關超然

    2001-01-01

    目的:研究肌浆网钙泵抑制是否参与H2O2诱导的大 鼠主动脉收缩反应。方法:离体主动脉环张力实验 比较H2O2及钙泵特异性抑制剂环匹阿尼酸(CPA)缩 血管效应及其信号机制的差异。结果:H2O2和CPA 均收缩去内皮主动脉环,但H2O2触发快速短暂相位 相收缩,而CPA诱导缓慢持续的张力相收缩。在无 钙液中,仅CPA 30 μmol/L而非H2O2 30μmol/L预 处理取消苯肾上腺素10 μmol/L缩血管效应。Thap- sigargin 30 μmol/L诱导最大收缩反应时,仅H2O2能 使血管环进一步收缩。另外,P2受体拮抗剂 suramin、RB-2(各100μmol/L)以及多种酶抑制剂包 括PLC、PKC、PLA2、COX和蛋白质酪氨酸激酶均 能抑制H2O2而非CPA诱导的缩血管效应,但2-APB 50μmol/L对两者都有抑制作用。结论:肌浆网钙 泵抑制不是H2O2收缩大鼠去内皮主动脉的机制。%AIM: To test whether inhibition of sarcoplasmic reticulum (SR) Ca2+ -pump is involved in H2O2-induced contraction of endothelium-denuded rat aorta. METH ODS: Isometric tension recording of H2O2 and cyclopia zonic acid (CPA)-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to discriminate their signaling path ways involved. RESULTS: Both H2O2 and CPA con tracted rat aortic rings, but with different contractile pat terns. H2O2 triggered a fast and phasic contraction, whereas CPA elicited a slow and sustained contraction. In Ca 2 +-free medium, pretreatment of aortic tings with CPA 30 μmol/L but not with H2O2 30 μmol/L nearly abolished phenylephrine (10 μmol/L)-induced contrac tion. In addition, upon the maximal contraction induced by thapsigargin 30 μ r mol/L, H2O2 but not CPA further contracted aortic rings. On the other hand, H2O2 (30 μmoL/L)- but not CPA (10 μmol/L)-induced contraction could be inhibited by suramin and RB-2 (each 100 μmol/L), two P2-purinoceptor antagonists. Further more, although

  4. 大鼠 H9c2心肌细胞缺氧/复氧损伤模型肌浆网钙调控相关蛋白表达及当归补血汤的干预作用%Danggui Buxue Decoction on expression of sarcoplasmic reticulum calcium regulatory protein in H9c2 myocardial cell model with hypoxia/reoxygenation damage

    Institute of Scientific and Technical Information of China (English)

    周春刚; 汤加; 李卿; 徐辰; 张志斌

    2016-01-01

    Objective To establish the model of hypoxia / reoxygenation damage in H9c2 rat myocardial cell and observe the expression of myocardial sarcoplasmic reticulum calcium regulatory protein and the effect of Danggui Bux-ue Decoction on calcium overload in H9c2 cells with hypoxia / reoxygenation injury. Methods JC-1 staining was used to detect the mitochondrial membrane potential by flow cytometry,Fluo-3 AM calcium fluorescence probe for the detection of intracellular calcium concentration,The expression level of sarcoplasmic reticulum Ca transport ATP en-zyme(SERCA2a),L type calcium channel(CAV1. 3),ryanodine receptor(RyR1,RyR2),phospholamban(PLB), calsequestrin(CASQ)mRNA were detected by RT-PCR. Results Compared with the normal control group,the early apoptosis rate and intracellular calcium concentration in model group increased significantly(P 0. 05),compared with the model group. Con-clusion There are obvious calcium overload and sarcoplasmic reticulum calcium regulating proteins adjustment disor-der in the model of H9c2 rat myocardial cell with hypoxia / reoxygenation damage,Danggui Buxue Decoction can in-crease the expression of the sarcoplasmic reticulumcalcium regulating protein SERCA2a and CASQ mRNA after reoxy-genation,alleviate calcium overload,and significantly reduce the early apoptosis rate of reoxygenation damage cells.%目的:建立缺氧/复氧大鼠 H9c2心肌细胞损伤模型,观察心肌细胞肌浆网钙调控相关蛋白表达及当归补血汤对 H9c2细胞缺氧/复氧损伤钙超载的影响。方法 JC-1染色流式细胞仪检测细胞线粒体膜电位, Fluo-3 AM 钙离子荧光探针检测细胞内钙离子浓度,RT-PCR 荧光相对定量检测大鼠心肌肌浆网 Ca 转运 ATP酶(SERCA2a)、L 型钙通道(CAV1.3)、兰尼碱受体(RyR1,RyR2)、受磷蛋白(PLB)、肌集钙蛋白( CASQ)的mRNA 表达水平。结果与正常对照组比较,模型组细胞早期凋亡显著增加(P <0.05),细胞内

  5. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

    DEFF Research Database (Denmark)

    Thastrup, Ole; Cullen, P J; Drøbak, B K

    1990-01-01

    Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca...... isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2...

  6. 肌浆网钙ATP酶基因转导对慢性心力衰竭犬心肌蛋白质组影响的初步研究%Overexpression of sarcoplasmic reticulum calcium ATPase induced hemodynamic and proteomic changes in a dog model of heart failure

    Institute of Scientific and Technical Information of China (English)

    付治卿; 李小鹰; 刘秀华; 孙胜; 刘涛; 米亚非; 周声安; 叶卫华; 王青松

    2008-01-01

    handling,myofibrils,and energy production in this dog model of chronic heart failure.%目的 分析心肌肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)基因转导对慢性心力衰竭(HF)犬心肌蛋白质组的影响,探讨SERCA2a基因转导改善心功能的机制.方法 快速右心室起搏建立HF犬模型并随机分为HF组、HF+绿色荧光蛋白(enhanced green fluorescent pmtein,EGFP)组、HF+SERCA2a组.后两组分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体.于基因转导30 d时停止起搏后进行超声心动图和血流动力学检查并制备心室肌双向电泳蛋白样品和心肌双向电泳图谱,图像分析软件分析蛋白表达差异点,MALDI-TOF-MS数据库搜索鉴定蛋白质.结果 基因转导30 d时,HF+SERCA2a组犬的症状、超声心动图和血流动力学指标与HF+EGFP组相比有显著好转(P<0.05);与对照组相比差异无统计学意义(P>0.05).挑选SERCA2a基因转导后表达量发生明显改变的10个蛋白点进行分析,经质谱鉴定分别为心肌收缩相关蛋白、线粒体能量代谢酶类和应激相关蛋白.结论 以rAAV为载体介导SERCA2a基因转导能够改善HF犬心脏的收缩和舒张功能,其可能的机制是恢复了心肌收缩相关蛋白正常表型或正常表达量,增加了心肌能量的产生,改变了应激相关蛋白的表达.

  7. Time Course of the Response of Myofibrillar and Sarcoplasmic Protein Metabolism to Unweighting of the Soleus Muscle

    Science.gov (United States)

    Munoz, Kathryn A.; Satarug, Soisungwan; Tischler, Marc E.

    1993-01-01

    Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of H-3-phenylaianine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

  8. Cooked sausage batter cohesiveness as affected by sarcoplasmic proteins.

    Science.gov (United States)

    Farouk, M M; Wieliczko, K; Lim, R; Turnwald, S; Macdonald, G A

    2002-05-01

    In the first trial, m. semitendinosus and m. biceps femoris were held at 0, 10 and 35 °C until they entered rigor, and in the second trial, minced m. semitendinosus was washed in water for 15, 30, 45 or 60 min. The samples from both the trials were then used to make a finely comminuted sausage batter. Soluble sarcoplasmic protein (SSP) levels decreased with increasing rigor temperature (P < 0.05) or washing (P < 0.01). Cooked batter shear stress was not affected by SSP level, but batter shear strain decreased with the decreasing SSP level associated with an increasing rigor temperature (P < 0.05) or washing (P < 0.01). Reducing the SSP content lowered the cook yield (P < 0.05) and emulsion stability (P < 0.01) of the batter from the washed samples compared to that of controls. The results suggest that sarcoplasmic proteins are important in determining the strain values (cohesiveness) of cooked sausage batter.

  9. Bioactive electrospun fish sarcoplasmic proteins as a drug delivery system

    DEFF Research Database (Denmark)

    Stephansen, Karen; Chronakis, Ioannis S.; Jessen, Flemming

    2014-01-01

    Nano-microfibers were made from cod (Gadus morhua) sarcoplasmic proteins (FSP) (Mwelectrospinning technique. The FSP fibers were studied by scanning electron microscopy, and thefiber morphology was found to be strongly dependent on FSP concentration. Interestingly, the FSP...

  10. Temperature dependence of cardiac sarcoplasmic reticulum and sarcolemma in the ventricle of catfish (Clarias gariepinus

    Directory of Open Access Journals (Sweden)

    El-Sabry Abu-Amra

    2015-10-01

    The sarcolemmal Ca2+ contribution of activator Ca2+ was greater at a test temperature of 30 °C as assessed by verapamil. Whereas the SR-Ca2+ contribution was higher at 20 and 30 °C and a frequency rate of 0.2 and 0.4 Hz as assessed by caffeine and adrenaline, respectively. Bradykinin potentiating factor (BPF7 which was isolated from jelly fish (Cassiopea andromeda decreased the cardiac force developed at a frequency rate of 0.2 Hz and a temperature of 20 °C, whereas it increased the force developed at frequency rates of 0.2 and 0.4 Hz at 30 °C. These results indicate that BPF7 may act like verapamil in reducing the cardiac force through blocking the sarcolemmal Ca2+ channels at low temperature and like adrenaline in an increase of the cardiac force developed at warm temperature and the high frequency rate through stimulation of SR-Ca2+ activator. Therefore, this study indicates that the sarcolemmal Ca2+ influx and the SR-Ca2+ release contributors of activator Ca2+ for cardiac force development in the catfish heart were significantly greater at warm temperature and at the pacing frequency rates of 0.2 and 0.4 Hz as assessed by verapamil, adrenaline, caffeine and BPF7. However, the relative contribution of the sarcolemmal Ca2+ influx in the development of cardiac force in the catfish heart was greater than that of SR-Ca2+ release.

  11. Role of glycogen availability in sarcoplasmic reticulum Ca2+ kinetics in human skeletal muscle

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Nielsen, Joachim; Saltin, Bengt

    2011-01-01

    Glucose is stored as glycogen in skeletal muscle. The importance of glycogen as a fuel during exercise has been recognized since the 1960s; however, little is known about the precise mechanism that relates skeletal muscle glycogen to muscle fatigue. We show that low muscle glycogen is associated...... that links energy utilization, i.e. muscle contraction, with the energy content in the muscle, thereby inhibiting a detrimental depletion of the muscle energy store....

  12. Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium-transport enzyme.

    Science.gov (United States)

    Ronzani, N; Stephan, L; Hasselbach, W

    1991-10-01

    The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7-14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200-400 ml.mol-1 depending on the composition of the reaction medium containing 5 microM and 20 microM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20-40 ml.mol-1. Vanadate binding is reduced in the presence of 30 microM calcium, and simultaneously both binding volumes are diminished by 100 ml.mol-1 and 20 ml.mol-1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.

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

    Science.gov (United States)

    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.

  14. Sarcoplasmic phospholamban protein is involved in the mechanisms of postresuscitation myocardial dysfunction and the cardioprotective effect of nitrite during resuscitation.

    Directory of Open Access Journals (Sweden)

    Yu Huang

    Full Text Available OBJECTIVES: Sarcoplasmic reticulum (SR Ca(2+-handling proteins play an important role in myocardial dysfunction after acute ischemia/reperfusion injury. We hypothesized that nitrite would improve postresuscitation myocardial dysfunction by increasing nitric oxide (NO generation and that the mechanism of this protection is related to the modulation of SR Ca(2+-handling proteins. METHODS: We conducted a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 µg/g. Nitrite (1.2 nmol/g or placebo was administered when chest compression was started. No cardiac arrest was induced in the sham group. Hemodynamic parameters were monitored invasively for 90 minutes after the return of spontaneous circulation (ROSC. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after ROSC. RESULTS: Myocardial function was significantly impaired in the nitrite and placebo groups after resuscitation, whereas cardiac function (i.e., ejection fraction and fractional shortening was significantly greater in the nitrite group than in the placebo group. Nitrite administration increased the level of nitric oxide in the myocardium 5 min after resuscitation compared to the other two groups. The levels of phosphorylated phospholamban (PLB were decreased after resuscitation, and nitrite increased the phosphorylation of phospholamban compared to the placebo. No significant differences were found in the expression of sarcoplasmic reticulum Ca(2+ ATPase (SERCA2a and ryanodine receptors (RyRs. CONCLUSIONS: postresuscitation myocardial dysfunction is associated with the impairment of PLB phosphorylation. Nitrite administered during resuscitation improves postresuscitation myocardial dysfunction by preserving phosphorylated PLB protein during resuscitation.

  15. Protein kinase C modulation of the regulation of sarcoplasmic reticular function by protein kinase A-mediated phospholamban phosphorylation in diabetic rats.

    Science.gov (United States)

    Watanuki, Satoko; Matsuda, Naoyuki; Sakuraya, Fumika; Jesmin, Subrina; Hattori, Yuichi

    2004-01-01

    1. The goal of this study was to elucidate the possible mechanisms by which protein kinase A (PKA)-mediated regulation of the sarcoplasmic reticulum (SR) via phospholambin protein phosphorylation is functionally impaired in streptozotocin-induced diabetic rats. 2. Phospholamban (PLB) protein and mRNA levels were 1.3-fold higher in diabetic than in control hearts, while protein expression of cardiac SR Ca(2+)-ATPase (SERCA2a) was unchanged. 3. Basal and isoprenaline-stimulated phosphorylation of PLB at Ser(16) or Thr(17) was unchanged in diabetic hearts. However, stronger immunoreactivity was observed at the basal level in diabetic hearts when antiphosphoserine antibody was used. 4. Basal (32)P incorporation into PLB was significantly higher in diabetic than in control SR vesicles, but the extent of the PKA-mediated increase in PLB phosphorylation was the same in the two groups of vesicles. 5. Stimulation of Ca(2+) uptake by PKA-catalyzed PLB phosphorylation was weaker in diabetic than in control SR vesicles. The PKA-induced increase in Ca(2+) uptake was attenuated when control SR vesicles were preincubated with protein kinase C (PKC). 6. PKC activities were increased by more than two-fold in the membranous fractions from diabetic hearts in comparison with control values, regardless of whether Ca(2+) was present. This was associated with increases in the protein content of PKCdelta, PKCeta, PKCiota, and PKClambda in diabetic membranous fractions. 7. The changes observed in diabetic rats were reversed by insulin therapy. 8. These results suggest that PKA-dependent phosphorylation may incompletely counteract the function of PLB as an inhibitor of SERCA2a activity in diabetes in which PKC expression and activity are enhanced.

  16. Chemical and Functional Characterization of Sarcoplasmic Proteins from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Rosa Linda Lopez-Enriquez

    2015-01-01

    Full Text Available Modification of pH and NaCl concentration changed the physicochemical properties of sarcoplasmic proteins (SP from jumbo squid mantle and consequently their functional properties. Better results of emulsifying activity index (EAI and foam capacity (FC were exhibited at pH 11 in NaCl absence due to higher solubility. But better emulsifying stability index (ESI was obtained at pH 11 in 0.5 M NaCl, while, foaming stability (FS was better at pH near to isoelectric point (pI. These results suggest that SP from jumbo squid may be a promising ingredient, whose functional properties can be manipulated by changing pH and NaCl concentration.

  17. Myopathy in Marinesco-Sjögren syndrome links endoplasmic reticulum chaperone dysfunction to nuclear envelope pathology.

    Science.gov (United States)

    Roos, Andreas; Buchkremer, Stephan; Kollipara, Laxmikanth; Labisch, Thomas; Gatz, Christian; Zitzelsberger, Manuela; Brauers, Eva; Nolte, Kay; Schröder, J Michael; Kirschner, Janbernd; Jesse, Christopher Marvin; Goebel, Hans Hilmar; Goswami, Anand; Zimmermann, Richard; Zahedi, René Peiman; Senderek, Jan; Weis, Joachim

    2014-05-01

    Marinesco-Sjögren syndrome (MSS) features cerebellar ataxia, mental retardation, cataracts, and progressive vacuolar myopathy with peculiar myonuclear alterations. Most MSS patients carry homozygous or compound heterozygous SIL1 mutations. SIL1 is a nucleotide exchange factor for the endoplasmic reticulum resident chaperone BiP which controls a plethora of essential processes in the endoplasmic reticulum. In this study we made use of the spontaneous Sil1 mouse mutant woozy to explore pathomechanisms leading to Sil1 deficiency-related skeletal muscle pathology. We found severe, progressive myopathy characterized by alterations of the sarcoplasmic reticulum, accumulation of autophagic vacuoles, mitochondrial changes, and prominent myonuclear pathology including nuclear envelope and nuclear lamina alterations. These abnormalities were remarkably similar to the myopathy in human patients with MSS. In particular, the presence of perinuclear membranous structures which have been reported as an ultrastructural hallmark of MSS-related myopathy could be confirmed in woozy muscles. We found that these structures are derived from the nuclear envelope and nuclear lamina and associate with proliferations of the sarcoplasmic reticulum. In line with impaired function of BiP secondary to loss of its nucleotide exchange factor Sil1, we observed activation of the unfolded protein response and the endoplasmic-reticulum-associated protein degradation-pathway. Despite initiation of the autophagy-lysosomal system, autophagic clearance was found ineffective which is in agreement with the formation of autophagic vacuoles. This report identifies woozy muscle as a faithful phenocopy of the MSS myopathy. Moreover, we provide a link between two well-established disease mechanisms in skeletal muscle, dysfunction of chaperones and nuclear envelope pathology.

  18. αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

    Science.gov (United States)

    Jiao, Qibin; Sanbe, Atsushi; Zhang, Xingwei; Liu, Jun-Ping; Minamisawa, Susumu

    2014-08-01

    Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

  19. Endoplasmic Reticulum Stress and Associated ROS

    Directory of Open Access Journals (Sweden)

    Hafiz Maher Ali Zeeshan

    2016-03-01

    Full Text Available The endoplasmic reticulum (ER is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS. Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI-endoplasmic reticulum oxidoreductin (ERO-1, glutathione (GSH/glutathione disuphide (GSSG, NADPH oxidase 4 (Nox4, NADPH-P450 reductase (NPR, and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.

  20. S100A1: A Regulator of Striated Muscle Sarcoplasmic Reticulum Ca2+ Handling, Sarcomeric, and Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Mirko Völkers

    2010-01-01

    S100A1 has further been detected at different sites within the cardiac sarcomere indicating potential roles in myofilament function. More recently, a study reported a mitochondrial location of S100A1 in cardiomyocytes. Additionally, normalizing the level of S100A1 protein by means of viral cardiac gene transfer in animal heart failure models resulted in a disrupted progression towards cardiac failure and enhanced survival. This brief review is confined to the physiological and pathophysiological relevance of S100A1 in cardiac and skeletal muscle Ca2+ handling with a particular focus on its potential as a molecular target for future therapeutic interventions.

  1. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca2+-dependence of SR Ca2+-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo, salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2...

  2. Doublet discharge stimulation increases sarcoplasmic reticulum Ca2+ release and improves performance during fatiguing contractions in mouse muscle fibres.

    Science.gov (United States)

    Cheng, Arthur J; Place, Nicolas; Bruton, Joseph D; Holmberg, Hans-Christer; Westerblad, Håkan

    2013-08-01

    Double discharges (doublets) of motor neurones at the onset of contractions increase both force and rate of force development during voluntary submaximal contractions. The purpose of this study was to examine the role of doublet discharges on force and myoplasmic free [Ca(2+)] ([Ca(2+)]i) during repeated fatiguing contractions, using a stimulation protocol mimicking the in vivo activation pattern during running. Individual intact fibres from the flexor digitorum brevis muscle of mice were stimulated at 33°C to undergo 150 constant-frequency (five pulses at 70 Hz) or doublet (an initial, extra pulse at 200 Hz) contractions at 300 ms intervals. In the unfatigued state, doublet stimulation resulted in a transient (∼10 ms) approximate doubling of [Ca(2+)]i, which was accompanied by a greater force-time integral (∼70%) and peak force (∼40%) compared to constant frequency contractions. Moreover, doublets markedly increased force-time integral and peak force during the first 25 contractions of the fatiguing stimulation. In later stages of fatigue, addition of doublets increased force production but the increase in force production corresponded to only a minor portion of the fatigue-induced reduction in force. In conclusion, double discharges at the onset of contractions effectively increase force production, especially in early stages of fatigue. This beneficial effect occurs without additional force loss in later stages of fatigue, indicating that the additional energy cost induced by doublet discharges to skeletal muscle is limited.

  3. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine

    2009-01-01

    .5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  4. Defective sarcoplasmic reticulum-mitochondria communication in aged heart and its effect on ischemia and reperfusion injury

    OpenAIRE

    Fernández Sanz, Celia

    2015-01-01

    Las alteraciones mitocondriales están vinculadas a la mayor vulnerabilidad de padecer enfermedades durante el envejecimiento. La edad avanzada es un factor determinante de la incidencia y gravedad de la cardiopatía isquémica. Estudios preclínicos sugieren la existencia de un daño celular intrínseco, por mecanismos no del todo establecidos, que contribuye a un incremento de la susceptibilidad del miocardio senescente al daño isquémico. Esta tesis investiga el papel de la comunicación mitoco...

  5. Defective sarcoplasmic reticulum-mitochondria communication in aged heart and its effect on ischemia and reperfusion injury

    OpenAIRE

    Fernández Sanz, Celia; Meseguer Navarro, Anna

    2015-01-01

    Las alteraciones mitocondriales están vinculadas a la mayor vulnerabilidad de padecer enfermedades durante el envejecimiento. La edad avanzada es un factor determinante de la incidencia y gravedad de la cardiopatía isquémica. Estudios preclínicos sugieren la existencia de un daño celular intrínseco, por mecanismos no del todo establecidos, que contribuye a un incremento de la susceptibilidad del miocardio senescente al daño isquémico. Esta tesis investiga el papel de la comunicación mitocondr...

  6. Effects of free oxygen radicals on Ca2+ release mechanisms in the sarcoplasmic reticulum of scallop (Pecten jacobaeus) adductor muscle.

    Science.gov (United States)

    Burlando, B; Viarengo, A; Pertica, M; Ponzano, E; Orunesu, M

    1997-08-01

    In vitro oxyradical effects on SR Ca2+ regulation were studied by using a SR-containing cell-free preparation from scallop (Pecten jacobaeus) adductor muscle. Ca2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe3+/ascorbate produced dose-dependent Ca2+ release from SR vesicles, eventually leading to massive Ca2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca2+ release but at a much slower rate. Pre-incubations with catalase or with the hydroxyl radical scavenger KMBA led to a significant decrease in the Fe3+/ascorbate-induced Ca2+ release rate and to a delay of massive Ca2+ loss. Pre-incubations with GSH or DTT strongly reduced the Ca2+ release caused by Fe3+/ascorbate and, moreover, they prevented massive Ca2+ loss from SR vesicles. Addition of GSH or DTT after Fe3+/ascorbate promptly reduced the Ca2+ release rate and delayed massive Ca2+ release. Pre-incubation with the SR Ca2+ channel blocker ruthenium red strongly reduced the Ca2+ release caused by Fe3+/ascorbate, and also prevented massive Ca2+ loss. In the presence of ruthenium red, Fe3+/ascorbate treatments followed by Ca2+ addition revealed that Ca2+ uptake inhibition was slower than Ca2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca2+ regulation. This mainly occurred through Ca2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca2+ channels is in line with their possible involvement in cell signaling.

  7. Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

    Science.gov (United States)

    Montague, Karli; Malik, Bilal; Gray, Anna L; La Spada, Albert R; Hanna, Michael G; Szabadkai, Gyorgy; Greensmith, Linda

    2014-07-01

    Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

  8. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates

    OpenAIRE

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2011-01-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h ...

  9. Role of Mitochondrial Enzymes and Sarcoplasmic ATPase in Cardioprotection Mediated by Aqueous Extract of Desmodium gangeticum (L) DC Root on Ischemic Reperfusion Injury.

    Science.gov (United States)

    Kurian, G A; Paddikkala, J

    2010-11-01

    The present study investigate the protective effect of aqueous root extract of Desmodium gangeticum in preserving mitochondrial and sarcoplasmic ATPase during ischemia reperfusion injury. The isolated rat hearts in both drug and control group were subjected to warm ischemia (37°), followed by reperfusion with the Langendorff perfusion system. The aqueous root extract of Desmodium gangeticum (L) at a dose of 50 mg/kg body weight was found to be effective in the rat heart for the management of ischemic reperfusion injury. Physiological parameters were significantly (PDesmodium gangeticum treated rat heart. These results suggest that Desmodium gangeticum aqueous root extract can preserve the mitochondrial and sarcoplasmic ATPase in the myocardium, resulting in the improvement of cardiac function after ischemia reperfusion injury.

  10. Effects of simvastatin on cardiac performance and expression of sarcoplasmic reticular calcium regulatory proteins in rat heart

    Institute of Scientific and Technical Information of China (English)

    Xia ZHENG; Shen-jiang HU

    2005-01-01

    Aim: To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA),phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Methods:Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 μmol/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax,and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 μmol/L) for 1 h or 24 h.The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay.Results: LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 μmol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 μmol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally,simvastatin (1-30 μmol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 μmol/L inhibited the cell viability. Conclusion:Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

  11. The sarcoplasmic calcium pump - a most efficient ion translocating system.

    Science.gov (United States)

    Hasselbach, W

    1977-04-21

    In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high afffinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca++, Mg++ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate who phosphoryl group can be transferred to ADP.

  12. Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

    Directory of Open Access Journals (Sweden)

    Caroline Ramspacher

    2015-06-01

    Full Text Available Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

  13. Endoplasmic reticulum stress and cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Xiaohui Duan; Yongfen Qi; Chaoshu Tang

    2009-01-01

    The endoplasmic reticulum (ER) serves several important functions, mainly post-translational modification, folding and assembly of newly synthesized secretary proteins, synthesizing lipids and cellular calcium storage. Various factors can disrupt ER homeostasis and disturb its functions, which leads to the accumulation of unfolded and misfolded proteins and to potential cellular dysfunction and pathological consequences, collectively termed ER stress. Recent progress suggests that ER stress plays a key role in the immune response, diabetes, tumor growth, and some neurodegenerative diseases. In particular, ER stress is involved in several processes of cardiovascular diseases, such as ischemia/reperfusion injury, cardiomyopathy, cardiac hypertrophy, heart failure, and atherosclerosis. Further research on the relation of ER stress to cardiovascular diseases will greatly enhance the understanding of these pathological processes and provide novel avenues to potential therapies.

  14. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin

    National Research Council Canada - National Science Library

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-01-01

    .... We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients...

  15. Endoplasmic reticulum stress in lung disease

    Directory of Open Access Journals (Sweden)

    Stefan J. Marciniak

    2017-06-01

    Full Text Available Exposure to inhaled pollutants, including fine particulates and cigarette smoke is a major cause of lung disease in Europe. While it is established that inhaled pollutants have devastating effects on the genome, it is now recognised that additional effects on protein folding also drive the development of lung disease. Protein misfolding in the endoplasmic reticulum affects the pathogenesis of many diseases, ranging from pulmonary fibrosis to cancer. It is therefore important to understand how cells respond to endoplasmic reticulum stress and how this affects pulmonary tissues in disease. These insights may offer opportunities to manipulate such endoplasmic reticulum stress pathways and thereby cure lung disease.

  16. Formalin evokes calcium transients from the endoplasmatic reticulum.

    Directory of Open Access Journals (Sweden)

    Michael J M Fischer

    Full Text Available The formalin test is the most widely used behavioral screening test for analgesic compounds. The cellular mechanism of action of formaldehyde, inducing a typically biphasic pain-related behavior in rodents is addressed in this study. The chemoreceptor channel TRPA1 was suggested as primary transducer, but the high concentrations used in the formalin test elicit a similar response in TRPA1 wildtype and knockout animals. Here we show that formaldehyde evokes a dose-dependent calcium release from intracellular stores in mouse sensory neurons and primary keratinocytes as well as in non-neuronal cell lines, and independent of TRPA1. The source of calcium is the endoplasmatic reticulum and inhibition of the sarco/endoplasmic reticulum calcium-ATPase has a major contribution. This TRPA1-independent mechanism may underlie formaldehyde-induced pan-neuronal excitation and subsequent inflammation.

  17. Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.

    Science.gov (United States)

    Zhang, Ying; Zhang, KaiQiang; Ma, Lin; Gu, HeFeng; Li, Jian; Lei, Shuang

    2016-09-01

    The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Endoplasmic reticulum stress in periimplantation embryos.

    Science.gov (United States)

    Michalak, Marek; Gye, Myung Chan

    2015-03-01

    Stress coping mechanisms are critical to minimize or overcome damage caused by ever changing environmental conditions. They are designed to promote cell survival. The unfolded protein response (UPR) pathway is mobilized in response to the accumulation of unfolded proteins, ultimately in order to regain endoplasmic reticulum (ER) homeostasis. Various elements of coping responses to ER stress including Perk, Ask1, Bip, Chop, Gadd34, Ire1, Atf4, Atf6, and Xbp1 have been identified and were found to be inducible in oocytes and preimplantation embryos, suggesting that, as a normal part of the cellular adaptive mechanism, these coping responses, including the UPR, play a pivotal role in the development of preimplantation embryos. As such, the UPR-associated molecules and pathways may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos. After implantation, ER stress-induced coping responses become physiologically important for a normal decidual response, placentation, and early organogenesis. Attenuation of ER stress coping responses by tauroursodeoxycholate and salubrinal was effective for prevention of cell death of cultured embryos. Further elucidation of new and relevant ER stress coping responses in periimplantation embryos might contribute to a comprehensive understanding of the regulation of normal development of embryonic development and potentiation of embryonic development in vitro.

  19. Studies on the Endoplasmic Reticulum

    Science.gov (United States)

    Porter, Keith R.; Machado, Raul D.

    1960-01-01

    Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO4. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO4-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell. PMID:14434278

  20. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    Science.gov (United States)

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  1. Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.

    Science.gov (United States)

    Dong, Yunzhou; Zhang, Miao; Wang, Shuangxi; Liang, Bin; Zhao, Zhengxing; Liu, Chao; Wu, Mingyuan; Choi, Hyoung Chul; Lyons, Timothy J; Zou, Ming-Hui

    2010-06-01

    The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo. ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet. Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet. We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.

  2. Influence of inorganic phosphate and pH on sarcoplasmic reticular ATPase in skinned muscle fibres of Xenopus laevis.

    Science.gov (United States)

    Stienen, G J; Papp, Z; Zaremba, R

    1999-08-01

    1. The influence of 30 mM inorganic phosphate (Pi) and pH (6.2-7.4) on the rate of ATP utilization was determined in mechanically skinned bundles of myofibrils from the iliofibularis muscle of Xenopus laevis at approximately 5 C. 2. BDM (2,3-butanedione monoxime; 10 mM) depressed isometric force production and actomyosin (AM) ATPase activity equally. Therefore sarcoplasmic reticular (SR) ATPase activity could be determined by extrapolation of the total ATPase activity to zero force. 3. The SR ATPase activity without added Pi at pH 7.1 was 42 +/- 2 % of the total ATPase activity. Addition of 30 mM Pi reduced SR ATPase activity slightly, by 9 +/- 5 %, and depressed force by 62 +/- 2 % and AM ATPase activity by 21 +/- 6 %. 4. At pH 6.2, force, SR ATPase activity and AM ATPase activity were reduced by 21 +/- 5, 61 +/- 5 and 10 +/- 4 % of their respective values at pH 7.1. 5. The SR ATPase activity at 30 mM Pi and pH 6.2 was reduced markedly to 20 +/- 6 % of the value under control conditions, suggesting that the maximum rate of Ca2+ uptake during muscle fatigue was strongly depressed. This reduction was larger than expected on the basis of the effects of Pi and pH alone.

  3. Targeting and retention of type 1 ryanodine receptors to the endoplasmic reticulum.

    Science.gov (United States)

    Meur, Gargi; Parker, Andrew K T; Gergely, Fanni V; Taylor, Colin W

    2007-08-10

    Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca(2+) release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.

  4. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes

    DEFF Research Database (Denmark)

    Boutrup Stephansen, Karen; Mattebjerg, Maria Ahlm; Wattjes, Jasper;

    2015-01-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged...... fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta...

  5. Association of cardiac injury with iron-increased oxidative and nitrative modifications of the SERCA2a isoform of sarcoplasmic reticulum Ca(2+)-ATPase in diabetic rats.

    Science.gov (United States)

    Li, Xueli; Li, Wenliang; Gao, Zhonghong; Li, Hailing

    2016-08-01

    The role of iron in the etiology of diabetes complications is not well established. Thus, this study was performed to test whether the iron-induced increase of oxidative/nitrative damage is involved in SERCA2a-related diabetic heart complication. Four randomly divided groups of rats were used: normal control group; iron overload group; diabetes group, and diabetic plus iron overload group. Iron supplementation stimulated cardiomyocyte hypertrophy and led to an increase in cardiac protein carbonyls, nitrotyrosine (3-NT) formation, and iNOS protein expression, thus resulting in abnormal myocardium calcium homeostasis of diabetic rats. The levels of SECA2a oxidation/nitration were significantly increased in the iron overload diabetic rats, along with a decrease in SECA2a expression and activity. In order to elucidate the possible role of iron in SERCA2a dysfunction, the effects of iron (Fe(3+) or hemin) on peroxynitrite (ONOO(-)) induced SERCA2a oxidation and nitration were further investigated in vitro. It was found that tyrosine nitration played more important role in SERCA2a inactivation than thiol oxidation. These results present a potential mechanism in which iron exacerbates the diabetes-induced oxidative/nitrative modification of SERCA2a, which may cause functional deficits in the myocyte associated with diabetic cardiac dysfunction. Our findings may help to further understand the role of iron in the pathogenesis of diabetic complications.

  6. Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed; Gaster, Michel

    2013-01-01

    lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C......-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin......, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS: NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel...

  7. Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs

    DEFF Research Database (Denmark)

    Winther, Anne-Marie Lund; Liu, Huizhen; Sonntag, Yonathan;

    2010-01-01

    transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S....... R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives...

  8. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca2+-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition

    OpenAIRE

    Fraqueza, Gil; de Carvalho, Luís A. E. Batista; Marques, M. Paula M.; Maia, Luisa; Ohlin, C. André; Casey, William H.; Aureliano, M.

    2012-01-01

    Recently we demonstrated that the decavanadate (V10) ion is a stronger Ca2+-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V10 interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the p...

  9. Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

    Science.gov (United States)

    Harmer, A R; Ruell, P A; Hunter, S K; McKenna, M J; Thom, J M; Chisholm, D J; Flack, J R

    2014-02-01

    Calcium cycling is integral to muscle performance during the rapid muscle contraction and relaxation of high-intensity exercise. Ca(2+) handling is altered by diabetes mellitus, but has not previously been investigated in human skeletal muscle. We investigated effects of high-intensity exercise and sprint training on skeletal muscle Ca(2+) regulation among men and women with type 1 diabetes (T1D, n = 8, 3F, 5M) and matched non-diabetic controls (CON, n = 8, 3F, 5M). Secondarily, we examined sex differences in Ca(2+) regulation. Subjects undertook 7 weeks of three times-weekly cycle sprint training. Before and after training, performance was measured, and blood and muscle were sampled at rest and after high-intensity exercise. In T1D, higher Ca(2+)-ATPase activity (+28%) and Ca(2+) uptake (+21%) than in CON were evident across both times and days (P women across both times and days. Intense exercise did not alter Ca(2+)-ATPase activity in T1D or CON. However, sex differences were evident: Ca(2+)-ATPase was reduced with exercise among men but increased among women across both days (time × sex interaction, P Sprint training reduced Ca(2+)-ATPase (-8%, P Sprint training reduced Ca(2+)-ATPase in T1D and CON. Sex differences in Ca(2+)-ATPase activity were evident and may be linked with fibre type proportion differences.

  10. Mapping the interactions between ATP and the sarcoplasmic reticulum Ca 2 + -ATPase with ATP and ATP analogs studied by Fourier transform infrared spectroscopy

    OpenAIRE

    Liu, Man

    2004-01-01

    Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beo...

  11. Glycoprotein folding in the endoplasmic reticulum

    NARCIS (Netherlands)

    Braakman, L.J.; Benham, A.M.

    2000-01-01

    Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of

  12. Protein transport into the human endoplasmic reticulum

    NARCIS (Netherlands)

    Dudek, Johanna; Pfeffer, Stefan; Lee, Po-Hsien; Jung, Martin; Cavalié, Adolfo; Helms, Volkhard; Förster, Friedrich; Zimmermann, Richard

    2015-01-01

    Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various trans

  13. Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Manfredi, Giovanni; Kawamata, Hibiki

    2016-06-01

    Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Water-mediated interactions influence the binding of thapsigargin to sarco/endoplasmic reticulum calcium adenosinetriphosphatase

    DEFF Research Database (Denmark)

    Paulsen, Eleonora S.; Villadsen, Jesper; Tenori, Eleonora;

    2013-01-01

    A crystal structure suggests four water molecules are present in the binding cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA). Computational chemistry indicates that three of these water molecules mediate an extensive hydrogen-bonding network between thapsigargin...

  15. Ca2+ and endoplasmic reticulum Ca2+-ATPase regulate the formation of silk fibers with favorable mechanical properties.

    Science.gov (United States)

    Wang, Xin; Li, Yi; Xie, Kang; Yi, Qiying; Chen, Quanmei; Wang, Xiaohuan; Shen, Hong; Xia, Qingyou; Zhao, Ping

    2015-02-01

    Calcium ions (Ca(2+)) are crucial for the conformational transition of silk fibroin in vitro, and silk fibroin conformations correlate with the mechanical properties of silk fibers. To investigate the relationship between Ca(2+) and mechanical properties of silk fibers, CaCl2 was injected into silkworms (Bombyx mori). Fourier-transform infrared spectroscopy (FTIR) analysis and mechanical testing revealed that injection of CaCl2 solution (7.5mg/g body weight) significantly increased the levels of α-helix and random coil structures of silk proteins. In addition, extension of silk fibers increased after CaCl2 injection. In mammals, sarcoplasmic reticulum Ca(2+)-ATPase in muscle and endoplasmic reticulum Ca(2+)-ATPase in other tissues (together denoted by SERCA) are responsible for calcium balance. Therefore, we analyzed the expression pattern of silkworm SERCA (BmSERCA) in silk glands and found that BmSERCA was abundant in the anterior silk gland (ASG). After injection of thapsigargin (TG) to block SERCA activity, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. Moreover, FTIR analysis revealed that the levels of α-helix and β-sheet structures increased in silk fibers from TG-injected silkworms compared to controls. The results provide evidence that BmSERCA has a key function in calcium transportation in ASG that is related to maintaining a suitable ionic environment. This ionic environment with a proper Ca(2+) concentration is crucial for the formation of silk fibers with favorable mechanical performances.

  16. Association of immunoproteasomes with the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Brooks, P.; Murray, R.Z.; Mason, G.G.F.;

    2000-01-01

    pathway. Two of the gamma-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7...... that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules....

  17. Endoplasmic Reticulum Stress and Insulin Biosynthesis: A Review

    Directory of Open Access Journals (Sweden)

    Mi-Kyung Kim

    2012-01-01

    Full Text Available Insulin resistance and pancreatic beta cell dysfunction are major contributors to the pathogenesis of diabetes. Various conditions play a role in the pathogenesis of pancreatic beta cell dysfunction and are correlated with endoplasmic reticulum (ER stress. Pancreatic beta cells are susceptible to ER stress. Many studies have shown that increased ER stress induces pancreatic beta cell dysfunction and diabetes mellitus using genetic models of ER stress and by various stimuli. There are many reports indicating that ER stress plays an important role in the impairment of insulin biosynthesis, suggesting that reduction of ER stress could be a therapeutic target for diabetes. In this paper, we reviewed the relationship between ER stress and diabetes and how ER stress controls insulin biosynthesis.

  18. Endoplasmic reticulum stress and insulin biosynthesis: a review.

    Science.gov (United States)

    Kim, Mi-Kyung; Kim, Hye-Soon; Lee, In-Kyu; Park, Keun-Gyu

    2012-01-01

    Insulin resistance and pancreatic beta cell dysfunction are major contributors to the pathogenesis of diabetes. Various conditions play a role in the pathogenesis of pancreatic beta cell dysfunction and are correlated with endoplasmic reticulum (ER) stress. Pancreatic beta cells are susceptible to ER stress. Many studies have shown that increased ER stress induces pancreatic beta cell dysfunction and diabetes mellitus using genetic models of ER stress and by various stimuli. There are many reports indicating that ER stress plays an important role in the impairment of insulin biosynthesis, suggesting that reduction of ER stress could be a therapeutic target for diabetes. In this paper, we reviewed the relationship between ER stress and diabetes and how ER stress controls insulin biosynthesis.

  19. Induction of Apoptosis by Hypertension Via Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Yingying Sun

    2015-02-01

    Full Text Available Background/Aims: Endoplasmic reticulum (ER stress is one of the intrinsic apoptosis pathways, and cardiac apoptosis can occur in cardiovascular diseases, such as hypertension. However, the mechanisms by which ER stress leads to apoptosis remain enigmatic, particularly in the progression from cardiac hypertrophy to diastolic heart failure due to hypertension. Methods: We used spontaneously hypertensive rats (SHRs to investigate possible signalling pathways for ER stress. Results: We found that cardiac protein and mRNA levels of glucose-regulated protein 78 were up-regulated. In addition, the CHOP- and caspase-12-dependent pathways, but not that of JNK, were activated in the SHR rats. Conclusions: These results suggest that ER stress can contribute to myocardial apoptosis during hypertensive disease.

  20. Hypnosis, suggestion, and suggestibility: an integrative model.

    Science.gov (United States)

    Lynn, Steven Jay; Laurence, Jean-Roch; Kirsch, Irving

    2015-01-01

    This article elucidates an integrative model of hypnosis that integrates social, cultural, cognitive, and neurophysiological variables at play both in and out of hypnosis and considers their dynamic interaction as determinants of the multifaceted experience of hypnosis. The roles of these variables are examined in the induction and suggestion stages of hypnosis, including how they are related to the experience of involuntariness, one of the hallmarks of hypnosis. It is suggested that studies of the modification of hypnotic suggestibility; cognitive flexibility; response sets and expectancies; the default-mode network; and the search for the neurophysiological correlates of hypnosis, more broadly, in conjunction with research on social psychological variables, hold much promise to further understanding of hypnosis.

  1. Endoplasmic reticulum stress and diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Toshiyuki Oshitari

    2008-02-01

    Full Text Available Toshiyuki Oshitari1,2, Natsuyo Hata1, Shuichi Yamamoto11Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba City, Chiba, Japan; 2Department of Ophthalmology, Kimitsu Central Hospital, Kisarazu City, Chiba, JapanAbstract: Endoplasmic reticulum (ER stress is involved in the pathogenesis of several diseases including Alzheimer disease and Parkinson disease. Many recent studies have shown that ER stress is related to the pathogenesis of diabetes mellitus, and with the death of pancreatic β-cells, insulin resistance, and the death of the vascular cells in the retina. Diabetic retinopathy is a major complication of diabetes and results in death of both neural and vascular cells. Because the death of the neurons directly affects visual function, the precise mechanism causing the death of neurons in early diabetic retinopathy must be determined. The ideal therapy for preventing the onset and the progression of diabetic retinopathy would be to treat the factors involved with both the vascular and neuronal abnormalities in diabetic retinopathy. In this review, we present evidence that ER stress is involved in the death of both retinal neurons and vascular cells in diabetic eyes, and thus reducing or blocking ER stress may be a potential therapy for preventing the onset and the progression of diabetic retinopathy.Keywords: endoplasmic reticulum stress, diabetic retinopathy, vascular cell death, neuronal cell death

  2. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    Science.gov (United States)

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%.

  3. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes.

    Science.gov (United States)

    Stephansen, Karen; Mattebjerg, Maria; Wattjes, Jasper; Milisavljevic, Ana; Jessen, Flemming; Qvortrup, Klaus; Goycoolea, Francisco M; Chronakis, Ioannis S

    2015-05-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 ± 3 nm, and the zeta potential was -42 ± 0.3 mV. The NCXs were stable in water, gastric buffer, intestinal buffer and HEPES buffered glycose, and at all pH values from 2 to 9 except pH 3, where they aggregated. When proteolytic enzymes were present in the buffer, the NCXs were degraded. Only at high concentrations the NCXs caused a decreased viability in HeLa and U2OS cell lines. The simple processing procedure and the high stability of the NCXs, makes them excellent candidates for use in the food and pharmaceutical industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

    Directory of Open Access Journals (Sweden)

    PA Possik

    2009-06-01

    Full Text Available We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2 in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER of cells.We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.

  5. THE RESPONSE OF DISSEMINATED RETICULUM CELL SARCOMA TO THE INTRAVENOUS INJECTION OF COLLOIDAL RADIOACTIVE GOLD

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Philip; Levitt, Seymour H.

    1963-06-15

    Case histories of two patients treated with colloidal radiogold for diffuse reticulum cell sarcoma are presented. Further analysis of the method is suggested by the unusually long survival time of one of the patients. It was concluded that, although external radiotherapy remains the treatment of choice in localized reticulum cell sarcoma, intravenous colloidal radiogold may be a useful agent in lymphosarcomas with diffuse minute neoplastic liver and spleen involvements. Intravenous colloidal radiogold can produce bone marrow depression and thrombocytopenia which can lead to death. This factor tends to argue against therapeutic use of the agent. It is suggested that no more than 50 mC Au/sup 198/ intravenously should be used for treatment of this disease. (R.M.G.)

  6. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    Science.gov (United States)

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  7. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders.

    Science.gov (United States)

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-02-11

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.

  8. Targeting endoplasmic reticulum stress in insulin resistance.

    Science.gov (United States)

    Salvadó, Laia; Palomer, Xavier; Barroso, Emma; Vázquez-Carrera, Manuel

    2015-08-01

    The endoplasmic reticulum (ER) is involved in the development of insulin resistance and progression to type 2 diabetes mellitus (T2DM). Disruption of ER homeostasis leads to ER stress, which activates the unfolded protein response (UPR). This response is linked to different processes involved in the development of insulin resistance (IR) and T2DM, including inflammation, lipid accumulation, insulin biosynthesis, and β-cell apoptosis. Understanding the mechanisms by which disruption of ER homeostasis leads to IR and its progression to T2DM may offer new pharmacological targets for the treatment and prevention of these diseases. Here, we examine ER stress, the UPR, and downstream pathways in insulin sensitive tissues, and in IR, and offer insights towards therapeutic strategies.

  9. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

    Science.gov (United States)

    Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

  10. Endoplasmic Reticulum (ER Stress and Endocrine Disorders

    Directory of Open Access Journals (Sweden)

    Daisuke Ariyasu

    2017-02-01

    Full Text Available The endoplasmic reticulum (ER is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR, which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI, Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2 are discussed in this article.

  11. Endoplasmic Reticulum Stress and Ethanol Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Fanmuyi Yang

    2015-10-01

    Full Text Available Ethanol abuse affects virtually all organ systems and the central nervous system (CNS is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK, inositol-requiring enzyme 1 (IRE1, and activating transcription factor 6 (ATF6. UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer’s disease (AD, Huntington’s disease (HD, Amyotrophic lateral sclerosis (ALS, and Parkinson’s disease (PD. However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity.

  12. Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injur y

    Institute of Scientific and Technical Information of China (English)

    Guo-zhu Sun; Fen-fei Gao; Zong-mao Zhao; Hai Sun; Wei Xu; Li-wei Wu; Yong-chang He

    2016-01-01

    Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo-plasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of lfuid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These ifndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.

  13. Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction.

    Science.gov (United States)

    Qin, Fuzhong; Yan, Chen; Patel, Ravish; Liu, Weimin; Dong, Erdan

    2006-05-15

    Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.

  14. Development of Endoplasmic Reticulum Stress during Experimental Oxalate Nephrolithiasis.

    Science.gov (United States)

    Motin, Yu G; Lepilov, A V; Bgatova, N P; Zharikov, A Yu; Motina, N V; Lapii, G A; Lushnikova, E L; Nepomnyashchikh, L M

    2016-01-01

    Morphological and ultrastructural study of the kidney was performed in rats with oxalate nephrolithiasis. Specific features of endoplasmic reticulum stress were evaluated during nephrolithiasis and treatment with α-tocopherol. We observed the signs of endoplasmic reticulum stress with activation of proapoptotic pathways and injury to the cell lining in nephron tubules and collecting ducts. Ultrastructural changes were found in the organelles, nuclei, and cell membranes of epitheliocytes. A relationship was revealed between endoplasmic reticulum stress and oxidative damage, which developed at the early state of lithogenesis.

  15. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

    Science.gov (United States)

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-09-01

    Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

  16. Theories of Suggestion.

    Science.gov (United States)

    Brown, W

    1928-02-01

    The word "suggestion" has been used in educational, scientific and medical literature in slightly different senses. In psychological medicine the use of suggestion has developed out of the earlier use of hypnotic influence.Charcot defined hypnosis as an artificial hysteria, Bernheim as an artificially increased suggestibility. The two definitions need to be combined to give an adequate account of hypnosis. Moreover, due allowance should be made for the factors of dissociation and of rapport in hypnotic phenomena.The relationships between dissociation, suggestibility, and hypnotizability.Theories of suggestion propounded by Pierre Janet, Freud, McDougall, Pawlow and others. Ernest Jones's theory of the nature of auto-suggestion. Janet explains suggestion in terms of ideo-motor action in which the suggested idea, because of the inactivity of competing ideas, produces its maximum effect. Freud explains rapport in terms of the sex instinct "inhibited in its aim" (transference) and brings in his distinction of "ego" and "ego-ideal" (or "super-ego") to supplement the theory. Jones explains auto-suggestion in terms of narcissism. McDougall explains hypnotic suggestion in terms of the instinct of self-abasement. But different instincts may supply the driving power to produce suggestion-effects in different circumstances. Such instincts as those of self-preservation (fear) and gregariousness may play their part. Auto-suggestion as a therapeutic factor is badly named. It supplements, but does not supplant the will, and makes complete volition possible.

  17. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

  18. An endoplasmic reticulum (ER)-directed fusion protein comprising a ...

    African Journals Online (AJOL)

    An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin ... which are used for the commercial production of therapeutic proteins. ... expression platforms) to purify recombinant proteins in crude plant extracts.

  19. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and

  20. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

  1. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

    Directory of Open Access Journals (Sweden)

    Jintao Zhang

    (siRNA targeting BIP and CHOP methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.

  2. Ultrafast glycerophospholipid-selective transbilayer motion mediated by a protein in the endoplasmic reticulum membrane.

    Science.gov (United States)

    Buton, X; Morrot, G; Fellmann, P; Seigneuret, M

    1996-03-22

    A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due

  3. Suggested safeguards an

    African Journals Online (AJOL)

    MJM Venter

    ... COORDINATION. (FACILITATION OR CASE MANAGEMENT) IN SOUTH AFRICA ... SUGGESTED SAFEGUARDS AND LIMITATIONS FOR EFFECTIVE AND .... professional practice.27 They have to assess the situation; educate the parents.

  4. Endoplasmic Reticulum Stress and Diabetic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Jiancheng Xu

    2012-01-01

    Full Text Available The endoplasmic reticulum (ER is an organelle entrusted with lipid synthesis, calcium homeostasis, protein folding, and maturation. Perturbation of ER-associated functions results in an evolutionarily conserved cell stress response, the unfolded protein response (UPR that is also called ER stress. ER stress is aimed initially at compensating for damage but can eventually trigger cell death if ER stress is excessive or prolonged. Now the ER stress has been associated with numerous diseases. For instance, our recent studies have demonstrated the important role of ER stress in diabetes-induced cardiac cell death. It is known that apoptosis has been considered to play a critical role in diabetic cardiomyopathy. Therefore, this paper will summarize the information from the literature and our own studies to focus on the pathological role of ER stress in the development of diabetic cardiomyopathy. Improved understanding of the molecular mechanisms underlying UPR activation and ER-initiated apoptosis in diabetic cardiomyopathy will provide us with new targets for drug discovery and therapeutic intervention.

  5. Manufacturer's Suggested Retail Prices

    NARCIS (Netherlands)

    Rosenkranz, S.

    2003-01-01

    Based on arguments of the `reference- dependent' theory of consumer choice we assume that a retailer's discount of a manufacturer's suggested retail price changes consumers' demand. We can show that the producer benefits from suggesting a retail price. If consumers are additionally sufficiently `los

  6. Research Suggestions for Students

    Science.gov (United States)

    Holland, John L.

    1974-01-01

    Describes how to perform accurate research. Also includes suggestions for specific research projects under such headings as: (1) types; (2) environments; (3) interactions; (4) classification; (5) hexagonal model; and (6) differentiation. (HMV)

  7. Open to Suggestion.

    Science.gov (United States)

    Journal of Reading, 1984

    1984-01-01

    Contributors offer suggestions concerning parents as reading stimulators, book discussions, a test bank for the secondary school/college reading lab, standardized reading tests, television reading, plagiarism, vocabulary development, and book reports. (FL)

  8. Open To Suggestion.

    Science.gov (United States)

    Journal of Reading, 1988

    1988-01-01

    Suggests class activities in three short articles including: (1) "Students Evaluate Reading," by Lenore Sandel; (2) "Solving Verbal Analogies," by Edward J. Dwyer; and (3) "Becoming Testwise," by Dean Schoen. (RS)

  9. Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood-Brain Barrier Damage.

    Science.gov (United States)

    Qie, Xiaojuan; Wen, Di; Guo, Hongyan; Xu, Guanjie; Liu, Shuai; Shen, Qianchao; Liu, Yi; Zhang, Wenfang; Cong, Bin; Ma, Chunling

    2017-01-01

    Methamphetamine (METH) abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood-brain barrier (BBB). Herein, we explored the potential mechanism of endoplasmic reticulum (ER) stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3) cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS) and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo) and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption.

  10. Unique defense strategy by the endoplasmic reticulum body in plants.

    Science.gov (United States)

    Yamada, Kenji; Hara-Nishimura, Ikuko; Nishimura, Mikio

    2011-12-01

    The endoplasmic reticulum (ER) is a site for the production of secretory proteins. Plants have developed ER subdomains for protein storage. The ER body is one such structure, which is observed in Brassicaceae plants. ER bodies accumulate in seedlings and roots or in wounded leaves in Arabidopsis. ER bodies contain high amounts of the β-glucosidases PYK10/BGLU23 in seedlings and roots or BGLU18 in wounded tissues. These results suggest that ER bodies are involved in the metabolism of glycoside molecules, presumably to produce repellents against pests and fungi. When Arabidopsis roots are homogenized, PYK10 formed large protein aggregates that include other β-glucosidases (BGLU21 and BGLU22), GDSL lipase-like proteins (GLL22) and cytosolic jacalin-related lectins (PBP1/JAL30, JAL31, JAL33, JAL34 and JAL35). Glucosidase activity increases by the aggregate formation. NAI1, a basic helix-loop-helix transcription factor, regulates the expression of the ER body proteins PYK10 and NAI2. Reduced expression of NAI2, PYK10 and BGLU21 resulted in abnormal ER body formation, indicating that these components regulate ER body formation. PYK10, BGLU21 and BGLU22 possess hydrolytic activity for scopolin, a coumaroyl glucoside that accumulates in the roots of Arabidopsis, and nai1 and pyk10 mutants are more susceptible to the symbiotic fungus Piriformospora indica. Therefore, it appears that the ER body is a unique organelle of Brassicaceae plants that is important for defense against pests and fungi.

  11. Selective export of autotaxin from the endoplasmic reticulum.

    Science.gov (United States)

    Lyu, Lin; Wang, Baolu; Xiong, Chaoyang; Zhang, Xiaotian; Zhang, Xiaoyan; Zhang, Junjie

    2017-04-28

    Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Altered localization of amyloid precursor protein under endoplasmic reticulum stress.

    Science.gov (United States)

    Kudo, Takashi; Okumura, Masayo; Imaizumi, Kazunori; Araki, Wataru; Morihara, Takashi; Tanimukai, Hitoshi; Kamagata, Eiichiro; Tabuchi, Nobuhiko; Kimura, Ryo; Kanayama, Daisuke; Fukumori, Akio; Tagami, Shinji; Okochi, Masayasu; Kubo, Mikiko; Tanii, Hisashi; Tohyama, Masaya; Tabira, Takeshi; Takeda, Masatoshi

    2006-06-02

    Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.

  13. Arachidonoyl-specific diacylglycerol kinase ε and the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Tomoyuki Nakano

    2016-11-01

    Full Text Available The endoplasmic reticulum (ER comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK that phosphorylates diacylglycerol (DG to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER.

  14. A Molecular Web: Endoplasmic Reticulum Stress, Inflammation and Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Namrata eChaudhari

    2014-07-01

    Full Text Available Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded protein response (UPR through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS. Toxic accumulation of ROS within ER and mitochondria disturb fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways has been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease and others. In this review we have discussed the UPR signaling pathways, and networking between ER stress induced inflammatory pathways, oxidative stress and mitochondrial signaling events which further induce or exacerbate ER stress.

  15. Attitudes to Suggestions

    Institute of Scientific and Technical Information of China (English)

    PETER; JOHNSON

    2007-01-01

    As an Australian expat teaching English in China for over four years, I often encourage my students to not only learn the English language but also try to understand Western culture. This includes the fact that Westerners frequently initiate proactive suggestions on any aspects of soci-

  16. Suggestions for Teaching Practice

    Institute of Scientific and Technical Information of China (English)

    ZHAN Na-na

    2013-01-01

    Teacher development and teaching practice(TP) have caught the eyes of researchers at home and abroad for many years. Many western scholars hold that reflective teaching is an efficient way to promote teacher development, but traditional TP is prevailing in China. Based on the merits and demerits of traditional TP and reflective TP, the author hopes to provide some suggestions for the people involved to promote the development of teacher education.

  17. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    Science.gov (United States)

    Gallego, Marta; Mora, Leticia; Concepción Aristoy, M; Toldrá, Fidel

    2016-04-01

    The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham.

  18. Lipid Transport between the Endoplasmic Reticulum and Mitochondria

    Science.gov (United States)

    Flis, Vid V.

    2013-01-01

    Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis. PMID:23732475

  19. Structural plasticity of the nuclear envelope and the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Sheval E. V.

    2014-09-01

    Full Text Available The nuclear envelope is a double membrane structure, continuous with endoplasmic reticulum, and the morphological organization of both these structures is quite conservative. However, nuclear envelope and endoplasmic reticulum demonstrate distinct structural plasticity, i. e., based on common organization, cells may form various non-canonical membrane structures that are observed only in specialized types of cells or appear in different pathologies. In this review, we will discuss the mechanisms of the biogenesis of such non-canonical structures, and the possible role of this plasticity in the development of pathological processes.

  20. Sulindac sulfide inhibits sarcoendoplasmic reticulum Ca2+ ATPase, induces endoplasmic reticulum stress response, and exerts toxicity in glioma cells: relevant similarities to and important differences from celecoxib.

    Science.gov (United States)

    White, M C; Johnson, G G; Zhang, W; Hobrath, J V; Piazza, G A; Grimaldi, M

    2013-03-01

    Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca(2+) homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca(2+) from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.

  1. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.

    Science.gov (United States)

    Sun, Wenxiang; Li, Yang; Chen, Lu; Chen, Huihui; You, Fuping; Zhou, Xiang; Zhou, Yi; Zhai, Zhonghe; Chen, Danying; Jiang, Zhengfan

    2009-05-26

    We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

  2. Disulfide Mispairing During Proinsulin Folding in the Endoplasmic Reticulum.

    Science.gov (United States)

    Haataja, Leena; Manickam, Nandini; Soliman, Ann; Tsai, Billy; Liu, Ming; Arvan, Peter

    2016-04-01

    Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Interestingly, formation of these two "interchain" disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes.

  3. Lipolysis Response to Endoplasmic Reticulum Stress in Adipose Cells*

    Science.gov (United States)

    Deng, Jingna; Liu, Shangxin; Zou, Liangqiang; Xu, Chong; Geng, Bin; Xu, Guoheng

    2012-01-01

    In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues. PMID:22223650

  4. Lipolysis response to endoplasmic reticulum stress in adipose cells.

    Science.gov (United States)

    Deng, Jingna; Liu, Shangxin; Zou, Liangqiang; Xu, Chong; Geng, Bin; Xu, Guoheng

    2012-02-24

    In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.

  5. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Suchita

    2011-01-01

    Full Text Available Abstract The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1. However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER, Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP, it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

  6. Transcriptional analysis implicates endoplasmic reticulum stress in bovine spongiform encephalopathy.

    Directory of Open Access Journals (Sweden)

    Yue Tang

    Full Text Available Bovine spongiform encephalopathy (BSE is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity.

  7. HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Ying [Department of Geriatric Medicine, Xiangya Hospital, Central South University, Changsha 410078 (China); Li, Shu-Jun [Department of Cardiovascular Medicine, Xiangya Hospital, Central South University, Changsha 410078 (China); Yang, Jian [Department of Geriatric Medicine, Xiangya Hospital, Central South University, Changsha 410078 (China); Qiu, Yuan-Zhen [Department of Otolaryngology, Xiangya Hospital, Central South University, Changsha 410078 (China); Chen, Fang-Ping, E-mail: xychenfp@163.com [Department of Hematology, Xiangya Hospital, Central South University, Changsha 410078 (China)

    2013-09-06

    Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

  8. Folding of viral envelope glycoproteins in the endoplasmic reticulum

    NARCIS (Netherlands)

    Braakman, L.J.; Anken, E. van

    2000-01-01

    Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality con

  9. A luminal flavoprotein in endoplasmic reticulum-associated degradation

    DEFF Research Database (Denmark)

    Riemer, Jan; Appenzeller-Herzog, Christian; Johansson, Linda

    2009-01-01

    The quality control system of the endoplasmic reticulum (ER) discriminates between native and nonnative proteins. The latter are degraded by the ER-associated degradation (ERAD) pathway. Whereas many cytosolic and membrane components of this system are known, only few luminal players have been id...

  10. PARM-1 is an endoplasmic reticulum molecule involved in endoplasmic reticulum stress-induced apoptosis in rat cardiac myocytes.

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    Koji Isodono

    Full Text Available To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1. While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER. In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.

  11. Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival

    NARCIS (Netherlands)

    Sun, S.; Shi, Guojun; Han, X.; Francisco, A.; Ji, Y.; Kersten, A.H.

    2014-01-01

    Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L’s physiological importance in mammalian ERAD, however, remains to be established. Her

  12. The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

    Science.gov (United States)

    Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

    2016-09-15

    Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q(2)cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as

  13. Endoplasmic Reticulum Stress and Insulin Biosynthesis: A Review

    OpenAIRE

    2012-01-01

    Insulin resistance and pancreatic beta cell dysfunction are major contributors to the pathogenesis of diabetes. Various conditions play a role in the pathogenesis of pancreatic beta cell dysfunction and are correlated with endoplasmic reticulum (ER) stress. Pancreatic beta cells are susceptible to ER stress. Many studies have shown that increased ER stress induces pancreatic beta cell dysfunction and diabetes mellitus using genetic models of ER stress and by various stimuli. There are many re...

  14. Prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine

    Directory of Open Access Journals (Sweden)

    Ravindra Kumar

    2017-09-01

    Full Text Available Background The endoplasmic reticulum plays an important role in many cellular processes, which includes protein synthesis, folding and post-translational processing of newly synthesized proteins. It is also the site for quality control of misfolded proteins and entry point of extracellular proteins to the secretory pathway. Hence at any given point of time, endoplasmic reticulum contains two different cohorts of proteins, (i proteins involved in endoplasmic reticulum-specific function, which reside in the lumen of the endoplasmic reticulum, called as endoplasmic reticulum resident proteins and (ii proteins which are in process of moving to the extracellular space. Thus, endoplasmic reticulum resident proteins must somehow be distinguished from newly synthesized secretory proteins, which pass through the endoplasmic reticulum on their way out of the cell. Approximately only 50% of the proteins used in this study as training data had endoplasmic reticulum retention signal, which shows that these signals are not essentially present in all endoplasmic reticulum resident proteins. This also strongly indicates the role of additional factors in retention of endoplasmic reticulum-specific proteins inside the endoplasmic reticulum. Methods This is a support vector machine based method, where we had used different forms of protein features as inputs for support vector machine to develop the prediction models. During training leave-one-out approach of cross-validation was used. Maximum performance was obtained with a combination of amino acid compositions of different part of proteins. Results In this study, we have reported a novel support vector machine based method for predicting endoplasmic reticulum resident proteins, named as ERPred. During training we achieved a maximum accuracy of 81.42% with leave-one-out approach of cross-validation. When evaluated on independent dataset, ERPred did prediction with sensitivity of 72.31% and specificity of 83

  15. The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane.

    Science.gov (United States)

    Honvo-Houéto, Edith; Henry, Céline; Chat, Sophie; Layani, Sarah; Truchet, Sandrine

    2016-10-01

    During lactation, mammary epithelial cells secrete huge amounts of milk from their apical side. The current view is that caseins are secreted by exocytosis, whereas milk fat globules are released by budding, enwrapped by the plasma membrane. Owing to the number and large size of milk fat globules, the membrane surface needed for their release might exceed that of the apical plasma membrane. A large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globule membranes was used to decipher the cellular origins of the milk fat globule membrane. Surprisingly, differential analysis of protein profiles of these two organelles strongly suggest that, in addition to the plasma membrane, the endoplasmic reticulum and the secretory vesicles contribute to the milk fat globule membrane. Analysis of membrane-associated and raft microdomain proteins reinforces this possibility and also points to a role for lipid rafts in milk product secretion. Our results provide evidence for a significant contribution of the endoplasmic reticulum to the milk fat globule membrane and a role for SNAREs in membrane dynamics during milk secretion. These novel aspects point to a more complex model for milk secretion than currently envisioned.

  16. Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.

    Science.gov (United States)

    Mund, Thomas; Gewies, Andreas; Schoenfeld, Nicole; Bauer, Manuel K A; Grimm, Stefan

    2003-04-01

    We have isolated Spike, a novel and evolutionary conserved BH3-only protein. BH3-only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3-only proteins, Spike could not interact with all tested Bcl-2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro-caspase-8 and Bcl-XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl-XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down-regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3-only and antiapoptotic Bcl-2 proteins regulate cell death.

  17. Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Emma Folch-Puy

    2016-05-01

    Full Text Available The endoplasmic reticulum (ER is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS. This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR, which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes.

  18. Distribution of an Ankyrin-repeat Protein on the Endoplasmic Reticulum in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Liqin Wei; Yan Li

    2009-01-01

    There are many ankyrin-repeat proteins in plant cells. However, the distribution and function of these proteins are mostly unclear. By reverse transcription-polymerase chain reaction, a gene encoding an ankyrin-like protein was cloned from Arabidopsis and named AtANK1 (GenBank accession no. NM_120340). The 6-His-tagged AtAnk1-N fusion protein was affinity-purified and its rabbit polyclonal antibody was obtained. Immuno-blotting with the purified anti-AtAnk1-N polyclonal antibody revealed that the relative molecular weight of the AtANK1 protein was about 76 kDa. By immunofluorescence labeling and immuno-gold labeling with the purified anti-AtAnk1-N polyclonal antibody, coupled with confocal and transmission electron microscopy observation, AtANK1 was found to be distributed on the membrane of the endoplaamic reticulum in Arabidopsis cells. Based on these results, we suggested that AtANK1 might be involved in endoplasmic reticulum-related protein localization and sorting in plant cells.

  19. 收缩潜伏期骨骼肌肌浆网的超微结构变化%Ultrastructural Change of Sarcoplasmic Reticulum in Skeletal Muscle during Contraction-Latency

    Institute of Scientific and Technical Information of China (English)

    邰艳红; 杨勇骥; 宋田斌; 汤莹; 吴越

    2000-01-01

    采用双向红外线探测--计算机控制的电刺激与超低温快速冷冻固定同步技术对收缩潜伏期的骨骼肌作实时固定,采用透射电镜对电刺激后0.8 ms,5.6 ms,8.4 ms及静息期的骨骼肌超微结构变化进行了对比研究,发现在骨骼肌收缩潜伏期(小于10 ms)内,肌浆网内的Ca2+从面向T-管的肌浆网前端流出.同时发现肌膜与肌纤维间的间隙大大变窄,肌浆网前端的膜产生两个圆孔,而静息时的骨骼肌却无上述改变.这些发现对揭示骨骼肌兴奋-收缩偶联的发生机制有重大意义.

  20. Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion.

    Science.gov (United States)

    Hurne, Alanna M; O'Brien, Jennifer J; Wingrove, Douglas; Cherednichenko, Gennady; Allen, Paul D; Beam, Kurt G; Pessah, Isaac N

    2005-11-04

    Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

  1. Endoplasmic reticulum stress as a primary pathogenic mechanism leading to age-related macular degeneration.

    Science.gov (United States)

    Libby, Richard T; Gould, Douglas B

    2010-01-01

    Age-related macular degeneration (AMD) is a multi-factorial disease and a leading cause of blindness. Proteomic and genetic data suggest that activation or de-repression of the alternate complement cascade of innate immunity is involved in end-stage disease. Several lines of evidence suggest that production of reactive oxygen species and chronic oxidative stress lead to protein and lipid modifications that initiate the complement cascade. Understanding the triggers of these pathogenic pathways and the site of the primary insult will be important for development of targeted therapeutics. Endoplasmic reticulum (ER) stress from misfolded mutant proteins and other sources are an important potential tributary mechanism. We propose that misfolded-protein-induced ER stress in the retinal-pigmented epithelium and/or choroid could lead to chronic oxidative stress, complement deregulation and AMD. Small molecules targeted to ER stress and oxidative stress could allow for a shift from disease treatment to disease prevention.

  2. The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Gagandip K. Poone

    2015-05-01

    Full Text Available Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP activation. In an in vitro and in vivo model of ischemic stroke, we investigated whether hypothermia regulates the unfolded protein response of CHOP and Endoplasmic reticulum oxidoreductin-α (Ero1-α, because Ero1-α is suggested to be a downstream CHOP target. The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C and hypothermic (31 °C conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course (p < 0.01, p < 0.0001, whereas Ero1-α expression peaked at three to six hours (p < 0.0001. Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001. On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001. Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia and treatment (hypothermia, we conclude that they are regulated independently.

  3. ILDR2: an endoplasmic reticulum resident molecule mediating hepatic lipid homeostasis.

    Directory of Open Access Journals (Sweden)

    Kazuhisa Watanabe

    Full Text Available Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time

  4. Emergence of the mitochondrial reticulum from fission and fusion dynamics.

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    Valerii M Sukhorukov

    Full Text Available Mitochondria form a dynamic tubular reticulum within eukaryotic cells. Currently, quantitative understanding of its morphological characteristics is largely absent, despite major progress in deciphering the molecular fission and fusion machineries shaping its structure. Here we address the principles of formation and the large-scale organization of the cell-wide network of mitochondria. On the basis of experimentally determined structural features we establish the tip-to-tip and tip-to-side fission and fusion events as dominant reactions in the motility of this organelle. Subsequently, we introduce a graph-based model of the chondriome able to encompass its inherent variability in a single framework. Using both mean-field deterministic and explicit stochastic mathematical methods we establish a relationship between the chondriome structural network characteristics and underlying kinetic rate parameters. The computational analysis indicates that mitochondrial networks exhibit a percolation threshold. Intrinsic morphological instability of the mitochondrial reticulum resulting from its vicinity to the percolation transition is proposed as a novel mechanism that can be utilized by cells for optimizing their functional competence via dynamic remodeling of the chondriome. The detailed size distribution of the network components predicted by the dynamic graph representation introduces a relationship between chondriome characteristics and cell function. It forms a basis for understanding the architecture of mitochondria as a cell-wide but inhomogeneous organelle. Analysis of the reticulum adaptive configuration offers a direct clarification for its impact on numerous physiological processes strongly dependent on mitochondrial dynamics and organization, such as efficiency of cellular metabolism, tissue differentiation and aging.

  5. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Science.gov (United States)

    Schuster, Frank; Müller, Rainer; Hartung, Edmund; Roewer, Norbert; Anetseder, Martin

    2005-01-01

    Background Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia. PMID:15946384

  6. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Directory of Open Access Journals (Sweden)

    Roewer Norbert

    2005-06-01

    Full Text Available Abstract Background Malignant hyperthermia (MH is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible, 7 MHE (equivocal and 16 MHN (non-susceptible classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM and halothane (0.11; 0.22; 0.44; 0.66 mM was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p Results Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.

  7. Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics.

    Science.gov (United States)

    Pizzo, Paola; Pozzan, Tullio

    2007-10-01

    Mitochondria and endoplasmic reticulum (ER) have different roles in living cells but they interact both physically and functionally. A key aspect of the mitochondria-ER relationship is the modulation of Ca(2+) signaling during cell activation, which thus affects a variety of physiological processes. We focus here on the molecular aspects that control the dynamics of the organelle-organelle interaction and their relationship with Ca(2+) signals, also discussing the consequences that these phenomena have, not only for cell physiology but also in the control of cell death.

  8. Connections between microtubules and endoplasmic reticulum in mitotic spindle

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    J. A. Tarkowska

    2015-01-01

    Full Text Available Dividing endosperm cells of Haemanthus katherinae Bak. were treated with an 0.025 per cent aqueous solution of an oleander glycosides mixture which produces severe disturtaances in the mitotic spindle and high hypertrophy of the endoplasmic reticulum (ER in the whole cells. There appear between the kinetochore microtubules (MTs numerous elongated and narrow ER cisterns, particularly well visible when the number of kinetochore MTs is reduced. Both these structures (MTs and ER are frequently connected by cross-bridges. The presumable role of these connections is discused.

  9. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    Science.gov (United States)

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  10. Salubrinal Alleviates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Endoplasmic Reticulum Stress Pathway

    Science.gov (United States)

    Rani, Shilpa; Sreenivasaiah, Pradeep Kumar; Cho, Chunghee; Kim, Do Han

    2017-01-01

    Pathological hypertrophy of the heart is closely associated with endoplasmic reticulum stress (ERS), leading to maladaptations such as myocardial fibrosis, induction of apoptosis, and cardiac dysfunctions. Salubrinal is a known selective inhibitor of protein phosphatase 1 (PP1) complex involving dephosphorylation of phospho-eukaryotic translation initiation factor 2 subunit (p-eIF2)-α, the key signaling process in the ERS pathway. In this study, the effects of salubrinal were examined on cardiac hypertrophy using the mouse model of transverse aortic constriction (TAC) and cell model of neonatal rat ventricular myocytes (NRVMs). Treatment of TAC-induced mice with salubrinal (0.5 mg·kg−1·day−1) alleviated cardiac hypertrophy and tissue fibrosis. Salubrinal also alleviated hypertrophic growth in endothelin 1 (ET1)-treated NRVMs. Therefore, the present results suggest that salubrinal may be a potentially efficacious drug for treating pathological cardiac remodeling. PMID:28152298

  11. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    Science.gov (United States)

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  12. Endoplasmic reticulum stress is induced in the human placenta during labour.

    Science.gov (United States)

    Veerbeek, J H W; Tissot Van Patot, M C; Burton, G J; Yung, H W

    2015-01-01

    Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics.

    Science.gov (United States)

    Smirle, Jeffrey; Au, Catherine E; Jain, Michael; Dejgaard, Kurt; Nilsson, Tommy; Bergeron, John

    2013-01-01

    Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.

  14. Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress.

    Directory of Open Access Journals (Sweden)

    Andrea D'Osualdo

    Full Text Available Perturbation of endoplasmic reticulum (ER homeostasis triggers the ER stress response (also known as Unfolded Protein Response, a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate NLRP1 gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to NLRP1 promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for NLRP1 induction. This study provides further evidence that links ER stress with innate.

  15. Pekinenin E Inhibits the Growth of Hepatocellular Carcinoma by Promoting Endoplasmic Reticulum Stress Mediated Cell Death

    Directory of Open Access Journals (Sweden)

    Lu Fan

    2017-06-01

    Full Text Available Hepatocellular carcinoma (HCC is a malignant primary liver cancer with poor prognosis. In the present study, we report that pekinenin E (PE, a casbane diterpenoid derived from the roots of Euphorbia pekinensis, has a strong antitumor activity against human HCC cells both in vitro and in vivo. PE suppressed the growth of human HCC cells Hep G2 and SMMC-7721. In addition, PE-mediated endoplasmic reticulum (ER stress caused increasing expressions of C/EBP homologous protein (CHOP, leading to apoptosis in HCC cells both in vitro and in vivo. Inhibition of ER stress with CHOP small interfering RNA or 4-phenyl-butyric acid partially reversed PE-induced cell death. Furthermore, PE induced S cell cycle arrest, which could also be partially reversed by CHOP knockdown. In all, these findings suggest that PE causes ER stress-associated cell death and cell cycle arrest, and it may serve as a potent agent for curing human HCC.

  16. Neuronal transport of acid hydrolases and peroxidase within the lysosomal system or organelles: involvement of agranular reticulum-like cisterns.

    Science.gov (United States)

    Broadwell, R D; Oliver, C; Brightman, M W

    1980-04-01

    degradation in the cell body. These observations extend our previously reported findings in the axon to the cell body and suggest that agranular reticulum-like cisterns in the perikaryon, like those in the axon, may be part of the lysosomal system rather than associated with the agranular reticulum. A diagram summarizing the lysosomal system of organelles and proposed transport of acid hydrolases and peroxidase in neurosecretory neurons specifically and in neurons in general is provided.

  17. CDIP1-BAP31 Complex Transduces Apoptotic Signals from Endoplasmic Reticulum to Mitochondria under Endoplasmic Reticulum Stress

    OpenAIRE

    Takushi Namba; Fang Tian; Kiki Chu; So-Young Hwang; Kyoung Wan Yoon; Sanguine Byun; Masatsugu Hiraki; Anna Mandinova; Sam W. Lee

    2013-01-01

    Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 i...

  18. Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders

    Science.gov (United States)

    Volpi, Vera G.; Touvier, Thierry; D'Antonio, Maurizio

    2017-01-01

    Reaching the correct three-dimensional structure is crucial for the proper function of a protein. The endoplasmic reticulum (ER) is the organelle where secreted and transmembrane proteins are synthesized and folded. To guarantee high fidelity of protein synthesis and maturation in the ER, cells have evolved ER-protein quality control (ERQC) systems, which assist protein folding and promptly degrade aberrant gene products. Only correctly folded proteins that pass ERQC checkpoints are allowed to exit the ER and reach their final destination. Misfolded glycoproteins are detected and targeted for degradation by the proteasome in a process known as endoplasmic reticulum-associated degradation (ERAD). The excess of unstructured proteins in the ER triggers an adaptive signal transduction pathway, called unfolded protein response (UPR), which in turn potentiates ERQC activities in order to reduce the levels of aberrant molecules. When the situation cannot be restored, the UPR drives cells to apoptosis. Myelin-forming cells of the central and peripheral nervous system (oligodendrocytes and Schwann cells) synthesize a large amount of myelin proteins and lipids and therefore are particularly susceptible to ERQC failure. Indeed, deficits in ERQC and activation of ER stress/UPR have been implicated in several myelin disorders, such as Pelizaeus-Merzbacher and Krabbe leucodystrophies, vanishing white matter disease and Charcot-Marie-Tooth neuropathies. Here we discuss recent evidence underlying the importance of proper ERQC functions in genetic disorders of myelinating glia. PMID:28101003

  19. Microorganisms in the rumen and reticulum of buffalo (Bubalus bubalis) fed two different feeding systems

    OpenAIRE

    Franzolin,Raul; Wright, André-Denis G.

    2016-01-01

    Abstract Background The community of microorganisms in the rumen and reticulum is influenced by feeding as well as the species and geographical distribution of ruminant animals. Bacteria, methanogenic archaea and ciliate protozoa existing in the rumen and reticulum were evaluated by real-time polymerase chain reaction and light microscopy in buffalo in two feeding systems, grazing and feedlot. ...

  20. Cytokeratin-positive interstitial reticulum cell tumors of lymph nodes: a case report and review of literature

    Institute of Scientific and Technical Information of China (English)

    DONG Ying-chun; WU Bo; SHENG Zhen; WANG Jian-dong; ZHOU Hang-bo; ZHOU Xiao-jun

    2008-01-01

    @@ Cytokeratin-positive interstitial reticulum cells(CIRCs)are considered to represent a subset of fibroblastic reticulum cells(FBRCs)belonging to accessory dendritic cells in lymph nodes,the spleen and tonsils.1-3

  1. Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

    Science.gov (United States)

    Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.

    2014-01-01

    The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132

  2. Advances in the mechanisms of atherosclerosis vulnerable plague and endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Zhong Zhang; Ruo-Lan Huang; Ru Mo; Ling Wang; Xiao Chang; Mu-Juan Xu

    2016-01-01

    Objective:Ischemic stroke and coronary heart disease occupy the first two place of world health economic burden, atherosclerotic vulnerable plaque rupture as the common factor of these diseases, is thought to be a key target of ischemic cardiovascular and cerebrovascular disease control. Endoplasmic reticulum stress is one of the classical pathway of cell apoptosis. More and more studies have indicated that the endoplasmic reticulum stress pathway was involved in the development of atherosclerotic plaque rupture. In this paper, the three main signal pathways of endoplasmic reticulum stress, including Protein kinase RNA-like ER kinase (PERK), Activating transcription factor 6 (ATF6) and Inositol-requiring protein 1αα(IRE1α) were reviewed. The relationship between the risk factors of atherosclerosis (including hyperlipidemia, hypertension and hyperglycemia) and endoplasmic reticulum stress, and the relationship between major cellular components (macrophages, vascular endothelial cells, vascular smooth muscle cells and vascular smooth muscle cells) of vulnerable plaque and endoplasmic reticulum stress were reviewed.

  3. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration.

    Science.gov (United States)

    Hoffmann, Christiane; Plocharski, Bartolome; Haferkamp, Ilka; Leroch, Michaela; Ewald, Ralph; Bauwe, Hermann; Riemer, Jan; Herrmann, Johannes M; Neuhaus, H Ekkehard

    2013-07-01

    The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO(2) concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.

  4. Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Jeffrey Thomas Cole

    2012-04-01

    Full Text Available Brain cells expend large amounts of energy sequestering calcium (Ca2+, while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P, a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA. Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1-10 mM. The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity

  5. The Herbal Medicine Cordyceps sinensis Protects Pancreatic Beta Cells from Streptozotocin-Induced Endoplasmic Reticulum Stress.

    Science.gov (United States)

    Liu, Hong; Cao, Diyong; Liu, Hua; Liu, Xinghai; Mai, Wenli; Lan, Haitao; Huo, Wen; Zheng, Qian

    2016-08-01

    Our previous work found that Cordyceps sinensis (CS) improves the activity and secretory function of pancreatic islet beta cells. The objective was to observe a further possible role of CS in the protection of insulin-secreting cells. A rat model of type 2 diabetes mellitus was developed with streptozotocin (STZ) and a high-energy fat diet (HFD). CS was administered in the successful model of rats with type 2 diabetes. After 4 weeks, the biochemistry index of blood samples was measured, and pathologic observation was performed by immunohistochemistry. In the rats with type 2 diabetes induced by a HFD and STZ, the levels of fasting blood glucose and fasting insulin were elevated, and the insulin sensitivity index was decreased. Pathologic examination found an increased number of apoptotic cells, an elevated protein expression of pro-apoptotic C/EBP homologous protein (CHOP) and an increased c-Jun level by means of JNK phosphorylation, responsive to the endoplasmic reticulum stress of islet beta cells. With treatment by CS for 4 weeks, the elevated levels of both fasting blood glucose and fasting insulin in the rats with type 2 diabetes were significantly lower, and the decreased insulin sensitivity index was reversed. Compared to the control rats with type 2 diabetes, CS application significantly reduced the number of apoptotic cells and decreased protein expression of both CHOP and c-Jun. The herbal compound CS could protect pancreatic beta cells from the pro-apoptotic endoplasmic reticulum stress induced by HFD-STZ. This suggests an alternative approach to treating type 2 diabetes. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  6. Depletion of cyclophilins B and C leads to dysregulation of endoplasmic reticulum redox homeostasis.

    Science.gov (United States)

    Stocki, Pawel; Chapman, Daniel C; Beach, Lori A; Williams, David B

    2014-08-15

    Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.

  7. Identification and functional characterization of an endoplasmic reticulum oxidoreductin 1-α gene in Litopenaeus vannamei.

    Science.gov (United States)

    Zhang, Ze-Zhi; Yuan, Kai; Yue, Hai-Tao; Yuan, Feng-Hua; Bi, Hai-Tao; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-04-01

    In the current study, full-length sequence of endoplasmic reticulum oxidoreductin 1-α (LvERO1-α) was cloned from Litopenaeus vannamei. Real-time RT-PCR results showed that LvERO1-α was highly expressed in hemocytes, gills, and intestines. White spot syndrome virus (WSSV) challenge was performed, and the expression of LvERO1-α and two other downstream genes of the double-stranded RNA-activated protein kinase-like ER kinase-eIF2α (PERK-α) pathway, namely, homocysteine-induced endoplasmic reticulum protein (LvHERP) and acylamino-acid-releasing enzyme (LvAARE), strongly increased in the hemocytes. Flow cytometry assay results indicated that the apoptosis rate of L. vannamei hemocytes in the LvERO1-α knockdown group was significantly lower than that of the controls. Moreover, shrimps with knockdown expression of LvERO1-α exhibited decreased cumulative mortality upon WSSV infection. Downregulation of L. vannamei immunoglobulin-binding protein (LvBip), which had been proven to induce unfolded protein response (UPR) in L. vannamei, did not only upregulate LvERO1-α, LvHERP, and LvAARE in hemocytes, but also increased their apoptosis rate, as well as the shrimp cumulative mortality. Furthermore, reporter gene assay results showed that the promoter of LvERO1-α was activated by L. vannamei activating transcription factor 4, thereby confirming that LvERO1-α was regulated by the PERK-eIF2α pathway. These results suggested that LvERO1-α plays a critical role in WSSV-induced apoptosis, which likely occurs through the WSSV-activated PERK-eIF2α pathway.

  8. Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Isomura, Midori; Kotake, Yaichiro, E-mail: yaichiro@hiroshima-u.ac.jp; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

    2013-10-01

    Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.

  9. Deepening Sleep by Hypnotic Suggestion

    Science.gov (United States)

    Cordi, Maren J.; Schlarb, Angelika A.; Rasch, Björn

    2014-01-01

    Study Objectives: Slow wave sleep (SWS) plays a critical role in body restoration and promotes brain plasticity; however, it markedly declines across the lifespan. Despite its importance, effective tools to increase SWS are rare. Here we tested whether a hypnotic suggestion to “sleep deeper” extends the amount of SWS. Design: Within-subject, placebo-controlled crossover design. Setting: Sleep laboratory at the University of Zurich, Switzerland. Participants: Seventy healthy females 23.27 ± 3.17 y. Intervention: Participants listened to an auditory text with hypnotic suggestions or a control tape before napping for 90 min while high-density electroencephalography was recorded. Measurements and Results: After participants listened to the hypnotic suggestion to “sleep deeper” subsequent SWS was increased by 81% and time spent awake was reduced by 67% (with the amount of SWS or wake in the control condition set to 100%). Other sleep stages remained unaffected. Additionally, slow wave activity was significantly enhanced after hypnotic suggestions. During the hypnotic tape, parietal theta power increases predicted the hypnosis-induced extension of SWS. Additional experiments confirmed that the beneficial effect of hypnotic suggestions on SWS was specific to the hypnotic suggestion and did not occur in low suggestible participants. Conclusions: Our results demonstrate the effectiveness of hypnotic suggestions to specifically increase the amount and duration of slow wave sleep (SWS) in a midday nap using objective measures of sleep in young, healthy, suggestible females. Hypnotic suggestions might be a successful tool with a lower risk of adverse side effects than pharmacological treatments to extend SWS also in clinical and elderly populations. Citation: Cordi MJ, Schlarb AA, Rasch B. Deepening sleep by hypnotic suggestion. SLEEP 2014;37(6):1143-1152. PMID:24882909

  10. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Naranmandura, Hua, E-mail: narenman@zju.edu.cn [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Xu, Shi [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Koike, Shota [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Pan, Li Qiang [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Bin [Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Wang, Yan Wei; Rehman, Kanwal; Wu, Bin [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Zhe [Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou (China); Suzuki, Noriyuki, E-mail: n-suzuki@p.chiba-u.ac.jp [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan)

    2012-05-01

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

  11. Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

    Directory of Open Access Journals (Sweden)

    Stephanie Jamison

    Full Text Available Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK in response to endoplasmic reticulum (ER stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A. Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2, to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

  12. Assembly of MHC class I molecules within the endoplasmic reticulum.

    Science.gov (United States)

    Zhang, Yinan; Williams, David B

    2006-01-01

    MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.

  13. Quality Control System of the Endoplasmic Reticulum and Related Diseases

    Institute of Scientific and Technical Information of China (English)

    Jun-Chao WU; Zhong-Qin LIANG; Zheng-Hong QIN

    2006-01-01

    The quality control (QC) system of the endoplasmic reticulum (ER) is an important monitoring mechanism in the protein maturation process, which ensures export of properly folded proteins from the ER.Incorrectly or incompletely folded proteins are retained in the ER for refolding or degradation by the ER-residing proteasome. The calnexin/calreticulin cycle and ER-associated degradation are the key elements in QC. These two mechanisms work together to allow incorrectly folded proteins have additional opportunities to achieve their native conformations. The QC dysfunction is involved in many diseases caused by mutant proteins, many of which are causes of neurodegenerative disorders. A better understanding of molecular regulation in the QC system will uncover the molecular pathogenic mechanisms of many diseases caused by protein misfolding and help discover novel strategies for preventing or treating these diseases.

  14. WLS retrograde transport to the endoplasmic reticulum during Wnt secretion.

    Science.gov (United States)

    Yu, Jia; Chia, Joanne; Canning, Claire Ann; Jones, C Michael; Bard, Frédéric A; Virshup, David M

    2014-05-12

    Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

  15. Plant transducers of the endoplasmic reticulum unfolded protein response

    KAUST Repository

    Iwata, Yuji

    2012-12-01

    The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.

  16. Homotypic fusion of endoplasmic reticulum membranes in plant cells

    Directory of Open Access Journals (Sweden)

    Junjie eHu

    2013-12-01

    Full Text Available The endoplasmic reticulum (ER is a membrane-bounded organelle whose membrane comprises a network of tubules and sheets. The formation of these characteristic shapes and maintenance of their continuity through homotypic membrane fusion appears to be critical for the proper functioning of the ER. The atlastins (ATLs, a family of ER-localized dynamin-like GTPases, have been identified as fusogens of the ER membranes in metazoans. Mutations of the ATL proteins in mammalian cells cause morphological defects in the ER, and purified Drosophila ATL mediates membrane fusion in vitro. Plant cells do not possess ATL, but a family of similar GTPases, named root hair defective 3 (RHD3, are likely the functional orthologs of ATLs. In this review, we summarize recent advances in our understanding of how RHD3 proteins play a role in homotypic ER fusion. We also discuss the possible physiological significance of forming a tubular ER network in plant cells.

  17. ANALYSIS OF ENDOPLASMIC RETICULUM OF TOBACCO CELLS USING CONFOCAL MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Barbora Radochová

    2011-05-01

    Full Text Available Image analysis techniques for preprocessing, segmentation and estimation of geometrical characteristics of fiber-like structures from 2-D or 3-D images captured by a confocal microscope are presented. Methods are demonstrated on fiber-like biological structure: endoplasmic reticulum (ER of tobacco cells. In the presented analysis of 2-D images of ER before and after the treatment of latrunculin B, ER and ER tubules were segmented and the area density of ER as well as the length density of ER tubules in the cell cortical layer were estimated by automatic image analysis algorithms. Images of 3-D arrangement of ER were reconstructed and rendered by various visualization techniques.

  18. Maternal obesity alters endoplasmic reticulum homeostasis in offspring pancreas.

    Science.gov (United States)

    Soeda, Jumpei; Mouralidarane, Angelina; Cordero, Paul; Li, Jiawei; Nguyen, Vi; Carter, Rebeca; Kapur, Sabrina R; Pombo, Joaquim; Poston, Lucilla; Taylor, Paul D; Vinciguerra, Manlio; Oben, Jude A

    2016-06-01

    The prevalence of non-alcoholic fatty pancreas disease (NAFPD) is increasing in parallel with obesity rates. Stress-related alterations in endoplasmic reticulum (ER), such as the unfolded protein response (UPR), are associated with obesity. The aim of this study was to investigate ER imbalance in the pancreas of a mice model of adult and perinatal diet-induced obesity. Twenty female C57BL/6J mice were assigned to control (Con) or obesogenic (Ob) diets prior to and during pregnancy and lactation. Their offspring were weaned onto Con or Ob diets up to 6 months post-partum. Then, after sacrifice, plasma biochemical analyses, gene expression, and protein concentrations were measured in pancreata. Offspring of Ob-fed mice had significantly increased body weight (p < 0.001) and plasma leptin (p < 0.001) and decreased insulin (p < 0.01) levels. Maternal obesogenic diet decreased the total and phosphorylated Eif2α and increased spliced X-box binding protein 1 (XBP1). Pancreatic gene expression of downstream regulators of UPR (EDEM, homocysteine-responsive endoplasmic reticulum-resident (HERP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP)) and autophagy-related proteins (LC3BI/LC3BII) were differently disrupted by obesogenic feeding in both mothers and offspring (from p < 0.1 to p < 0.001). Maternal obesity and Ob feeding in their offspring alter UPR in NAFPD, with involvement of proapoptotic and autophagy-related markers. Upstream and downstream regulators of PERK, IRE1α, and ATF6 pathways were affected differently following the obesogenic insults.

  19. The overexpression of nuclear envelope protein Lap2β induces endoplasmic reticulum reorganisation via membrane stacking

    Directory of Open Access Journals (Sweden)

    Ekaterina G. Volkova

    2012-06-01

    Some nuclear envelope proteins are localised to both the nuclear envelope and the endoplasmic reticulum; therefore, it seems plausible that even small amounts of these proteins can influence the organisation of the endoplasmic reticulum. A simple method to study the possible effects of nuclear envelope proteins on endoplasmic reticulum organisation is to analyze nuclear envelope protein overexpression. Here, we demonstrate that Lap2β overexpression can induce the formation of cytoplasmic vesicular structures derived from endoplasmic reticulum membranes. Correlative light and electron microscopy demonstrated that these vesicular structures were composed of a series of closely apposed membranes that were frequently arranged in a circular fashion. Although stacked endoplasmic reticulum cisternae were highly ordered, Lap2β could readily diffuse into and out of these structures into the surrounding reticulum. It appears that low-affinity interactions between cytoplasmic domains of Lap2β can reorganise reticular endoplasmic reticulum into stacked cisternae. Although the effect of one protein may be insignificant at low concentrations, the cumulative effect of many non-specialised proteins may be significant.

  20. [Suggestion and hypnosis in hysteria].

    Science.gov (United States)

    Berner, P

    1995-12-15

    Suggestive influences allow to resolve ambiguities. Normally they are only accepted if they correspond with the knowledge and believes of the subject. Under hypnosis or under the impact of serious psychic perturbations one may take up reality constructions which are not in conformity with these criteria. The restriction of consciousness and the ignoring of certain functions permitting this are the common basis of hypnosis and hysteria. But suggestions do not cause the later; they may only shape the symptomatology. Hypnosis can create a terrain facilitating the resolution of the problems underlying hysteria but it does not represent the treatment of hysteria.

  1. A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria.

    Science.gov (United States)

    Lahiri, Sujoy; Chao, Jesse T; Tavassoli, Shabnam; Wong, Andrew K O; Choudhary, Vineet; Young, Barry P; Loewen, Christopher J R; Prinz, William A

    2014-10-01

    Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

  2. Emergence of endoplasmic reticulum stress and activated microglia in Purkinje cell degeneration mice.

    Science.gov (United States)

    Kyuhou, Shin-ichi; Kato, Nobuo; Gemba, Hisae

    2006-03-27

    In the current studies, we characterized the molecular and cellular mechanism of cell death in Purkinje cell degeneration (pcd) mice using real-time quantitative PCR, immunohistochemistry, and Western blotting. It appears that endoplasmic reticulum (ER) stress is involved in this degeneration of Purkinje cells because ER stress-related substrates, such as CHOP and caspase 12, were strongly activated in Purkinje cells of pcd mice during the third postnatal (P) week. A significant increase in the expression of the ER-specific chaperone BiP suggested that unfolded protein responses were induced. We also found that Purkinje cells underwent apoptosis via the activation of caspase 3 and subsequent fragmentation of DNA. In addition to the activation of apoptosis in Purkinje cells, many activated microglial cells are found to be present in the molecular layer of the cerebellar cortex. In the later phase of degeneration, there was conspicuous expression of inducible nitric oxide synthase (iNOS), and some Purkinje cells were strongly labeled with an antibody to nitrotyrosine, suggesting that Purkinje cells in pcd mice are damaged by nitric oxide released from microglial cells. Administration of minocycline, which may inhibit iNOS expression, delayed the death of Purkinje cells in pcd mice and mildly improved their motor abilities. These findings suggest that ER stress participates in the degeneration of Purkinje cells and that activation of microglia accelerates Purkinje cell death in pcd mice.

  3. A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

    Science.gov (United States)

    Tavassoli, Shabnam; Wong, Andrew K. O.; Choudhary, Vineet; Young, Barry P.; Loewen, Christopher J. R.; Prinz, William A.

    2014-01-01

    Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth. PMID:25313861

  4. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  5. Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber.

    Science.gov (United States)

    Hagedorn, Monica; Ziegler, Andreas

    2002-07-01

    In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.

  6. Suggestions on photons and fermions

    CERN Document Server

    Alvargonzalez, R

    2007-01-01

    In this paper we suggest a configuration of photons consistent with a spin $\\hbar$, and a configuration of the fermions coherent with a spin $\\hbar/2$. These suggested configurations open the way to further analyses which lead to the following conclusions: - There cannot exist elementary particles of spin $\\hbar/2$ with a mass inferior to $1m_e$ or with a radius greater than $1l_e$. - The electrostatic force derives from the centrifugal forces inherent to the spin and are propagated by photons. - The derivation of the electrostatic force explains the existence of positive and negative charges and Coulomb's law. - The enormous differences between the centrifugal forces and the centripetal forces at the surface of the protons give rise to quantic fluctuations of space which generate the energy flows necessary for equilibrium. These energy flows can explain gravitation and the strong force. - The mass of the proton, $m_p$, and the mass of the neutron, $m_n$, must each have a concrete value required for the cohes...

  7. Microorganisms in the rumen and reticulum of buffalo (Bubalus bubalis) fed two different feeding systems

    National Research Council Canada - National Science Library

    Franzolin, Raul; Wright, André-Denis G

    2016-01-01

    .... Bacteria, methanogenic archaea and ciliate protozoa existing in the rumen and reticulum were evaluated by real-time polymerase chain reaction and light microscopy in buffalo in two feeding systems, grazing and feedlot...

  8. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

    Science.gov (United States)

    Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

    2003-07-30

    Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

  9. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress.

    Science.gov (United States)

    Mecha, M; Torrao, A S; Mestre, L; Carrillo-Salinas, F J; Mechoulam, R; Guaza, C

    2012-06-28

    Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation.

  10. Effect of amiloride on endoplasmic reticulum stress response in the injured spinal cord of rats.

    Science.gov (United States)

    Kuroiwa, Masahiro; Watanabe, Masahiko; Katoh, Hiroyuki; Suyama, Kaori; Matsuyama, Daisuke; Imai, Takeshi; Mochida, Joji

    2014-10-01

    After traumatic spinal cord injury (SCI), endoplasmic reticulum (ER) stress exacerbates secondary injury, leading to expansion of demyelination and reduced remyelination due to oligodendrocyte precursor cell (OPC) apoptosis. Although recent studies have revealed that amiloride controls ER stress and leads to improvement in several neurological disorders including SCI, its mechanism is not completely understood. Here, we used a rat SCI model to assess the effects of amiloride on functional recovery, secondary damage expansion, ER stress-induced cell death and OPC survival. Hindlimb function in rats with spinal cord contusion significantly improved after amiloride administration. Amiloride significantly decreased the expression of the pro-apoptotic transcription factor CHOP in the injured spinal cord and significantly increased the expression of the ER chaperone GRP78, which protects cells against ER stress. In addition, amiloride treatment led to a significant decrease in ER stress-induced apoptosis and a significant increase of NG2-positive OPCs in the injured spinal cord. Furthermore, in vitro experiments performed to investigate the direct effect of amiloride on OPCs revealed that amiloride reduced CHOP expression in OPCs cultured under ER stress. These results suggest that amiloride controls ER stress in SCI and inhibits cellular apoptosis, contributing to OPC survival. The present study suggests that amiloride may be an effective treatment to reduce ER stress-induced cell death in the acute phase of SCI.

  11. Overexpressed PLTP in macrophage may promote cholesterol accumulation by prolonged endoplasmic reticulum stress.

    Science.gov (United States)

    Yang, Xinquan; Yu, Yang; Wang, Daxin; Qin, Shucun

    2017-01-01

    It is well known that phospholipid transfer protein (PLTP) is involved in the lipid metabolism and development of atherosclerosis (AS). Abundant PLTP is considered to be expressed on the foam cells derived from monocyte/macrophages in atherosclerotic plaques, suggesting that high level of active PLTP may promote the formation of foam cells. However, the exact role of PLTP on the process of macrophage derived foam cell formation remains unclear. The accumulation of free cholesterol (FC) in the cytoplasm may lead to the prolonged endoplasmic reticulum stress (ERs) and the imbalance of intracellular cholesterol homeostasis. Different PLTP level definitely alternates the phospholipids (PL) and cholesterol level in plasma, strongly suggesting that active PLTP may change the level of FC and PL intracellularly, which subsequently induced the ERs in macrophage. Thus, we hypothesize that high level of PLTP may promote the accumulation of cholesterol in macrophage via the alteration ratio of FC to PL. Therefore, validating this hypothesis may clarify the role of PLTP in macrophage ERs in AS and also raise a novel strategy in the regression of AS plaques via restoring intracellular membrane lipid homeostasis and attenuating ERs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

    OpenAIRE

    Jintao Zhang; Man Yi; Longying Zha; Siqiang Chen; Zhijia Li; Cheng Li; Mingxing Gong; Hong Deng; Xinwei Chu; Jiehua Chen; Zheqing Zhang; Limei Mao; Suxia Sun

    2016-01-01

    Purpose Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated au...

  13. Placental endoplasmic reticulum stress and acidosis: relevant aspects in gestational diabetes.

    Science.gov (United States)

    Jawerbaum, Alicia

    2016-10-01

    In this issue, Yung and colleagues (doi: 10.1007/s00125-016-4040-2 ) report endoplasmic reticulum stress in the placenta of patients with gestational diabetes mellitus. With the use of a trophoblast-like cell line, these authors identify putative mechanisms involved in, and treatments to prevent the induction of endoplasmic reticulum stress. Here, the relevance and possible implications of these findings and areas for further research are discussed.

  14. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness.

    Science.gov (United States)

    Gill, David J; Tham, Keit Min; Chia, Joanne; Wang, Shyi Chyi; Steentoft, Catharina; Clausen, Henrik; Bard-Chapeau, Emilie A; Bard, Frederic A

    2013-08-20

    Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-density glycosylation, is critical for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.

  15. Interaction between endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLV-ts1-infected astrocytes.

    Science.gov (United States)

    Liu, Na; Scofield, Virginia L; Qiang, Wenan; Yan, Mingshan; Kuang, Xianghong; Wong, Paul K Y

    2006-05-10

    The murine retrovirus, MoMuLV-ts1, induces progressive paralysis and immune deficiency in FVB/N mice. We have reported previously that ts1 infection causes apoptosis in astrocytes via endoplasmic reticulum (ER) and mitochondrial stress (Liu, N., Kuang, X., Kim, H.T., Stoica, G., Qiang, W., Scofield, V.L., Wong, P.K.Y. Wong. 2004. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes. J. NeuroVirol. 10, 189-198). In the present study, we show that caspase 8 activation in these cells is mediated through ER stress-associated elevation of death receptor DR5 and the C/EBP homologous protein (GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNFalpha and TNF-R1 interactions on the cell surface. Treatment with Z-IETD-FMK, a specific inhibitor of caspase 8 enzymatic activity, reduced ER stress by two mechanisms: by inhibiting caspase 8 activation, and by preventing cleavage of the ER-associated membrane protein BAP31 into BAP20, which exacerbates the ER stress response. These findings suggest that caspase 8- and ER stress-associated apoptotic pathways are linked in ts1-infected astrocytes.

  16. The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum.

    Science.gov (United States)

    Breckenridge, David G; Nguyen, Mai; Kuppig, Stephan; Reth, Michael; Shore, Gordon C

    2002-04-02

    BAP31 is an integral protein of the endoplasmic reticulum membrane and a substrate of caspase-8. Here, we describe the procaspase-8 isoform, procaspase-8L, which is ubiquitously expressed and selectively recruited to the BAP31 complex in response to apoptotic signaling by E1A. Procaspase-8L is characterized by the N-terminal extension (Nex) domain, which extends procaspase-8/a at the N terminus and is required for selective association of procaspase-8L with the BAP31 complex. Gene deletion identified BAP31 and related BAP29 as required for processing of procaspase-8L in response to E1A, by a FADD-independent mechanism that was blocked by BCL-2. Further, Bap29,31 deletion, as well as a Nex-domain dominant-negative mutant, curtailed the activation of downstream caspases (IETDase and DEVDase) and cell death in response to E1A. Preferential recruitment of procaspase-8L by the BAP31 complex at the endoplasmic reticulum suggests an additional pathway for regulating initiator caspase-8 during apoptosis.

  17. Endurance exercise training ameliorates insulin resistance and reticulum stress in adipose and hepatic tissue in obese rats.

    Science.gov (United States)

    da Luz, Gabrielle; Frederico, Marisa J S; da Silva, Sabrina; Vitto, Marcelo F; Cesconetto, Patricia A; de Pinho, Ricardo A; Pauli, José R; Silva, Adelino S R; Cintra, Dennys E; Ropelle, Eduardo R; De Souza, Cláudio T

    2011-09-01

    Obesity-induced endoplasmatic reticulum (ER) stress has been demonstrated to underlie the induction of obesity-induced JNK and NF-κB activation inflammatory responses, and generation of peripheral insulin resistance. On the other hand, exercise has been used as a crucial tool in obese and diabetic patients, and may reduce inflammatory pathway stimulation. However, the ability of exercise training to reverse endoplasmatic reticulum stress in adipose and hepatic tissue in obesity has not been investigated in the literature. Here, we demonstrate that exercise training ameliorates ER stress and insulin resistance in DIO-induced rats. Rats were fed with standard rodent chow (3,948 kcal kg(-1)) or high-fat diet (5,358 kcal kg(-1)) for 2 months. After that rats were submitted to swimming training (1 h per day, 5 days for week with 5% overload of the body weight for 8 weeks). Samples from epididymal fat and liver were obtained and western blot analysis was performed. Our results showed that swimming protocol reduces pro-inflammatory molecules (JNK, IκB and NF-κB) in adipose and hepatic tissues. In addition, exercise leads to reduction in ER stress, by reducing PERK and eIF2α phosphorylation in these tissues. In parallel, an increase in insulin pathway signaling was observed, as confirmed by increases in IR, IRSs and Akt phosphorylation following exercise training in DIO rats. Thus, results suggest that exercise can reduce ER stress, improving insulin resistance in adipose and hepatic tissue.

  18. Stress responses from the endoplasmic reticulum in cancer

    Directory of Open Access Journals (Sweden)

    Hironori eKato

    2015-04-01

    Full Text Available The endoplasmic reticulum (ER is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR. The UPR also contributes to the regulation of various intracellular signalling pathways such as calcium signalling and lipid signalling. More recently, the mitochondria-associated ER membrane (MAM, which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signalling, inflammatory signalling, the autophagic response, and the UPR. Interestingly, in cancer, these signalling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signalling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM.

  19. Chemical chaperones mitigate experimental asthma by attenuating endoplasmic reticulum stress.

    Science.gov (United States)

    Makhija, Lokesh; Krishnan, Veda; Rehman, Rakhshinda; Chakraborty, Samarpana; Maity, Shuvadeep; Mabalirajan, Ulaganathan; Chakraborty, Kausik; Ghosh, Balaram; Agrawal, Anurag

    2014-05-01

    Endoplasmic reticulum (ER) stress and consequent unfolded protein response (UPR) are important in inflammation but have been poorly explored in asthma. We used a mouse model of allergic airway inflammation (AAI) with features of asthma to understand the role of ER stress and to explore potential therapeutic effects of inhaled chemical chaperones, which are small molecules that can promote protein folding and diminish UPR. UPR markers were initially measured on alternate days during a 7-day daily allergen challenge model. UPR markers increased within 24 hours after the first allergen challenge and peaked by the third challenge, before AAI was fully established (from the fifth challenge onward). Three chemical chaperones-glycerol, trehalose, and trimethylamine-N-oxide (TMAO)-were initially administered during allergen challenge (preventive regimen). TMAO, the most effective of these chemical chaperones and 4-phenylbutyric acid, a chemical chaperone currently in clinical trials, were further tested for potential therapeutic activities after AAI was established (therapeutic regimen). Chemical chaperones showed a dose-dependent reduction in UPR markers, airway inflammation, and remodeling in both regimens. Our results indicate an early and important role of the ER stress pathway in asthma pathogenesis and show therapeutic potential for chemical chaperones.

  20. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    Science.gov (United States)

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  1. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  2. Mechanisms of CFTR folding at the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Soo Jung Kim

    2012-12-01

    Full Text Available In the past decade much has been learned about how CFTR folds and misfolds as the etiologic cause of cystic fibrosis (CF. CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM segments into the endoplasmic reticulum (ER membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jigsaw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s caused by ∆F508 that provides a molecular target(s for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.

  3. Regulation of endoplasmic reticulum turnover by selective autophagy.

    Science.gov (United States)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-06-18

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

  4. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Xue Yu

    Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

  5. Endoplasmic reticulum stress in adipose tissue augments lipolysis.

    Science.gov (United States)

    Bogdanovic, Elena; Kraus, Nicole; Patsouris, David; Diao, Li; Wang, Vivian; Abdullahi, Abdikarim; Jeschke, Marc G

    2015-01-01

    The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca(2+) homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24-48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.

  6. Hyperhomocysteinemia,endoplasmic reticulum stress,and alcoholic liver injury

    Institute of Scientific and Technical Information of China (English)

    Cheng Ji; Neil Kaplowitz

    2004-01-01

    Deficiencies in vitamins or other factors (B6, B12, folic acid,betaine) and genetic disorders for the metabolism of the non-protein amino acid-homocysteine (Hcy) lead to hyperhomocysteinemia (Hhcy). Hhcy is an integral component of several disorders including cardiovascular disease, neurodegeneration, diabetes and alcoholic liver disease. Hhcy unleashes mediators of inflammation such as NFκB, IL-1β, IL-6, and IL-8, increases production of intracellular superoxide anion causing oxidative stress and reducing intracellular level of nitric oxide (NO), and induces endoplasmic reticulum (ER) stress which can explain many processes of Hcy-promoted cell injury such as apoptosis,fat accumulation, and inflammation. Animal models have played an important role in determining the biological effects of Hhcy. ER stress may also be involved in other liver diseases such as α1-antitrypsin (α1-AT) deficiency and hepatitis C and/or B virus infection. Future research should evaluate the possible potentiative effects of alcohol and hepatic virus infection on ER stress-induced liver injury, study potentially beneficial effects of lowering Hcy and preventing ER stress in alcoholic humans,and examine polymorphism of Hcy metabolizing enzymes as potential risk-factors for the development of Hhcy and liver disease.

  7. Coordination of Endoplasmic Reticulum (ER) Signaling During Maize Seed Development

    Energy Technology Data Exchange (ETDEWEB)

    Boston, Rebecca S.

    2010-11-20

    Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesize storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies. Our underlying hypothesis is that the endoplasmic reticulum (ER) stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presence of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly. The capacity of the ER to deposit these aberrant proteins in protein bodies during a period that extends several weeks provides an excellent model for deconvoluting the ER stress response of plants. We have focused in this project on the means by which the ER senses and responds to functional perturbations and the underlying intracellular communication that occurs among biosynthetic, trafficking and degradative pathways for proteins during seed development.

  8. Endoplasmic reticulum stress, diabetes mellitus, and tissue injury.

    Science.gov (United States)

    Huang, Liu; Xie, Hong; Liu, Hao

    2014-01-01

    Endoplasmic reticulum (ER) stress is characterized by the accumulation of unfolded and misfolded proteins in the ER lumen. Unfolded and misfolded protein accumulation interferes with the ER function and triggers ER stress response. Thus, ER stress response, also called unfolded protein response (UPR), is an adaptive process that controls the protein amount in the ER lumen and the downstream protein demand. In normal conditions, the role of ER stress is to maintain ER homeostasis, restore ER function, and protect stressed cells from apoptosis, by coordinating gene expression, protein synthesis, and accelerating protein degradation through several molecular pathways. However, prolonged ER stress response plays a paradoxical role, which leads to cell damage, apoptosis, and concomitant tissue injuries. A number of tissue alterations are involved with diabetes mellitus progress and its comorbidities via ER stress. However, certain pharmacological agents affecting ER stress have been identified. In this review, we summarized the relationship between ER stress and insulin resistance development. Moreover, we aim to explain how ER stress influences type 2 diabetes mellitus (T2DM) development. In addition, we reviewed the literature on ER stress and UPR in three kinds of tissue injuries induced by T2DM. Finally, a retrospective analysis of the effects of anti-diabetes medications on ER stress is presented.

  9. Heme oxygenase-1 comes back to endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hong Pyo [School of Biological Sciences, Ulsan University (Korea, Republic of); Pae, Hyun-Ock [Department of Immunology, Wonkwang University School of Medicine (Korea, Republic of); Back, Sung Hun; Chung, Su Wol [School of Biological Sciences, Ulsan University (Korea, Republic of); Woo, Je Moon [Department of Opthalmology, Ulasn University Hospital (Korea, Republic of); Son, Yong [Department of Anesthesiology and Pain Medicine, Wonkwang University School of Medicine (Korea, Republic of); Chung, Hun-Taeg, E-mail: chung@ulsan.ac.kr [School of Biological Sciences, Ulsan University (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  10. Small GTPases and Brucella entry into the endoplasmic reticulum.

    Science.gov (United States)

    de Bolle, Xavier; Letesson, Jean-Jacques; Gorvel, Jean-Pierre

    2012-12-01

    A key determinant for intracellular pathogenic bacteria to ensure their virulence within host cells is their ability to bypass the endocytic pathway and to reach a safe niche of replication. In the case of Brucella, the bacterium targets the ER (endoplasmic reticulum) to create a replicating niche called the BCV (Brucella-containing vacuole). The ER is a suitable strategic place for pathogenic Brucella. Indeed, bacteria can be hidden from host cell defences to persist within the host, and they can take advantage of the membrane reservoir delivered by the ER to replicate. Interaction with the ER leads to the presence on the BCV of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the small GTPase Rab2 known to be located on secretory vesicles that traffic between the ER and the Golgi apparatus. GAPDH and the small GTPase Rab2 controls Brucella replication at late times post-infection. A specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA was identified. Altered kinetics of intracellular trafficking and faster proliferation of the Brucella abortus ΔricA mutant was observed compared with the wild-type strain. RicA is the first reported effector with a proposed function for B. abortus.

  11. The Endoplasmic Reticulum: A Social Network in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Jun Chen; Caitlin Doyle; Xingyun Qi; Huanquan Zheng

    2012-01-01

    The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules.The ER plays crucial roles in the biosynthesis and transport of proteins and lipids,and in calcium (Ca2+) regulation in compartmentalized eukaryotic cells including plant cells.To support its well-segregated functions,the shape of the ER undergoes notable changes in response to both developmental cues and outside influences.In this review,we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER,and the importance of the interconnected ER network in cell polarity.In animal and yeast cells,two family proteins,the reticulons and DP1/Yop1,are required for shaping high-curvature ER tubules,while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network.In plant cells,recent data also indicate that the reticulons are involved in shaping ER tubules,while RHD3,a plant member of the atlastin GTPases,is required for the generation of an interconnected ER network.We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles,with a focus on how the ER and Golgi interplay in plant cells.

  12. Endoplasmic Reticulum-Mediated Protein Quality Control in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jianming eLi

    2014-04-01

    Full Text Available A correct three-dimensional structure is crucial for the physiological functions of a protein, yet the folding of proteins to acquire native conformation is a fundamentally error-prone process. Eukaryotic organisms have evolved a highly conserved endoplasmic reticulum-mediated protein quality control (ERQC mechanism to monitor folding processes of secretory and membrane proteins, allowing export of only correctly folded proteins to their physiological destinations, retaining incompletely/mis-folded ones in the ER for additional folding attempts, marking and removing terminally-misfolded ones via a unique multiple-step degradation process known as ER-associate degradation (ERAD. Most of our current knowledge on ERQC and ERAD came from genetic and biochemical investigations in yeast and mammalian cells. Recent studies in the reference plant Arabidopsis thaliana uncovered homologous components and similar mechanisms in plants for monitoring protein folding and for retaining, repairing, and removing misfolded proteins. These studies also revealed critical roles of the plant ERQC/ERAD systems in regulating important biochemical/physiological processes, such as abiotic stress tolerance and plant defense. In this review, we discuss our current understanding about the molecular components and biochemical mechanisms of the plant ERQC/ERAD system in comparison to yeast and mammalian systems.

  13. Protein bodies in leaves exchange contents through the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Reza eSaberianfar

    2016-05-01

    Full Text Available Protein bodies (PBs are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP, or hydrophobin-I (HFBI. In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

  14. Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.

    Science.gov (United States)

    Missiroli, Sonia; Morganti, Claudia; Giorgi, Carlotta; Pinton, Paolo

    2016-01-01

    Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.

  15. Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.

    Science.gov (United States)

    Rieusset, Jennifer

    2017-06-01

    The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca(2+)) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca(2+) signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.

    Science.gov (United States)

    Xue, Yong; Schmollinger, Stefan; Attar, Narsis; Campos, Oscar A; Vogelauer, Maria; Carey, Michael F; Merchant, Sabeeha S; Kurdistani, Siavash K

    2017-08-11

    The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

    Science.gov (United States)

    Tufi, R; Panaretakis, T; Bianchi, K; Criollo, A; Fazi, B; Di Sano, F; Tesniere, A; Kepp, O; Paterlini-Brechot, P; Zitvogel, L; Piacentini, M; Szabadkai, G; Kroemer, G

    2008-02-01

    Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

  18. The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Michiko [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Hayashi, Teruo, E-mail: thayashi@mail.nih.gov [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Su, Tsung-Ping, E-mail: tsu@intra.nida.nih.gov [Cellular Pathobiology Section, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer The endoplasmic reticulum subdomain termed MAM associates with mitochondria. Black-Right-Pointing-Pointer The biophysical role of lipids in the MAM-mitochondria association is unknown. Black-Right-Pointing-Pointer The in vitro membrane association assay was used to examine the role of lipids. Black-Right-Pointing-Pointer Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP{sub 3} receptor-mediated Ca{sup 2+} influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-{beta}-cyclodextrin (M{beta}C) significantly increases the association between MAMs and mitochondria, whereas M{beta}C saturated with cholesterol does not change the association. {sup 14}C-Serine pulse-labeling demonstrated that the treatment of living cells with M{beta}C decreases the level of de novo synthesized {sup 14}C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of

  19. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  20. The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum.

    Science.gov (United States)

    Lipschutz, Joshua H; Lingappa, Vishwanath R; Mostov, Keith E

    2003-06-01

    We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259-4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of m

  1. CDIP1-BAP31 Complex Transduces Apoptotic Signals from Endoplasmic Reticulum to Mitochondria under Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Takushi Namba

    2013-10-01

    Full Text Available Resolved endoplasmic reticulum (ER stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31 as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

  2. CDIP1-BAP31 complex transduces apoptotic signals from endoplasmic reticulum to mitochondria under endoplasmic reticulum stress.

    Science.gov (United States)

    Namba, Takushi; Tian, Fang; Chu, Kiki; Hwang, So-Young; Yoon, Kyoung Wan; Byun, Sanguine; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W

    2013-10-31

    Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

  3. Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib.

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    Ramón Coca

    Full Text Available Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+-pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of

  4. ER reorganization is remarkably induced in COS-7 cells accumulating transmembrane protein receptors not competent for export from the endoplasmic reticulum.

    Science.gov (United States)

    D'Agostino, Massimo; Crespi, Arianna; Polishchuk, Elena; Generoso, Serena; Martire, Gianluca; Colombo, Sara Francesca; Bonatti, Stefano

    2014-11-01

    The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.

  5. Evolutionary Constraints on the Planet-Hosting Subgiant Epsilon Reticulum from its White Dwarf Companion

    CERN Document Server

    Farihi, J; Holberg, J B; Casewell, S L; Barstow, M A

    2011-01-01

    The planet-hosting and Sirius-type binary system epsilon Reticulum (HD 27442) is examined from the perspective of its more evolved white dwarf secondary. The stellar parameters are determined from a combination of Balmer line spectroscopy, gravitational redshift, and solid angle. These three methods conspire to yield the most accurate physical description of the companion to date: Teff=15,310 \\pm 350 K and M=0.60 \\pm 0.02 Msol. Post-main sequence mass loss indicates the current binary separation has increased by a factor of 1.6 from its primordial state when the current primary was forming its planet(s), implying a0 > 150 AU and constraining stable planets to within 15-20 AU for a binary eccentricity of e=0.5. Almost 80 years have passed since the first detection of the stellar companion, and marginal orbital motion may be apparent in the binary, suggesting a near edge-on configuration with i > 70 deg, albeit with substantial uncertainty. If correct, the mass of the planet HD 27442b is bound between 1.66 and ...

  6. KUS121, a VCP modulator, attenuates ischemic retinal cell death via suppressing endoplasmic reticulum stress

    Science.gov (United States)

    Hata, Masayuki; Ikeda, Hanako O.; Kikkawa, Chinami; Iwai, Sachiko; Muraoka, Yuki; Hasegawa, Tomoko; Kakizuka, Akira; Yoshimura, Nagahisa

    2017-01-01

    Ischemic neural damages cause several devastating diseases, including brain stroke and ischemic retinopathies, and endoplasmic reticulum (ER) stress has been proposed to be the underlying mechanism of the neuronal cell death of these conditions. We previously synthesized Kyoto University substances (KUSs) as modulators of valosin-containing protein (VCP); KUSs inhibit VCP ATPase activity and protect cells from different cell death-inducing insults. Here, we examined the efficacy of KUS121 in a rat model of retinal ischemic injury. Systemic administration of KUS121 to rats with ischemic retinal injury significantly suppressed inner retinal thinning and death of retinal ganglion and amacrine cells, with a significant functional maintenance of visual functions, as judged by electroretinography. Furthermore, intravitreal injection of KUS121, which is the clinically preferred route of drug administration for retinal diseases, appeared to show an equal or better neuroprotective efficacy in the ischemic retina compared with systemic administration. Indeed, induction of the ER stress marker C/EBP homologous protein (CHOP) after the ischemic insult was significantly suppressed by KUS121 administration. Our study suggests VCP modulation by KUS as a promising novel therapeutic strategy for ischemic neuronal diseases. PMID:28317920

  7. Complete Element Abundances of Nine Stars in the r-process Galaxy Reticulum II

    Science.gov (United States)

    Ji, Alexander P.; Frebel, Anna; Simon, Joshua D.; Chiti, Anirudh

    2016-10-01

    We present chemical abundances derived from high-resolution Magellan/Magellan Inamori Kyocera Echelle spectra of the nine brightest known red giant members of the ultra-faint dwarf galaxy Reticulum II (Ret II). These stars span the full metallicity range of Ret II (‑3.5 date. The other two stars are the most metal-poor stars in the system ([Fe/H] up to the iron peak are otherwise similar to abundances of stars in the halo and in other ultra-faint dwarf galaxies. However, the scatter in abundance ratios is large enough to suggest that inhomogeneous metal mixing is required to explain the chemical evolution of this galaxy. The presence of low amounts of neutron-capture elements in other ultra-faint dwarf galaxies may imply the existence of additional r-process sites besides the source of r-process elements in Ret II. Galaxies like Ret II may be the original birth sites of r-process enhanced stars now found in the halo. This paper includes data gathered with the 6.5 m Magellan Telescopes located at Las Campanas Observatory, Chile.

  8. Altered Ca2+ Homeostasis and Endoplasmic Reticulum Stress in Myotonic Dystrophy Type 1 Muscle Cells

    Directory of Open Access Journals (Sweden)

    Gyorgy Szabadkai

    2013-06-01

    Full Text Available The pathogenesis of Myotonic Dystrophy type 1 (DM1 is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.

  9. Endoplasmic reticulum stress regulates inflammation and insulin resistance in skeletal muscle from pregnant women.

    Science.gov (United States)

    Liong, Stella; Lappas, Martha

    2016-04-15

    Sterile inflammation and infection are key mediators of inflammation and peripheral insulin resistance associated with gestational diabetes mellitus (GDM). Studies have shown endoplasmic reticulum (ER) stress to induce inflammation and insulin resistance associated with obesity and type 2 diabetes, however is paucity of studies investigating the effects of ER stress in skeletal muscle on inflammation and insulin resistance associated with GDM. ER stress proteins IRE1α, GRP78 and XBP-1s were upregulated in skeletal muscle of obese pregnant women, whereas IRE1α was increased in GDM women. Suppression of ER stress, using ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or siRNA knockdown of IRE1α and GRP78, significantly downregulated LPS-, poly(I:C)- or IL-1β-induced production of IL-6, IL-8, IL-1β and MCP-1. Furthermore, LPS-, poly(I:C)- or TNF-α-induced insulin resistance was improved following suppression of ER stress, by increasing insulin-stimulated phosphorylation of IR-β, IRS-1, GLUT-4 expression and glucose uptake. In summary, our inducible obesity and GDM-like models suggests that the development of GDM may be involved in activating ER stress-induced inflammation and insulin resistance in human skeletal muscle.

  10. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    Science.gov (United States)

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  11. Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL.

    Science.gov (United States)

    Rosati, Emanuela; Sabatini, Rita; Rampino, Giuliana; De Falco, Filomena; Di Ianni, Mauro; Falzetti, Franca; Fettucciari, Katia; Bartoli, Andrea; Screpanti, Isabella; Marconi, Pierfrancesco

    2010-10-14

    A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8-mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

  12. Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum.

    Science.gov (United States)

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2008-10-15

    To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention "receptor" since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

  13. New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum.

    Science.gov (United States)

    Rudner, J; Jendrossek, V; Belka, C

    2002-10-01

    The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.

  14. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    Science.gov (United States)

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  15. A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection.

    Directory of Open Access Journals (Sweden)

    Mengding Qian

    2009-06-01

    Full Text Available The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism.

  16. The Ca(2+)-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

    Science.gov (United States)

    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.

  17. Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Fenglei Chen

    2015-08-01

    Full Text Available Zearalenone (ZEA is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78 and CCAAT/enhancer binding protein homologous protein (CHOP, two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs, significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

  18. Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

    Science.gov (United States)

    Chen, Fenglei; Li, Qian; Zhang, Zhe; Lin, Pengfei; Lei, Lanjie; Wang, Aihua; Jin, Yaping

    2015-08-20

    Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

  19. Adaptive endoplasmic reticulum stress alters cellular responses to the extracellular milieu.

    Science.gov (United States)

    Liu, Yiting; Neely, Elizabeth; Simmons, Zachary; Connor, James R

    2015-05-01

    The ability to respond to perturbations in endoplasmic reticulum (ER) function is a critical property for all cells. In the presence of chronic ER stress, the cell must adapt so that cell survival is favored or the stress may promote apoptosis. In some pathological processes, such as neurodengeneration, persistent ER stress can be tolerated for an extended period, but eventually cell death occurs. It is not known how an adaptive response converts from survival into apoptosis. To gain a better understanding of the role of adaptive ER stress in neurodegeneration, in this study, with a neuronal cell line SH-SY5Y and primary motor neuron-glia cell mixed cultures, we induced adaptive ER stress and modified the extracellular environment with physiologically relevant changes that alone did not activate ER stress. Our data demonstrate that an adaptive ER stress favored neuronal cell survival, but when cells were exposed to additional physiological insults the level of ER stress was increased, followed by activation of the caspase pathway. Our results indicate that an adaptive ER stress response could be converted to apoptosis when the external cellular milieu changed, suggesting that the conversion from prosurvival to proapoptotic pathways can be driven by the external milieu. This conversion was due at least partially to an increased level of ER stress. © 2015 Wiley Periodicals, Inc.

  20. Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate.

    Science.gov (United States)

    Brookes, Steven J; Barron, Martin J; Boot-Handford, Ray; Kirkham, Jennifer; Dixon, Michael J

    2014-05-01

    Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease.

  1. Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress.

    Science.gov (United States)

    Li, Jian-Shuang; Wang, Wen-Jun; Sun, Yu; Zhang, Yu-Hao; Zheng, Ling

    2015-05-01

    Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10-30 μg ml(-1)) significantly decreased palmitic acid induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD treatment.

  2. Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: from Infection to Cancer

    Directory of Open Access Journals (Sweden)

    Doriana Fruci

    2012-07-01

    Full Text Available Endoplasmic reticulum (ER aminopeptidases ERAP1 and ERAP2 (ERAPs are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8+ T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases.

  3. ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants.

    Science.gov (United States)

    Le Bars, Romain; Marion, Jessica; Le Borgne, Rémi; Satiat-Jeunemaitre, Béatrice; Bianchi, Michele Wolfe

    2014-06-20

    Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

  4. Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency.

    LENUS (Irish Health Repository)

    Kelly, Emer

    2009-06-19

    Z alpha(1)-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFkappaB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFkappaB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFkappaB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.

  5. Endoplasmic reticulum stress promotes amyloid-beta peptides production in RGC-5 cells.

    Science.gov (United States)

    Liu, Bingqian; Zhu, Yingting; Zhou, Jiayi; Wei, Yantao; Long, Chongde; Chen, Mengfei; Ling, Yunlan; Ge, Jian; Zhuo, Yehong

    2014-11-01

    Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including Alzheimer's disease. We have previously observed amyloid production in the retina of the Tg2576 transgenic mouse model of Alzheimer's disease. In this study, we used tunicamycin-induced ER stress in RGC-5 cells, a cell line identical to the photoreceptor cell line 661W, to investigate the effect of ER stress on production of amyloid-beta (Abeta) peptides. We found that the mRNA level of amyloid-beta precursor protein (APP) remained stable, while the protein level of amyloid-beta precursor protein (APP) was decreased, the amyloid-beta precursor protein cleaving enzymes beta-site APP-cleaving enzyme 1 and presenilin 1 were upregulated, Abeta1-40 and Abeta1-42 production were increased, and reactive oxygen species production and apoptosis markers were elevated following induction of ER stress. The protein level of Abeta degradation enzymes, neprilysin, endothelin-converting enzyme 1, and endothelin-converting enzyme 2 remained unchanged during the prolonged ER stress, showing that the generation of Abeta did not result from reduction of proteolysis by these enzymes. Inclusion of group II caspase inhibitor, Z-DEVD-FMK, increased the ER stress mediated Abeta production, suggesting that they are generated by a caspase-independent mechanism. Our findings provided evidence of a role of ER stress in Abeta peptide overproduction and apoptotic pathway activation in RGC-5 cells.

  6. Tremorgenic mycotoxins increase gastric smooth muscle activity of sheep reticulum and rumen in vitro.

    Science.gov (United States)

    Wang, L; Cross, A L; Allen, K L; Smith, B L; McLeay, L M

    2003-02-01

    Reticulum and rumen strips (consisting of both muscle layers and the myenteric plexus) were superfused with Tyrode Ringer and their contractions recorded isometrically. The strips were subjected to exogenous acetylcholine and electrical field stimulation (EFS) resulting in contractions that could be blocked by atropine. Responses to the tremorgenic mycotoxin penitrem A and others thought to be involved in ryegrass staggers, paxilline and lolitrem B (10(-10)-10(-6)M), were compared with those of control vehicle (0.1% DMSO). The tremorgens were without effect on quiescent preparations. Penitrem A and paxilline enhanced spontaneously active preparations and the amplitude of contractions in response to EFS. Responses to paxilline had a shorter latency than to penitrem A. Responses of spontaneously active preparations were resistant to atropine. Penitrem A, but not paxilline, increased responses to exogenous acetylcholine. Lolitrem B (10(-6)M) increased responses to EFS, but many responses were equivocal, possibly due to the lower solubility of lolitrem B in aqueous solutions compared to the other tremorgens. The results show that these mycotoxins have peripheral excitatory effects on the reticulorumen and it is suggested that such activity in vivo may reflexly affect centrally derived cyclical contractions.

  7. Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44*

    Science.gov (United States)

    Hampe, Lutz; Radjainia, Mazdak; Xu, Cheng; Harris, Paul W. R.; Bashiri, Ghader; Goldstone, David C.; Brimble, Margaret A.; Wang, Yu; Mitra, Alok K.

    2015-01-01

    Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys39 located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12–18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys39 and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control. PMID:26060250

  8. Fenofibrate treatment attenuated chronic endoplasmic reticulum stress in the liver of nonalcoholic fatty liver disease mice.

    Science.gov (United States)

    Zhang, Nan; Lu, Yunxia; Shen, Xinru; Bao, Yingying; Cheng, Jingjing; Chen, Li; Li, Bao; Zhang, Qiu

    2015-01-01

    Fenofibrate is widely used in clinical practice, but its influence on chronic endoplasmic reticulum (ER) stress induced by feeding a high-calorie and high-cholesterol diet (HCD) has still not been studied. We thus investigated its effects on the liver of the nonalcoholic fatty liver disease (NAFLD) mouse model. Male C57BL/6 mice fed an HCD for 3 months were treated with fenofibrate (HCD + FF, 40 mg/kg, once daily) via gavage for 4 weeks. Insulin sensitivity, serum lipid and inflammatory cytokines were measured. Liver tissues were procured for histological examination as well as analysis of hepatic triglyceride levels, distribution of inflammatory cytokines and genes involved in ER stress. Our results showed that chronic feeding of an HCD successfully induced an NAFLD model accompanied by inflammatory activation, apoptosis and severe ER stress in the liver. Fenofibrate administration significantly improved symptoms of NAFLD and decreased apoptosis, expression of inflammatory cytokines and genes involved in ER stress, such as inositol-requiring enzyme 1α (IRE1α), X-box binding protein 1 (XBP1) and JNK phosphorylation. Thus, our study suggests that fenofibrate protected against inflammatory injury and apoptosis, maybe alleviating ER stress through the IRE1α-XBP1-JNK pathway in the liver of NAFLD mice. © 2015 S. Karger AG, Basel

  9. The Endoplasmic Reticulum Unfolded Protein Response in Neurodegenerative Disorders and Its Potential Therapeutic Significance

    Directory of Open Access Journals (Sweden)

    Paolo Remondelli

    2017-06-01

    Full Text Available In eukaryotic cells, the endoplasmic reticulum (ER is the cell compartment involved in secretory protein translocation and quality control of secretory protein folding. Different conditions can alter ER function, resulting in the accumulation of unfolded or misfolded proteins within the ER lumen. Such a condition, known as ER stress, elicits an integrated adaptive response known as the unfolded protein response (UPR that aims to restore proteostasis within the secretory pathway. Conversely, in prolonged cell stress or insufficient adaptive response, UPR signaling causes cell death. ER dysfunctions are involved and contribute to neuronal degeneration in several human diseases, including Alzheimer, Parkinson and Huntington disease and amyotrophic lateral sclerosis. The correlations between ER stress and its signal transduction pathway known as the UPR with neuropathological changes are well established. In addition, much evidence suggests that genetic or pharmacological modulation of UPR could represent an effective strategy for minimizing the progressive neuronal loss in neurodegenerative diseases. Here, we review recent results describing the main cellular mechanisms linking ER stress and UPR to neurodegeneration. Furthermore, we provide an up-to-date panoramic view of the currently pursued strategies for ameliorating the toxic effects of protein unfolding in disease by targeting the ER UPR pathway.

  10. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    2016-10-01

    Full Text Available Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS. Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.

  11. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayoshi [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2013-02-01

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  12. Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection.

    Science.gov (United States)

    Grangeon, Romain; Agbeci, Maxime; Chen, Jun; Grondin, Gilles; Zheng, Huanquan; Laliberté, Jean-François

    2012-09-01

    The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.

  13. Optimal microdomain crosstalk between endoplasmic reticulum and mitochondria for Ca2+ oscillations.

    Science.gov (United States)

    Qi, Hong; Li, Linxi; Shuai, Jianwei

    2015-01-23

    A Ca(2+) signaling model is proposed to consider the crosstalk of Ca(2+) ions between endoplasmic reticulum (ER) and mitochondria within microdomains around inositol 1, 4, 5-trisphosphate receptors (IP3R) and the mitochondrial Ca(2+) uniporter (MCU). Our model predicts that there is a critical IP3R-MCU distance at which 50% of the ER-released Ca(2+) is taken up by mitochondria and that mitochondria modulate Ca(2+) signals differently when outside of this critical distance. This study highlights the importance of the IP3R-MCU distance on Ca(2+) signaling dynamics. The model predicts that when MCU are too closely associated with IP3Rs, the enhanced mitochondrial Ca(2+) uptake will produce an increase of cytosolic Ca(2+) spike amplitude. Notably, the model demonstrates the existence of an optimal IP3R-MCU distance (30-85 nm) for effective Ca(2+) transfer and the successful generation of Ca(2+) signals in healthy cells. We suggest that the space between the inner and outer mitochondria membranes provides a defense mechanism against occurrences of high [Ca(2+)]Cyt. Our results also hint at a possible pathological mechanism in which abnormally high [Ca(2+)]Cyt arises when the IP3R-MCU distance is in excess of the optimal range.

  14. Osteoclast precursor differentiation by MCPIP via oxidative stress, endoplasmic reticulum stress,and autophagy

    Institute of Scientific and Technical Information of China (English)

    Kangkai Wang; Jianli Niu; Hyunbae Kim; Pappachan E. Kolattukudy

    2011-01-01

    Osteoclasts (OCs) are responsible for bone resorption in inflammatory joint diseases.Monocyte chemotactic protein-t (MCP-t) has been shown to induce differentiation of monocytes to OC precursors,but nothing is known about the underlying mechanisms.Here,we elucidate how MCPIP,induced by MCP-1,mediates this differentiation.Knockdown of MCPIP abolished MCP-1-mediated expression of OC markers,tartrate-resistant acid phosphatase,and serine protease cathepsin K.Expression of MCPIP induced p47PHOX and its membrane translocation,reactive oxygen species formation,and induction of endoplasmic reticulum (ER) stress chaperones,up-regulation of autophagy marker,Beclin-1,and lipidation of LC3,and induction of OC markers.Inhibition of oxidative stress attenuated ER stress and autophagy,and suppressed expression of OC markers.Inhibition of ER stress by a specific inhibitor or by knockdown of I REt blocked autophagy and induction of OC markers.ER stress inducers,tunicamycin and thapsigargin,induced expression of OC markers.Autophagy inhibition by 3'-methyladenine,LY294002,wortmannin or by knockdown of Beclin-1 or Atg 7 inhibited MCPIP-induced expression of OC markers.These results strongly suggest that MCP-1-induced differentiation of OC precursor cells is mediated via MCPIP-induced oxidative stress that causes ER stress leading to autophagy,revealing a novel mechanistic insight into the role of MCP-1 in OCs differentiation.

  15. Protective Effect of Gomisin N against Endoplasmic Reticulum Stress-Induced Hepatic Steatosis.

    Science.gov (United States)

    Jang, Min-Kyung; Yun, Ye-Rang; Kim, Seon Hoo; Kim, Ji Ha; Jung, Myeong Ho

    2016-05-01

    Gomisin N is a physiological substance derived from Schisandra chinensis. In the present study, the in vitro and in vivo effects of gomisin N on endoplasmic reticulum (ER) stress and hepatic steatosis were investigated. We quantified the expression of markers of ER stress, including glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homolog protein (CHOP), and X-box-binding protein-1 (XBP-1), and triglyceride (TG) accumulation, in HepG2 cells treated with tunicamycin or palmitate. Tunicamycin treatment in HepG2 cells induced expression of markers of ER stress and increased TG levels; Gomisin N reversed these effects, reducing the expression of markers of ER stress and TG levels. Similar effects were seen following palmitate pretreatment of HepG2 cells. The inhibitory effects of gomisin N were further confirmed in mice injected with tunicamycin. Gomisin N reduced expression of markers of ER stress and decreased TG levels in mouse liver after tunicamycin injection. Furthermore, gomisin N decreased expression of inflammatory and lipogenic genes in palmitate-incubated HepG2 cells. These results suggest that gomisin N inhibits ER stress and ameliorates hepatic steatosis induced by ER stress.

  16. Critical Role of Endoplasmic Reticulum Stress in Cognitive Impairment Induced by Microcystin-LR

    Directory of Open Access Journals (Sweden)

    Fei Cai

    2015-11-01

    Full Text Available Recent studies showed that cyanobacteria-derived microcystin-leucine-arginine (MCLR can cause hippocampal pathological damage and trigger cognitive impairment; but the underlying mechanisms have not been well understood. The objective of the present study was to investigate the mechanism of MCLR-induced cognitive deficit; with a focus on endoplasmic reticulum (ER stress. The Morris water maze test and electrophysiological study demonstrated that MCLR caused spatial memory injury in male Wistar rats; which could be inhibited by ER stress blocker; tauroursodeoxycholic acid (TUDCA. Meanwhile; real-time polymerase chain reaction (real-time PCR and immunohistochemistry demonstrated that the expression level of the 78-kDa glucose-regulated protein (GRP78; C/EBP homologous protein (CHOP and caspase 12 were significantly up-regulated. These effects were rescued by co-administration of TUDCA. In agreement with this; we also observed that treatment of rats with TUDCA blocked the alterations in ER ultrastructure and apoptotic cell death in CA1 neurons from rats exposed to MCLR. Taken together; the present results suggested that ER stress plays an important role in potential memory impairments in rats treated with MCLR; and amelioration of ER stress may serve as a novel strategy to alleviate damaged cognitive function triggered by MCLR.

  17. Methyl jasmonate affects morphology, number and activity of endoplasmic reticulum bodies in Raphanus sativus root cells.

    Science.gov (United States)

    Gotté, Maxime; Ghosh, Rajgourab; Bernard, Sophie; Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Hara-Nishimura, Ikuko; Driouich, Azeddine

    2015-01-01

    The endoplasmic reticulum (ER) bodies are ER-derived structures that are found in Brassicaceae species and thought to play a role in defense. Here, we have investigated the occurrence, distribution and function of ER bodies in root cells of Raphanus sativus using a combination of microscopic and biochemical methods. We have also assessed the response of ER bodies to methyl jasmonate (MeJA), a phytohormone that mediates plant defense against wounding and pathogens. Our results show that (i) ER bodies do occur in different root cell types from the root cap region to the differentiation zone; (ii) they do accumulate a PYK10-like protein similar to the major marker protein of ER bodies that is involved in defense in Arabidopsis thaliana; and (iii) treatment of root cells with MeJA causes a significant increase in the number of ER bodies and the activity of β-glucosidases. More importantly, MeJA was found to induce the formation of very long ER bodies that results from the fusion of small ones, a phenomenon that has not been reported in any other study so far. These findings demonstrate that MeJA impacts the number and morphology of functional ER bodies and stimulates ER body enzyme activities, probably to participate in defense responses of radish root. They also suggest that these structures may provide a defensive system specific to root cells.

  18. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Nozomi Kawazoe

    2017-06-01

    Full Text Available Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER and unfolded protein response (UPR has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v. Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid and mild ethanol stress (5% ethanol induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

  19. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    Science.gov (United States)

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-01-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro. PMID:28145529

  20. KLF15 is a molecular link between endoplasmic reticulum stress and insulin resistance.

    Directory of Open Access Journals (Sweden)

    Dae Young Jung

    Full Text Available Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER, resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15, as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15 (-/- mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1, a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15 (-/- liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.

  1. Stellar Kinematics and Metallicities in the Ultra-Faint Dwarf Galaxy Reticulum II

    CERN Document Server

    Simon, J D; Li, T S; Nord, B; Geha, M; Bechtol, K; Balbinot, E; Buckley-Geer, E; Lin, H; Marshall, J; Santiago, B; Strigari, L; Wang, M; Wechsler, R H; Yanny, B; Abbott, T; Bauer, A H; Bernstein, G M; Bertin, E; Brooks, D; Burke, D L; Capozzi, D; Rosell, A Carnero; Kind, M Carrasco; D'Andrea, C B; da Costa, L N; DePoy, D L; Desai, S; Diehl, H T; Dodelson, S; Cunha, C E; Estrada, J; Evrard, A E; Neto, A Fausti; Fernandez, E; Finley, D A; Flaugher, B; Frieman, J; Gaztanaga, E; Gerdes, D; Gruen, D; Gruendl, R A; Honscheid, K; James, D; Kuehn, K; Kuropatkin, N; Lahav, O; Maia, M A G; March, M; Martini, P; Miller, C J; Miquel, R; Ogando, R; Romer, A K; Roodman, A; Rykoff, E S; Sako, M; Sanchez, E; Schubnell, M; Sevilla, I; Smith, R C; Soares-Santos, M; Sobreira, F; Suchyta, E; Swanson, M E C; Tarle, G; Thaler, J; Tucker, D; Vikram, V; Walker, A R; Wester, W

    2015-01-01

    We present Magellan/M2FS, VLT/GIRAFFE, and Gemini South/GMOS spectroscopy of the newly discovered Milky Way satellite Reticulum II. Based on the spectra of 25 Ret II member stars selected from Dark Energy Survey imaging, we measure a mean heliocentric velocity of 62.8 +/- 0.5 km/s and a velocity dispersion of 3.3 +/- 0.7 km/s. The mass-to-light ratio of Ret II within its half-light radius is 470 +/- 210 Msun/Lsun, demonstrating that it is a strongly dark matter-dominated system. Despite its spatial proximity to the Magellanic Clouds, the radial velocity of Ret II differs from that of the LMC and SMC by 199 and 83 km/s, respectively, suggesting that it is not gravitationally bound to the Magellanic system. The likely member stars of Ret II span 1.3 dex in metallicity, with a dispersion of 0.28 +/- 0.09 dex, and we identify several extremely metal-poor stars with [Fe/H] < -3. In combination with its luminosity, size, and ellipticity, these results confirm that Ret II is an ultra-faint dwarf galaxy. With a me...

  2. Endoplasmic reticulum remodeling tunes IP₃-dependent Ca²+ release sensitivity.

    Directory of Open Access Journals (Sweden)

    Lu Sun

    Full Text Available The activation of vertebrate development at fertilization relies on IP₃-dependent Ca²⁺ release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP₃ receptors within ER patches have a higher sensitivity to IP₃ than those in the neighboring reticular ER. The lateral diffusion rate of IP₃ receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP₃ receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP₃ receptors through ER remodeling is sufficient to sensitize IP₃-dependent Ca²⁺ release. Mathematical modeling supports this concept of 'geometric sensitization' of IP₃ receptors as a population, and argues that it depends on enhanced Ca²⁺-dependent cooperativity at sub-threshold IP₃ concentrations. This represents a novel mechanism of tuning the sensitivity of IP₃ receptors through ER remodeling during meiosis.

  3. Exogenous taurine attenuates mitochondrial oxidative stress and endoplasmic reticulum stress in rat cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yujie Yang; Yue Zhang; Xiaoyu Liu; Ji Zuo; Keqiang Wang; Wen Liu; Junbo Ge

    2013-01-01

    Taurine,a conditionally essential amino acid,plays a critical role in cardiovascular function.Here we examined the effect of taurine on mitochondria and endoplasmic reticulum in rat cardiomyocytes during glucose deprivation (GD).Data showed that cell viability,intracellular taurine contents,and taurine transporter expression were decreased during GD.In contrast,an increase in reactive oxygen species and intracellular Ca2+ contents was observed.GD also caused disrupted mitochondrial membrane potential,apoptotic cell death,and dissociation of unfolded protein response (UPR)-relative proteins in cardiomyocytes.Signal transduction analysis showed that Bcl-2 family protein balance was disturbed,caspase-12 was activated and UPR-relative protein levels were up-regulated.Moreover,pre-treatment with 80 mM exogenous taurine attenuated GD effect in cardiomyocytes.Our results suggest that taurine have beneficial effects on inhibiting mitochondria-dependent cell apoptosis and UPR-associated cell apoptosis and might have clinical impfications on acute myocardial infarction in future.

  4. Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells.

    Science.gov (United States)

    Li, Tianliang; Su, Ling; Zhong, Ning; Hao, Xuexi; Zhong, Diansheng; Singhal, Sunil; Liu, Xiangguo

    2013-07-01

    Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

  5. Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kawazoe, Nozomi; Kimata, Yukio; Izawa, Shingo

    2017-01-01

    Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER) and unfolded protein response (UPR) has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v). Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid) and mild ethanol stress (5% ethanol) induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.

  6. Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration.

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    Glenn P Lobo

    Full Text Available Inherited retinal disorders (IRDs result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP and Leber congenital amaurosis (LCA. TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.

  7. Loss of endoplasmic reticulum Ca homeostasis:contribution to neuronal cell death during cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Ankur BODALIA; Hongbin LI; Michael F JACKSON

    2013-01-01

    The loss of Ca2+ homeostasis during cerebral ischemia is a hallmark of impending neuronal demise.Accordingly,considerable cellular resources are expended in maintaining low resting cytosolic levels of Ca2+.These include contributions by a host of proteins involved in the sequestration and transport of Ca2+,many of which are expressed within intracellular organelles,including lysosomes,mitochondria as well as the endoplasmic reticulum (ER).Ca2+ sequestration by the ER contributes to cytosolic Ca2+ dynamics and homeostasis.Furthermore,within the ER Ca2+ plays a central role in regulating a host of physiological processes.Conversely,impaired ER Ca2+ homeostasis is an important trigger of pathological processes.Here we review a growing body of evidence suggesting that ER dysfunction is an important factor contributing to neuronal injury and loss post-ischemia.Specifically,the contribution of the ER to cytosolic Ca2+ elevations during ischemia will be considered,as will the signalling cascades recruited as a consequence of disrupting ER homeostasis and function.

  8. MET Suppresses Epithelial VEGFR2 via Intracrine VEGF-induced Endoplasmic Reticulum-associated Degradation

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    Tom T. Chen

    2015-05-01

    Full Text Available Hepatocyte growth factor (HGF and vascular endothelial growth factor (VEGF drive cancer through their respective receptors, MET and VEGF receptor 2 (VEGFR2. VEGFR2 inhibits MET by promoting MET dephosphorylation. However, whether MET conversely regulates VEGFR2 remains unknown. Here we show that MET suppresses VEGFR2 protein by inducing its endoplasmic-reticulum-associated degradation (ERAD, via intracrine VEGF action. HGF–MET signaling in epithelial cancer cells promoted VEGF biosynthesis through PI3-kinase. In turn, VEGF and VEGFR2 associated within the ER, activating inositol-requiring enzyme 1α, and thereby facilitating ERAD-mediated depletion of VEGFR2. MET disruption upregulated VEGFR2, inducing compensatory tumor growth via VEGFR2 and MEK. However, concurrent disruption of MET and either VEGF or MEK circumvented this, enabling more profound tumor inhibition. Our findings uncover unique cross-regulation between MET and VEGFR2—two RTKs that play significant roles in tumor malignancy. Furthermore, these results suggest rational combinatorial strategies for targeting RTK signaling pathways more effectively, which has potentially important implications for cancer therapy.

  9. Recombinant Wheat Endoplasmic Reticulum Oxidoreductin 1 Improved Wheat Dough Properties and Bread Quality.

    Science.gov (United States)

    Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Zhang, Ya-Ping; Li, Cunzhi; Li, Lin; Hu, Song-Qing

    2017-03-15

    Recombinant wheat endoplasmic reticulum oxidoreductin 1 (wEro1) with considerable ability was expressed in Escherichia coli. The functional roles of wEro1 in flour processing quality were investigated by farinographic, rheological, texture profile analysis, electrophoresis, size exclusion chromatography, scanning electron microscopy, and Fourier transform infrared spectroscopy. wEro1 exhibited an obvious oxidation activity of sulfhydryl groups in small molecule and protein. Addition of wEro1 could strengthen the processing quality of dough, indicated by the improved mixing characteristics, viscoelastic properties, and bread qualities. These improvement effects of wEro1 could be attributed to the formation of macromolecular gluten polymers and massive gluten networks by disulfide cross-linking. Additionally, the increased β-turn structure further demonstrated the enhancement of dough strength. Moreover, the amount of peroxide in dough was improved significantly from 2.36 to 2.82 μmol/g of flour with 0.15% wEro1 treatment. Therefore, the results suggested that wEro1 is a promising novel flour improver.

  10. Contribution of mitochondria and endoplasmic reticulum dysfunction in insulin resistance: Distinct or interrelated roles?

    Science.gov (United States)

    Rieusset, J

    2015-11-01

    Mitochondria and the endoplasmic reticulum (ER) regulate numerous cellular processes, and are critical contributors to cellular and whole-body homoeostasis. More important, mitochondrial dysfunction and ER stress are both closely associated with hepatic and skeletal muscle insulin resistance, thereby playing crucial roles in altered glucose homoeostasis in type 2 diabetes mellitus (T2DM). The accumulated evidence also suggests a potential interrelationship between alterations in both types of organelles, as mitochondrial dysfunction could participate in activation of the unfolded protein response, whereas ER stress could influence mitochondrial function. The fact that mitochondria and the ER are physically and functionally interconnected via mitochondria-associated membranes (MAMs) supports their interrelated roles in the pathophysiology of T2DM. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance to the control of glucose homoeostasis, are still unknown. This review evaluates the involvement of mitochondria and ER independently in the development of peripheral insulin resistance, as well as their potential roles in the disruption of organelle crosstalk at MAM interfaces in the alteration of insulin signalling. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

    Science.gov (United States)

    Gunduz, Nuray; Ceylan, Hakan; Guler, Mustafa O.; Tekinay, Ayse B.

    2017-02-01

    Understanding the toxicity of nanomaterials remains largely limited to acute cellular response, i.e., short-term in vitro cell-death based assays, and analyses of tissue- and organ-level accumulation and clearance patterns in animal models, which have produced very little information about how these materials (from the toxicity point of view) interact with the complex intracellular machinery. In particular, understanding the mechanism of toxicity caused by the gradual accumulation of nanomaterials due to prolonged exposure times is essential yet still continue to be a largely unexplored territory. Herein, we show intracellular accumulation and the associated toxicity of gold nanoparticles (AuNPs) for over two-months in the cultured vascular endothelial cells. We observed that steady exposure of AuNPs at low (non-lethal) dose leads to rapid intracellular accumulation without causing any detectable cell death while resulting in elevated endoplasmic reticulum (ER) stress. Above a certain intracellular AuNP threshold, inhibition of macropinocytosis mechanism ceases further nanoparticle uptake. Interestingly, the intracellular depletion of nanoparticles is irreversible. Once reaching the maximum achievable intracellular dose, a steady depletion is observed, while no cell death is observed at any stage of this overall process. This depletion is important for reducing the ER stress. To our knowledge, this is the first report suggesting active regulation of nanoparticle uptake by cells and the impact of long-term exposure to nanoparticles in vitro.

  12. Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos.

    Science.gov (United States)

    Cui, Jingqiu; Chen, Wei; Sun, Jinhong; Guo, Huan; Madley, Rachel; Xiong, Yi; Pan, Xingyi; Wang, Hongliang; Tai, Andrew W; Weiss, Michael A; Arvan, Peter; Liu, Ming

    2015-11-20

    Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.

  13. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

    2016-09-19

    Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

  14. Targeted induction of endoplasmic reticulum stress induces cartilage pathology.

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    M Helen Rajpar

    2009-10-01

    Full Text Available Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS, a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS-causing mutation (COL10A1 p.Asn617Lys to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER stress and a robust unfolded protein response (UPR due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF-mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress-inducing protein (the cog mutant of thyroglobulin in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant

  15. Endoplasmic reticulum stress is chronically activated in chronic pancreatitis.

    Science.gov (United States)

    Sah, Raghuwansh P; Garg, Sushil K; Dixit, Ajay K; Dudeja, Vikas; Dawra, Rajinder K; Saluja, Ashok K

    2014-10-03

    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

  16. Klotho Ameliorates Chemically Induced Endoplasmic Reticulum (ER Stress Signaling

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    Srijita Banerjee

    2013-05-01

    Full Text Available Background: Both endoplasmic reticulum (ER stress, a fundamental cell response associated with stress-initiated unfolded protein response (UPR, and loss of Klotho, an anti-aging hormone linked to NF-κB-induced inflammation, occur in chronic metabolic diseases such as obesity and type 2 diabetes. We investigated if the loss of Klotho is causally linked to increased ER stress. Methods: We treated human renal epithelial HK-2, alveolar epithelial A549, HEK293, and SH-SH-SY5Y neuroblastoma cells with ER stress-inducing agents, thapsigargin and/or tunicamycin. Effects of overexpression or siRNA-mediated knockdown of Klotho on UPR signaling was investigated by immunoblotting and Real-time PCR. Results: Elevated Klotho levels in HK-2 cells decreased expression of ER stress markers phospho-IRE1, XBP-1s, BiP, CHOP, pJNK, and phospho-p38, all of which were elevated in response to tunicamycin and/or thapsigargin. Similar results were observed using A549 cells for XBP-1s, BiP, and CHOP in response to thapsigargin. Conversely, knockdown of Klotho in HEK 293 cells using siRNA caused further thapsigargin-induced increases in pIRE-1, XBP-1s, and BiP. Klotho overexpression in A549 cells blocked thapsigargin-induced caspase and PARP cleavage and improved cell viability. Conclusion: Our data indicate that Klotho has an important role in regulating ER stress and that loss of Klotho is causally linked to ER stress-induced apoptosis.

  17. Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

    Science.gov (United States)

    Okeke, Emmanuel; Dingsdale, Hayley; Parker, Tony; Voronina, Svetlana; Tepikin, Alexei V

    2016-06-01

    Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  18. Sertraline induces endoplasmic reticulum stress in hepatic cells.

    Science.gov (United States)

    Chen, Si; Xuan, Jiekun; Couch, Letha; Iyer, Advait; Wu, Yuanfeng; Li, Quan-Zhen; Guo, Lei

    2014-08-01

    Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity.

  19. Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.

    Science.gov (United States)

    Melnik, Bodo C

    2014-12-01

    Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population.

  20. PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana.

    Science.gov (United States)

    Nakano, Ryohei T; Piślewska-Bednarek, Mariola; Yamada, Kenji; Edger, Patrick P; Miyahara, Mado; Kondo, Maki; Böttcher, Christoph; Mori, Masashi; Nishimura, Mikio; Schulze-Lefert, Paul; Hara-Nishimura, Ikuko; Bednarek, Paweł

    2017-01-01

    The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of β-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant β-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  1. PKR-like endoplasmic reticulum kinase is necessary for lipogenic activation during HCMV infection.

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    Yongjun Yu

    Full Text Available PKR-like endoplasmic reticulum (ER kinase (PERK is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1, resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV infection in human fibroblasts (HF induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA, resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1 protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP

  2. PKR-like endoplasmic reticulum kinase is necessary for lipogenic activation during HCMV infection.

    Science.gov (United States)

    Yu, Yongjun; Pierciey, Francis J; Maguire, Tobi G; Alwine, James C

    2013-01-01

    PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for

  3. The endoplasmic reticulum stress response is associated with insulin resistance-mediated drug resistance in HepG2 cells.

    Science.gov (United States)

    Li, L; Li, G; Wei, H; Sun, J; Chen, J; Xie, B; Wang, B; Gu, J; Li, C; Tian, B; Wang, F

    2015-01-01

    Insulin resistance has a close relationship with tumorigenesis, tumor progression, and cancer prognosis. Importantly, the liver is the main target tissue of insulin, and the resistance to chemotherapeutic agents has been reported in hepatocarcinoma. However, little is known about the relationship between drug resistance and insulin resistance in hepatocarcinoma. Therefore, we treated HepG2 cells (a human hepatoma cell line) with high concentrations of insulin to establish a cell-based model of insulin resistance (HepG2/IR cells) to define the relationship between insulin resistance and the resistance to chemotherapy. We identified that HepG2/IR cells exhibited stable insulin resistance, with decreased glucose consumption, reduced glycogen synthesis, and decreased expression of the insulin receptor gene. HepG2/IR cells also exhibited endoplasmic reticulum (ER) dilatation and degranulation. Molecular markers of endoplasmic reticulum stress, including glucose-regulated protein78 (GRP78) and phosphorylated protein kinase R-like ER kinase (p-PERK), increased significantly, which was accompanied by increased reactive oxygen metabolism and decreased mitochondrial membrane potential. In addition, HepG2/IR cells were resistant to the chemotherapy agent Adriamycin, which was accompanied by the upregulation of multidrug resistance gene 1/ P-glycoprotein (P-gp; an endoplasmic reticulum chaperone that plays a role in ER stress), and enhanced drug efflux. These data suggest that the endoplasmic reticulum (ER) stress response was active in HepG2/IR cells, and that insulin resistance was related to drug resistance in HepG2 cells. Interestingly, the ER stress and chemotherapy resistance observed in HepG2/IR cells could be reversed by treatment with the insulin sensitizer pioglitazone. Therefore, our study suggests that there is a close relationship between the resistance to chemotherapy and insulin resistance in HepG2 cells, and that the ER stress response play a role in insulin

  4. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells

    DEFF Research Database (Denmark)

    Cardozo, Alessandra K; Ortis, Fernanda; Storling, Joachim

    2005-01-01

    , beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol......Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown......, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding...

  5. Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis.

    Science.gov (United States)

    Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo

    2016-12-20

    Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis.

  6. Endoplasmic reticulum stress in pathogenesis of diabetic retinopathy and effect of calcium dobesilate

    Institute of Scientific and Technical Information of China (English)

    Yu-Min Gui; Ming Zhao; Jie Ding

    2016-01-01

    Objective:To study the mechanism of endoplasmic reticulum stress in the pathogenesis of diabetic retinopathy and effect of calcium dobesilate.Methods:A total of 120 diabetic retinopathy patients treated in our hospital from January 2010 to September 2015 were enrolled in this article. The serum endoplasmic reticulum stress protein and interleukin protein expression levels were analyzed before and after calcium dobesilate treatment. A total of 55 cases of healthy subjects receiving physical examination in our hospital during the same period were taken as control group.Results:Serum endoplasmic reticulum stress proteins PERK, CHOP and IRE as well as interleukin proteins IL1, IL2, IL6 and IL10 expression significantly increased, serum MDA level significantly increased while SOD, CAT and GSHpx levels significantly decreased in diabetic retinopathy patients, and compared with control group (P<0.01); after calcium dobesilate treatment, above factors were significantly restored (P<0.01).Conclusions: Diabetic retinopathy is closely related to endoplasmic reticulum stress and calcium dobesilate treatment may improve diabetic retinopathy by inhibiting endoplasmic reticulum stress.

  7. Caspase-4 directly activates caspase-9 in endoplasmic reticulum stress-induced apoptosis in SH-SY5Y cells.

    Science.gov (United States)

    Yamamuro, Akiko; Kishino, Takashi; Ohshima, Yu; Yoshioka, Yasuhiro; Kimura, Tomoki; Kasai, Atsushi; Maeda, Sadaaki

    2011-01-01

    The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress-induced neuronal apoptosis.

  8. Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response Constitutes a Pathogenic Strategy of group A Streptococcus

    Directory of Open Access Journals (Sweden)

    Emanuel eHanski

    2014-08-01

    Full Text Available The connection between bacterial pathogens and unfolded protein response (UPR is poorly explored. In this review we highlight the evidence showing that group A streptococcus (GAS induces endoplasmic reticulum (ER stress and UPR through which it captures the amino acid asparagine (ASN from the host. GAS acts extracellularly and during adherence to host cells it delivers the hemolysin toxins; streptolysin O (SLO and streptolysin S (SLS. By poorly understood pathways, these toxins trigger UPR leading to the induction of the transcriptional regulator ATF4 and consequently to the upregulation of asparagine synthetase (ASNS transcription leading to production and release of ASN. GAS senses ASN and alters gene expression profile accordingly, and increases the rate of multiplication. We suggest that induction of UPR by GAS and by other bacterial pathogens represent means through which bacterial pathogens gain nutrients from the host, obviating the need to become internalized or inflict irreversible cell damage.

  9. Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Christen, Verena [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Capelle, Martinus [Crucell, P.O. Box 2048, NL-2301 Leiden (Netherlands); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich, Department of Environmental Systems Science, CH-8092 Zürich (Switzerland)

    2013-10-15

    Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL and Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes.

  10. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury.

    Science.gov (United States)

    Fan, H; Tang, H-B; Kang, J; Shan, L; Song, H; Zhu, K; Wang, J; Ju, G; Wang, Y-Z

    2015-12-17

    Microglia/macrophages play a crucial role in inflammation after spinal cord injury (SCI). Although extensive studies have been performed on the mechanisms of microglia/macrophage activation and recruitment, how microglia/macrophages are eliminated remains unclear. In the present study, we observed a high-level expression of mixed lineage kinase domain-like protein (MLKL), a key molecule in the execution of necroptosis, in microglia/macrophages after SCI in mice. In vivo PI-labeling and Necrostatin-1 treatment confirmed the necroptosis of microglia/macrophages. Interestingly, our electronic microscopic (EM) study revealed that MLKL localized not only at the membrane but also on the endoplasmic reticulum (ER) of necroptotic microglia/macrophages. Furthermore, receptor-interacting protein 3 (RIP3), another necrosome component, was also found on the ER of necroptotic microglia/macrophages. And Glucose-regulated protein 78 (GRP78), an ER stress sensor, was up-regulated in MLKL-positive microglia/macrophages after SCI, suggesting a possible link between necroptosis and ER stress. In vitro, oxygen-glucose deprivation (OGD) stress induced ER stress and necroptosis in microglia. Inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly blocked the OGD-induced necroptosis of microglia. In the end, our data showed that, GRP78 and phosphorylated MLKL were co-expressed by the microglia/macrophages in the injured human spinal cord. Taken together, these results suggested that microglia/macrophages undergo an ER-stress involved necroptosis after SCI, implying that ER stress and necroptosis could be manipulated for modulating inflammation post-SCI.

  11. Endoplasmic reticulum protein (ERp) 29 binds as strongly as protein disulfide isomerase (PDI) to bisphenol A.

    Science.gov (United States)

    Miyake, Yuka; Hashimoto, Shoko; Sasaki, Yoshie; Kudo, Tomohiro; Oguro, Ami; Imaoka, Susumu

    2014-04-21

    Bisphenol A (BPA), which is used in polycarbonate and epoxy resins, affects the development or function of the central nervous system. Previously, we isolated a BPA-binding protein from rat brain, identified it as protein disulfide isomerase (PDI), and found that BPA binds to the b' domain of PDI and inhibits its activity. There are 20 kinds of PDI family proteins in mammalian endoplasmic reticulum. The member proteins each have a different length and domain arrangement. Here we investigated the binding of BPA and T3 to ERp29, ERp57, and ERp72, which each have the b or b' domain. BPA/T3 binding of ERp57 and that of ERp72 were lower than that of PDI, and BPA did not inhibit the oxidase or reductase activity of these proteins. On the other hand, BPA and T3 bound to ERp29 as strongly as to PDI. The CD spectrum of PDI was changed in the presence of BPA in a dose-dependent manner, while that of ERp29 was not, suggesting that BPA did not affect the conformation of ERp29. We found that PDI suppresses GH expression in rat GH3 cells stimulated by thyroid hormone (T3) overexpression of PDI and that ERp57 reduced the GH level, but overexpression of ERp29 did not change GH expression. These results suggested that affinity to T3 does not involve the reduction of the T3 response. In this study, ERp29 was first identified as a BPA-binding protein but is not involved in the T3 response of GH3 cells.

  12. The SGK1 inhibitor SI113 induces autophagy, apoptosis, and endoplasmic reticulum stress in endometrial cancer cells.

    Science.gov (United States)

    Conza, Domenico; Mirra, Paola; Calì, Gaetano; Tortora, Teresa; Insabato, Luigi; Fiory, Francesca; Schenone, Silvia; Amato, Rosario; Beguinot, Francesco; Perrotti, Nicola; Ulianich, Luca

    2017-12-01

    Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here, we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B-II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase-9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real-Time RT-PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer. © 2017 Wiley Periodicals, Inc.

  13. Fructus Broussonetae extract improves cognitive function and endoplasmic reticulum stress in Alzheimer's disease models

    Institute of Scientific and Technical Information of China (English)

    Yinghong Li; Li Hu; Zhengzhi Wu; Zhiling Yu; Meiqun Cao; Kehuan Sun; Yu Jin; Anmin Wu; Andrew CJ Huang

    2011-01-01

    This study investigated the effects and possible targets of Fructus Broussonetiae extract, a traditional Chinese medicinal herb, on a model of Alzheimer's disease induced by beta-amyloid peptide 25-35 and D-galactose. The results revealed that intragastric administration of Fructus Broussonetiae significantly increased the expression of immunoglobulin-binding protein, a key factor in the endoplasmic reticulum stress-signaling pathway in rat hippocampus. In contrast, the treatment significantly decreased expression levels of PKR-like endoplasmic reticulum kinase and C/EBP homologous protein, and substantially improved learning, memory and spatial recognition dysfunction in rats. This evidence indicates that Fructus Broussonetiae extract improves spatial learning and memory abilities in rats by affecting the regulation of hippocampal endoplasmic reticulum stress and activation of the apoptosis pathway.

  14. Dark matter annihilation and decay profiles for the Reticulum II dwarf spheroidal galaxy

    CERN Document Server

    Bonnivard, V; Maurin, D; Geringer-Sameth, A; Koushiappas, S M; Walker, M G; Mateo, M; Olszewski, E; Bailey, J I

    2015-01-01

    The dwarf spheroidal galaxies (dSph) of the Milky Way are among the most attractive targets for indirect searches of dark matter. In this work, we reconstruct the dark matter annihilation (J-factor) and decay profiles for the newly discovered dSph Reticulum~II. This is done using an optimized spherical Jeans analysis of kinematic data obtained from the Michigan/Magellan Fiber System (M2FS). We find Reticulum~II to have one of the highest J-factor when compared to the other Milky Way dSphs. We have also checked the robustness of this result against several ingredients of the analysis. Unless it suffers from tidal disruption or significant inflation of its velocity dispersion from binary stars, Reticulum~II may provide a unique window on dark matter particle properties.

  15. Dark Matter Annihilation and Decay Profiles for the Reticulum II Dwarf Spheroidal Galaxy

    Science.gov (United States)

    Bonnivard, Vincent; Combet, Céline; Maurin, David; Geringer-Sameth, Alex; Koushiappas, Savvas M.; Walker, Matthew G.; Mateo, Mario; Olszewski, Edward W.; Bailey, John I., III

    2015-08-01

    The dwarf spheroidal galaxies (dSph) of the Milky Way are among the most attractive targets for indirect searches of dark matter (DM). In this work, we reconstruct the DM annihilation (J-factor) and decay profiles for the newly discovered dSph Reticulum II. Using an optimized spherical Jeans analysis of kinematic data obtained from the Michigan/Magellan Fiber System, we find Reticulum II’s J-factor to be among the largest of any Milky Way dSph. We have checked the robustness of this result against several ingredients of the analysis. Unless it suffers from tidal disruption or significant inflation of its velocity dispersion from binary stars, Reticulum II may provide a unique window on DM particle properties.

  16. Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Liping Yang; Lemeng Wu; Dongmei Wang; Ying Li; Hongliang Dou; Mark OMTso; Zhizhong Ma

    2013-01-01

    Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in-creased, which was associated with retinal ganglion celldeath in diabetic retinas. The C/ERB ho-mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in-dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu-ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

  17. Effects of pH-treated Fish Sarcoplasmic Proteins on the Functional Properties of Chicken Myofibrillar Protein Gel Mediated by Microbial Transglutaminase.

    Science.gov (United States)

    Hemung, Bung-Orn; Chin, Koo Bok

    2014-01-01

    pH adjustment would be of advantage in improving the water holding capacity of muscle proteins. The objective of this study was to evaluate the addition of fish sarcoplasmic protein (SP) solution, which was adjusted to pH 3.0 or 12.0, neutralized to pH 7.0, and lyophilized to obtain the acid- and alkaline-treated SP samples, on the functional properties of the chicken myofibrillar protein induced by microbial transglutaminase (MTG). The solubility of alkaline-treated SP was higher than that of the acid counterpart; however, those values of the two pH-treated samples were lower than that of normal SP (pproteins (MP) extracted from chicken breast, and incubated with MTG. The shear stresses of MP with acid- and alkaline-treated SP were higher than that of normal SP. The thermal stability of MP mixture reduced upon adding SP, regardless of the pH treatment. The breaking force of MP gels with acid-treated SP increased more than those of alkaline-treated SP, while normal SP showed the highest value. The MP gel lightness increased, but cooking loss reduced, with the addition of SP. Smooth microstructure of the gel surface was observed. These results indicated that adjusting the pH of SP improved the water holding capacity of chicken myofibrillar proteins induced by MTG.

  18. The endoplasmic reticulum:A dynamic and well-connected organelle

    Institute of Scientific and Technical Information of China (English)

    Chris Hawes; Petra Kiviniemi; Verena Kriechbaumer

    2015-01-01

    The endoplasmic reticulum forms the first compart-ment in a series of organel es which comprise the secretory pathway. It takes the form of an extremely dynamic and pleomorphic membrane-bounded network of tubules and cisternae which have numerous different cel ular functions. In this review, we discuss the nature of endoplasmic reticulum structure and dynamics, its relationship with closely associated organel es, and its possible function as a highway for the distribution and delivery of a diverse range of structures from metabolic complexes to viral particles.

  19. Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after feeding amino acids

    DEFF Research Database (Denmark)

    Rojek, Aleksandra; Füchtbauer, Ernst-Martin; Füchtbauer, Annette

    2013-01-01

    of the mice (24 h) induced modest dilatation of the rough endoplasmic reticulum (RER) in the periportal hepatocytes. Refeeding with standard mouse chow induced rapid generation of large RER-derived vacuoles in Aqp11 KO mice hepatocytes. Similar effects were observed following oral administration of pure...... protein or larger doses of various amino acids. The fasting/refeeding challenge is associated with increased expression of markers of ER stress Grp78 and GADD153 and decreased glutathione levels, suggesting that ER stress may play role in the development of vacuoles in the AQP11-deficient hepatocytes. NMR...

  20. Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum.

    Science.gov (United States)

    Gajate, C; Matos-da-Silva, M; Dakir, el-H; Fonteriz, R I; Alvarez, J; Mollinedo, F

    2012-05-24

    Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspase- and mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH(2)-terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 α-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER.

  1. Calcineurin is involved in cardioprotection induced by ischemic postconditioning through attenuating endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    CHEN Yi-hong; WU Xu-dong; YAO Shu-tong; SUN Seng; LIU Xiu-hua

    2011-01-01

    Background Ischemic postconditioning (I-postC) is a newly discovered and more amenable cardioprotective strategy capable of protecting the myocardium from ischemia/reperfusion (I/R) injury.Endoplasmic reticulum (ER) is a principal site for secretary protein synthesis and calcium storage.Myocardial I/R causes ER stress and emerging studies suggest that the cardioprotection has been linked to the modulation of ER stress.The aim of the present study was to determine whether cardioprotection of I-postC involves reduction in ER stress through calcineurin pathway.Methods In the in vivo model of rat myocardial I/R,myocardial infarct size was measured by triphenyltetrazolium chloride (TTC) staining and apoptosis was detected using terminal eoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay.Expression of calreticulin,C/EBP homologous protein (CHOP),caspase-12,and activation of caspase-12 in myocardium were detected by Western blotting.The activity and expression of calcineurin in myocardium were also detected.Results I-postC protected the I/R heart against apoptosis,myocardial infarction,and leakage of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB).I-postC suppressed I/R-induced ER stress,as shown by a decrease in the expression of calreticulin and CHOP,and caspase-12 activation.I-postC downregulated calcineurin activation in myocardium subjected to I/R.Conclusion I-postC protects myocardium from I/R injury by suppressing ER stress and calcineurin pathways are not associated with the I-postC-induced suppression of ER stress-related apoptosis.

  2. The yeast p5 type ATPase, spf1, regulates manganese transport into the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Yifat Cohen

    Full Text Available The endoplasmic reticulum (ER is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+ in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+ we could observe concurrent reduction in many Mn(2+-related process in the ER lumen. Conversely, cytosolic Mn(2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+-dependent neurological disorders.

  3. Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

    Science.gov (United States)

    Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

    2017-08-01

    Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese

  4. Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response

    Science.gov (United States)

    Hansen, Henning Gram; Schmidt, Jonas Damgård; Søltoft, Cecilie Lützen; Ramming, Thomas; Geertz-Hansen, Henrik Marcus; Christensen, Brian; Sørensen, Esben Skipper; Juncker, Agnieszka Sierakowska; Appenzeller-Herzog, Christian; Ellgaard, Lars

    2012-01-01

    Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell. PMID:23027870

  5. Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

    Directory of Open Access Journals (Sweden)

    Héctor M Mora-Montes

    2010-02-01

    Full Text Available Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.

  6. Structures linking the myonemes, endoplasmic reticulum, and surface membranes in the contractile ciliate Vorticella.

    Science.gov (United States)

    Allen, R D

    1973-02-01

    An electron microscope investigation of the interface between the myonemes of Vorticella convallaria and their associated endoplasmic reticulum (ER) has revealed structures of a complex morphology linking these two organelles. These structures are named "linkage complexes". Each complex contains a spindle-shaped midpiece which lies in a groove of the ER membrane. Microfilaments splay out from the tips of the midpiece and may come in contact with the inner alveolar sac membrane. Three to six raillike structures lie on each side of the midpiece and parallel it. The ER membrane appears to pass through the sides of the rails. In the lumen of the ER these rails are associated with a meshwork of filaments. A cradle of five rods lies within the groove under the midpiece. The ER membrane also passes through these rods which contact the same meshwork. In the scopular region and in the stalk the microfilaments from the midpiece form a bundle which passes into the lumen of modified basal bodies. These basal bodies are connected to the alveolar sac which, in the stalk, passes as a flattened tube along its length. The parts of the dissociated linkage complex are scattered throughout the spasmoneme of the stalk along membranes of the intraspasmonemal tubules. Thus, both stalk and body contractile bundles have linkage complexes that link their associated membrane systems to the microfibrils and, in turn, connect this membrane-microfibrillar interface to the pellicular membranes. The arrangement of the linkage complex suggests an involvement in the control of the transport of calcium ions between ER and microfibrils, and possibly the transfer of a message from the surface membranes to the sites of calcium release to trigger myonemal contraction.

  7. Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

    Directory of Open Access Journals (Sweden)

    Andrea Janz Moreira

    Full Text Available Hepatocellular carcinoma (HCC is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP ribose polymerase (PARP cleavage, and Bcl-associated X protein (Bax/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6, C/EBP-homologous protein (CHOP and immunoglobulin heavy chain-binding protein (BiP, while cyclooxygenase (COX-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.

  8. The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

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    Wu, Dan, E-mail: danw@bjmu.edu.cn [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Liu, Jing; Wu, Baiyan [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Tu, Bo; Zhu, Weiguo [Department of Biochemistry and Molecular Biology, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Luo, Jianyuan, E-mail: jluo@som.umaryland.edu [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Department of Medical and Research Technology, School of Medicine, University of Maryland, Baltimore 21201 (United States)

    2014-04-25

    Highlights: • The work reveals a protective properties of CLN3 towards TM-induced apoptosis. • CLN3 regulates expression of the GRP78 and the CHOP in response to the ER stress. • CLN3 plays a specific role in the ERS response. - Abstract: Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

  9. Polyamine Depletion Attenuates Isoproterenol-Induced Hypertrophy and Endoplasmic Reticulum Stress in Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Yan Lin

    2014-10-01

    Full Text Available Background/Aim: Polyamines (putrescine, spermidine and spermine play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO. Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO. Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC and spermidine/spermine N1-acetyltransferase (SSAT were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH and malondialdehyde (MDA level in the culture medium. In addition, DFMO (0.5 mM down regulated the expression of ODC, glucose-regulated protein 78 (GRP78, C/EBP homologous protein (CHOP, cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

  10. Vertical sleeve gastrectomy reduces blood pressure and hypothalamic endoplasmic reticulum stress in mice

    Directory of Open Access Journals (Sweden)

    Anne K. McGavigan

    2017-03-01

    Full Text Available Bariatric surgery, such as vertical sleeve gastrectomy (VSG, causes remarkable improvements in cardiometabolic health, including hypertension remission. However, the mechanisms responsible remain undefined and poorly studied. Therefore, we developed and validated the first murine model of VSG that recapitulates the blood pressure-lowering effect of VSG using gold-standard radiotelemetry technology. We used this model to investigate several potential mechanisms, including body mass, brain endoplasmic reticulum (ER stress signaling and brain inflammatory signaling, which are all critical contributors to the pathogenesis of obesity-associated hypertension. Mice fed on a high-fat diet underwent sham or VSG surgery and radiotelemeter implantation. Sham mice were fed ad libitum or were food restricted to match their body mass to VSG-operated mice to determine the role of body mass in the ability of VSG to lower blood pressure. Blood pressure was then measured in freely moving unstressed mice by radiotelemetry. VSG decreased energy intake, body mass and fat mass. Mean arterial blood pressure (MAP was reduced in VSG-operated mice compared with both sham-operated groups. VSG-induced reductions in MAP were accompanied by a body mass-independent decrease in hypothalamic ER stress, hypothalamic inflammation and sympathetic nervous system tone. Assessment of gut microbial populations revealed VSG-induced increases in the relative abundance of Gammaproteobacteria and Enterococcus, and decreases in Adlercreutzia. These results suggest that VSG reduces blood pressure, but this is only partly due to the reduction in body weight. VSG-induced reductions in blood pressure may be driven by a decrease in hypothalamic ER stress and inflammatory signaling, and shifts in gut microbial populations.

  11. Endoplasmic reticulum stress-sensing mechanism is activated in Entamoeba histolytica upon treatment with nitric oxide.

    Directory of Open Access Journals (Sweden)

    Julien Santi-Rocca

    Full Text Available The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR. The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii induction of DNA repair and redox gene expression and iii up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis.

  12. Analysis of the Endoplasmic Reticulum Subproteome in the Livers of Type 2 Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Sang-Oh Kwon

    2012-12-01

    Full Text Available Type 2 diabetes is a chronic metabolic disease that results from insulin resistance in the liver, muscle, and adipose tissue and relative insulin deficiency. The endoplasmic reticulum (ER plays a crucial role in the regulation of the cellular response to insulin. Recently, ER stress has been known to reduce the insulin sensitivity of the liver and lead to type 2 diabetes. However, detailed mechanisms of ER stress response that leads to type 2 diabetes remains unknown. To obtain a global view of ER function in type 2 diabetic liver and identify proteins that may be responsible for hepatic ER stress and insulin resistance, we performed proteomics analysis of mouse liver ER using nano UPLC-MSE. A total of 1584 proteins were identified in control C57 and type 2 diabetic db/db mice livers. Comparison of the rER and sER proteomes from normal mice showed that proteins involved in protein synthesis and metabolic process were enriched in the rER, while those associated with transport and cellular homeostasis were localized to the sER. In addition, proteins involved in protein folding and ER stress were found only in the rER. In the livers of db/db mice, however, the functions of the rER and sER were severely disrupted, including the capacity to resolve ER stress. These results provide new insight into the research on hepatic insulin resistance and type 2 diabetes and are suggestive of the potential use of the differentially expressed hepatic ER proteins as biomarkers for hepatic insulin resistance and type 2 diabetes.

  13. Stellar Kinematics and Metallicities in the Ultra-faint Dwarf Galaxy Reticulum II

    Science.gov (United States)

    Simon, J. D.; Drlica-Wagner, A.; Li, T. S.; Nord, B.; Geha, M.; Bechtol, K.; Balbinot, E.; Buckley-Geer, E.; Lin, H.; Marshall, J.; Santiago, B.; Strigari, L.; Wang, M.; Wechsler, R. H.; Yanny, B.; Abbott, T.; Bauer, A. H.; Bernstein, G. M.; Bertin, E.; Brooks, D.; Burke, D. L.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; D'Andrea, C. B.; da Costa, L. N.; DePoy, D. L.; Desai, S.; Diehl, H. T.; Dodelson, S.; Cunha, C. E.; Estrada, J.; Evrard, A. E.; Fausti Neto, A.; Fernandez, E.; Finley, D. A.; Flaugher, B.; Frieman, J.; Gaztanaga, E.; Gerdes, D.; Gruen, D.; Gruendl, R. A.; Honscheid, K.; James, D.; Kent, S.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Maia, M. A. G.; March, M.; Martini, P.; Miller, C. J.; Miquel, R.; Ogando, R.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sako, M.; Sanchez, E.; Schubnell, M.; Sevilla, I.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Tucker, D.; Vikram, V.; Walker, A. R.; Wester, W.; DES Collaboration

    2015-07-01

    We present Magellan/M2FS, Very Large Telescope/GIRAFFE, and Gemini South/GMOS spectroscopy of the newly discovered Milky Way satellite Reticulum II. Based on the spectra of 25 Ret II member stars selected from Dark Energy Survey imaging, we measure a mean heliocentric velocity of 62.8+/- 0.5 {km} {{{s}}}-1 and a velocity dispersion of 3.3+/- 0.7 {km} {{{s}}}-1. The mass-to-light ratio of Ret II within its half-light radius is 470+/- 210 {M}⊙ /{L}⊙ , demonstrating that it is a strongly dark matter-dominated system. Despite its spatial proximity to the Magellanic Clouds, the radial velocity of Ret II differs from that of the LMC and SMC by 199 and 83 {km} {{{s}}}-1, respectively, suggesting that it is not gravitationally bound to the Magellanic system. The likely member stars of Ret II span 1.3 dex in metallicity, with a dispersion of 0.28 ± 0.09 dex, and we identify several extremely metal-poor stars with {{[Fe/H]}}\\lt -3. In combination with its luminosity, size, and ellipticity, these results confirm that Ret II is an ultra-faint dwarf galaxy. With a mean metallicity of {{[Fe/H]}}=-2.65+/- 0.07, Ret II matches Segue 1 as the most metal-poor galaxy known. Although Ret II is the third-closest dwarf galaxy to the Milky Way, the line-of-sight integral of the dark matter density squared is {{log}}10(J)=18.8+/- 0.6 {GeV}{ }2 {{cm}}-5 within 0.°2, indicating that the predicted gamma-ray flux from dark matter annihilation in Ret II is lower than that of several other dwarf galaxies. Based on data obtained from the ESO Science Archive Facility under request number 157689.

  14. Hepatic Amiodarone Lipotoxicity Is Ameliorated by Genetic and Pharmacological Inhibition of Endoplasmatic Reticulum Stress.

    Science.gov (United States)

    Erez, Noam; Hubel, Einav; Avraham, Roy; Cohen, Raya; Fishman, Sigal; Bantel, Heike; Manns, Michael; Tirosh, Boaz; Zvibel, Isabel; Shibolet, Oren

    2017-10-01

    Amiodarone is a commonly used antiarrhythmic drug and can cause liver steatosis. We investigated the role of endoplasmic reticulum (ER) stress/unfolded protein response in the pathogenesis of amiodarone-induced steatosis. Amiodarone-induced liver injury was obtained by 1 intraperitoneal injection to wild-type (WT) or C/EBP homologous protein knock-out mice (Ddit3-/-). Amiodarone directly reduced intracellular ATP and Ca2+ in hepatocytes invitro, inducing ER stress and lipid accumulation. In vivo, amiodarone-driven liver damage and lipid accumulation was accompanied by activation of ER stress/unfolded protein response, as demonstrated by up-regulation of genes encoding key ER stress mediators and by phosphorylation of eIF2α. In contrast to WT mice, Ddit3-/- mice were protected from amiodarone-induced ER stress and lipid accumulation. Importantly, amiodarone-induced lipid accumulation was not mediated by de novo hepatic lipogenesis, increased adipose tissue lipolysis or increased hepatic uptake of triglycerides or free fatty acids. Rather, amiodarone strongly increased hepatic mRNA expression of lipid droplet proteins, particularly Cidea and Cidec, in WT, but less so in Ddit3-/- mice, suggesting a link between ER stress and increased triglyceride storage. Moreover, while insulin attenuated amiodarone-induced phosphorylation of hormone sensitive lipase (HSL) in WT, it did not affect pHSL in Ddit3-/-, indicating increased lipolysis and therefore reduced lipid accumulation in these mice. Finally, ER stress attenuation using 2 different pharmacological chaperones reduced lipid accumulation, accompanied by reduced mRNA expression of Cidec. In conclusion, amiodarone-induced ER stress drives liver steatosis and may be considered for therapeutic targeting. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

    Science.gov (United States)

    Luan, Qi; Jin, Lei; Jiang, Chen Chen; Tay, Kwang Hong; Lai, Fritz; Liu, Xiao Ying; Liu, Yi Lun; Guo, Su Tang; Li, Chun Ying; Yan, Xu Guang; Tseng, Hsin-Yi; Zhang, Xu Dong

    2015-01-01

    Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

  16. Protrudin regulates endoplasmic reticulum morphology and function associated with the pathogenesis of hereditary spastic paraplegia.

    Science.gov (United States)

    Hashimoto, Yutaka; Shirane, Michiko; Matsuzaki, Fumiko; Saita, Shotaro; Ohnishi, Takafumi; Nakayama, Keiichi I

    2014-05-09

    Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.

  17. Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Yoke-Chen; Wang, James D. [Rutgers University, Pharmacology and Toxicology, 170 Frelinghuysen Rd, Piscataway, NJ 08854 (United States); Svoboda, Kathy K. [Texas A and M University, Baylor College of Dentistry, Center for Craniofacial Research 3302 Gaston Ave, Dallas, Texas 75246 (United States); Casillas, Robert P. [MRIGlobal, 425 Volker Boulevard, Kansas City, MO 64110 (United States); Laskin, Jeffrey D. [UMDNJ-Robert Wood Johnson Medical School, Environmental and Occupational Medicine, 170 Frelinghuysen Rd, Piscataway, NJ 08854 (United States); Gordon, Marion K. [Rutgers University, Pharmacology and Toxicology, 170 Frelinghuysen Rd, Piscataway, NJ 08854 (United States); Gerecke, Donald R., E-mail: gerecke@eohsi.rutgers.edu [Rutgers University, Pharmacology and Toxicology, 170 Frelinghuysen Rd, Piscataway, NJ 08854 (United States)

    2013-04-15

    The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24 h to 168 h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72 h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24 h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury. - Highlights: ► We demonstrated ER stress response in the mouse ear vesicant model. ► We described the asymmetrical nature of wound repair in the MEVM. ► We identified the distribution of various ER stress markers in the MEVM.

  18. Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Christoph Mueller; Joerg Emmrich; Robert Jaster; Dagmar Braun; Stefan Liebe; Gisela Sparmann

    2006-01-01

    AIM: To investigate the biological effects of cishydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms.METHODS: The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence.Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering.RESULTS: In addition to inhibition of proliferation,incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions,followed by a loss of cell adherence. Simultaneously,we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.CONCLUSION: Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.

  19. TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways.

    Science.gov (United States)

    Lee, Dae-Hee; Sung, Ki Sa; Guo, Zong Sheng; Kwon, William Taehyung; Bartlett, David L; Oh, Sang Cheul; Kwon, Yong Tae; Lee, Yong J

    2016-05-01

    It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

  20. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

    Science.gov (United States)

    Ng, F W; Nguyen, M; Kwan, T; Branton, P E; Nicholson, D W; Cromlish, J A; Shore, G C

    1997-10-20

    We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

  1. C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions.

    Science.gov (United States)

    Casals, Cristina; Johansson, Hanna; Saenz, Alejandra; Gustafsson, Magnus; Alfonso, Carlos; Nordling, Kerstin; Johansson, Jan

    2008-02-01

    Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

  2. Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Haslund, Mads Lyhne; Robach, Paul

    2010-01-01

    As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O(2). With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-(13)C-...

  3. Aluminium induced endoplasmic reticulum stress mediated cell death in SH-SY5Y neuroblastoma cell line is independent of p53.

    Science.gov (United States)

    Mustafa Rizvi, Syed Husain; Parveen, Arshiya; Verma, Anoop K; Ahmad, Iqbal; Arshad, Md; Mahdi, Abbas Ali

    2014-01-01

    Aluminium (Al) is the third most abundant element in the earth's crust and its compounds are used in the form of house hold utensils, medicines and in antiperspirant etc. Increasing number of evidences suggest the involvement of Al+3 ions in a variety of neurodegenerative disorders including Alzheimer's disease. Here, we have attempted to investigate the role of Al in endoplasmic reticulum stress and the regulation of p53 during neuronal apoptosis using neuroblastoma cell line. We observed that Al caused oxidative stress by increasing ROS production and intracellular calcium levels together with depletion of intracellular GSH levels. We also studied modulation of key pro- and anti-apoptotic proteins and found significant alterations in the levels of Nrf2, NQO1, pAKT, p21, Bax, Bcl2, Aβ1-40 and Cyt c together with increase in endoplasmic reticulum (ER) stress related proteins like CHOP and caspase 12. However, with respect to the role of p53, we observed downregulation of its transcript as well as protein levels while analysis of its ubiquitination status revealed no significant changes. Not only did Al increase the activities of caspase 9, caspase 12 and caspase 3, but, by the use of peptide inhibitors of specific and pan-caspases, we observed significant protection against neuronal cell death upon inhibition of caspase 12, demonstrating the prominent role of endoplasmic reticulum stress generated responses in Al toxicity. Overall our findings suggest that Al induces ER stress and ROS generation which compromises the antioxidant defenses of neuronal cells thereby promoting neuronal apoptosis in p53 independent pathway.

  4. Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast

    DEFF Research Database (Denmark)

    Hou, Jin; Tang, Hongting; Liu, Zihe

    2014-01-01

    In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects...

  5. Comparing Galactic Center MSSM dark matter solutions to the Reticulum II gamma-ray data

    NARCIS (Netherlands)

    Achterberg, A.; van Beekveld, M.; Beenakker, W.; Caron, S.; Hendriks, L.

    2015-01-01

    Observations with the Fermi Large Area Telescope (LAT) indicate a possible small photon signal originating from the dwarf galaxy Reticulum II that exceeds the expected background between 2 GeV and 10 GeV . We have investigated two specific scenarios for annihilating WIMP dark matter within the pheno

  6. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy

    NARCIS (Netherlands)

    Grumati, Paolo; Morozzi, Giulio; Hölper, Soraya; Mari, Muriel; Harwardt, Marie-Lena I. E.; Yan, Riqiang; Müller, Stefan; Reggiori, Fulvio; Heilemann, Mike; Dikic, Ivan

    2017-01-01

    The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that

  7. Grab a Golgi: Laser trapping of golgi bodies reveals in vivo Interactions with the endoplasmic reticulum

    NARCIS (Netherlands)

    Sparkes, I.A.; Ketelaar, T.; Ruijter, de N.C.A.; Hawes, C.

    2009-01-01

    In many vacuolate plant cells individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi atta

  8. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

    NARCIS (Netherlands)

    Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D

    1998-01-01

    Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by mani

  9. Effects of ginger extract on smooth muscle activity of sheep reticulum and rumen

    Directory of Open Access Journals (Sweden)

    Amin Mamaghani

    2013-06-01

    Full Text Available Reticulorumen hypomotility leads to the impaired physiologic functions of the digestive tract. Prokinetic action of ginger has been demonstrated in the laboratory animals and human. The aim of this study was to evaluate the effect of hydroalcoholic extract of ginger on contraction and motility of reticulum and rumen of ruminants. Collected samples of reticulum and rumen from eight sheep were investigated in vitro. The extract at the concentration of 0.1 and 1.0 mg L-1 had no effect on any preparations. Contraction of reticulum and rumen preparations was occurred at 10.0 and 100 mg L-1 concentrations (p < 0.05. Concentration of 1000 mg L-1 caused a relaxation in preparations contracted with 10.0 and 100 mg L-1. Likewise, the concentration of 1000 mg L-1 significantly (p < 0.05 inhibited ACh-induced contraction in both tissues. Six sheep were involved in electromyographic study. Administration of 40 mg kg-1 of the extract increased the overall frequency of contractions of the reticulum and rumen at the subsequent three days with the prominent increase at the second day (p < 0.05. Results of in vitro study indicated that hydroalcoholic extract of ginger contained spasmogenic and spasmolytic constituents. The results in vivo study represented evidences that the extract may have stimulant effect on reticulorumen motility in 40 mg kg-1 concentration.

  10. Ethanol stress impairs protein folding in the endoplasmic reticulum and activates Ire1 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Miyagawa, Ken-Ichi; Ishiwata-Kimata, Yuki; Kohno, Kenji; Kimata, Yukio

    2014-01-01

    Impaired protein folding in the endoplasmic reticulum (ER) evokes the unfolded protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane protein Ire1. Here, we report that ethanol stress damages protein folding in the ER, causing activation of Ire1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.

  11. Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

    Science.gov (United States)

    Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom

    2017-03-20

    Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

  12. IRE1alpha is an endogenous substrate of endoplasmic reticulum-associated degradation

    NARCIS (Netherlands)

    Sun, Shengyi; Shi, Guojun; Sha, Haibo; Ji, Yewei; Han, Xuemei; Shu, Xin; Ma, Hongming; Takamasa, Inoue; Gao, Beixue; Bu, Pengcheng; Guber, Robert D.; Shen, Xiling; Lee, Ann H.; Iwawaki, Takao; Paton, Adrienne W.; Paton, James C.; Fang, Deyu; Tsai, Billy; Yates III, John R.; Wu, Haoquan; Kersten, Sander; Long, Qiaoming; Duhamel, Gerald E.; Simpson, Kenneth W.; Qi, Ling

    2015-01-01

    Endoplasmic reticulum-associated degradation (ERAD) represents a principle quality control (QC) mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unknown. Here we discover that IRE1alpha, the sensor of

  13. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

    DEFF Research Database (Denmark)

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida

    2016-01-01

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regula...

  14. The structure of the PERK kinase domain suggests the mechanism for its activation

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Wenjun; Li, Jingzhi [University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Ron, David [University of Cambridge, Cambridge CB2 0QQ (United Kingdom); Sha, Bingdong, E-mail: bdsha@uab.edu [University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2011-05-01

    The endoplasmic reticulum-localized transmembrane kinase PERK is one of three major ER stress transducers. The crystal structure of PERK’s kinase domain has been determined to 2.8 Å resolution. The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK’s N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK’s kinase domain has been determined to 2.8 Å resolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a ‘line-up’ model for PERK activation triggered by oligomerization of its luminal domain.

  15. Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

    Science.gov (United States)

    Chen, Wen-Juan; Xiong, Zheng-Ai; Zhang, Min; Yao, Chen-Guo; Zhao, Zhong-Yong; Hua, Yuan-Yuan; Zhou, Wei

    2013-03-01

    The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

  16. Molecular characterisation of the smooth endoplasmic reticulum Ca(2+)-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle.

    Science.gov (United States)

    Hagedorn, Monica; Weihrauch, Dirk; Towle, David W; Ziegler, Andreas

    2003-07-01

    The anterior sternal epithelial cells of the terrestrial isopod Porcellio scaber transport large amounts of calcium during the formation and resorption of intermittent calcium carbonate deposits. Recent investigations on epithelia involved in mineralisation processes suggest a role of the smooth endoplasmic reticulum Ca(2+)-ATPase (SERCA) in transcellular calcium transport. We present the first molecular characterisation of a SERCA within a crustacean mineralising epithelium. We cloned the SERCA from a cDNA library of the anterior sternal epithelium and used in situ hybridisation to compare the expression of the SERCA mRNA between three different moulting stages. The full-length SERCA cDNA has an open reading frame of 3006 nucleotides. The deduced 1002 amino-acid polypeptide has a predicted molecular mass of 109.7 kDa and 87% identity to the SERCA of Procambarus clarkii axial muscle isoform. In situ hybridisation confirmed expression within the anterior sternal epithelium and revealed an increase in SERCA mRNA abundance from the non-transporting, early premoult stage to the calcium transporting, late premoult and intramoult stage. The results support previous indications of a contribution by the smooth endoplasmic reticulum to transcellular calcium transport and suggest a transcriptional regulation of SERCA activity.

  17. The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis.

    Science.gov (United States)

    Wang, Jing; Kirby, Celeste E; Herbst, Ronald

    2002-11-29

    PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics.

  18. Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo.

    Science.gov (United States)

    Bergman, Zane J; Mclaurin, Justin D; Eritano, Anthony S; Johnson, Brittany M; Sims, Amanda Q; Riggs, Blake

    2015-01-01

    Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope.

  19. Retention of chimeric Tat2-Gap1 permease in the endoplasmic reticulum induces unfolded protein response in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mochizuki, Takahiro; Kimata, Yukio; Uemura, Satoshi; Abe, Fumiyoshi

    2015-08-01

    In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.

  20. Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

    Science.gov (United States)

    Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

    2015-01-01

    Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

  1. Sphingosine inhibits the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity.

    Science.gov (United States)

    Benaim, Gustavo; Pimentel, Adriana A; Felibertt, Pimali; Mayora, Adriana; Colman, Laura; Sojo, Felipe; Rojas, Héctor; De Sanctis, Juan B

    2016-04-29

    The increase in the intracellular Ca(2+) concentration ([Ca(2+)]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(2+)]i by inhibiting the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca(2+) pump. The results showed that addition of sphingosine produced a release of Ca(2+) from the endoplasmic reticulum followed by a Ca(2+) entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca(2+)]I in mammalian cells.

  2. CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response

    Science.gov (United States)

    Caputo, Manuela; Balzerano, Alessio; Arisi, Ivan; D’Onofrio, Mara; Brandi, Rossella; Bongiorni, Silvia; Brancorsini, Stefano; Frontini, Mattia; Proietti-De-Santis, Luca

    2017-01-01

    The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome. PMID:28253359

  3. RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase.

    Science.gov (United States)

    Fairbank, Maria; Huang, Kun; El-Husseini, Alaa; Nabi, Ivan R

    2012-07-30

    Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. An Involvement of Oxidative Stress in Endoplasmic Reticulum Stress and Its Associated Diseases

    Directory of Open Access Journals (Sweden)

    Bidur Bhandary

    2012-12-01

    Full Text Available The endoplasmic reticulum (ER is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS. Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress.

  5. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

    Directory of Open Access Journals (Sweden)

    Tomasz J. Wodzicki

    2015-01-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Benaim, Gustavo, E-mail: gbenaim@idea.gob.ve [Instituto de Estudios Avanzados (IDEA), Caracas (Venezuela, Bolivarian Republic of); Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Pimentel, Adriana A., E-mail: adriana.pimentel@ucv.ve [Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Felibertt, Pimali [Facultad de Ciencias, Universidad de Carabobo, Valencia (Venezuela, Bolivarian Republic of); Mayora, Adriana [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Colman, Laura [Instituto Pasteur de Montevideo, Montevideo (Uruguay); Sojo, Felipe [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Rojas, Héctor [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas (Venezuela, Bolivarian Republic of); De Sanctis, Juan B. [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of)

    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 that 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.

  7. The SAT Protein of Porcine Parvovirus Accelerates Viral Spreading through Induction of Irreversible Endoplasmic Reticulum Stress.

    Science.gov (United States)

    Mészáros, István; Tóth, Renáta; Olasz, Ferenc; Tijssen, Peter; Zádori, Zoltán

    2017-08-15

    The SAT protein (SATp) of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER), and SAT deletion induces the slow-spreading phenotype. The in vitro comparison of the wild-type Kresse strain and its SAT knockout (SAT(-)) mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT(-) virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences in the quantity and the localization of CHOP were detected, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of the induction of irreversible ER stress in porcine testis (PT) cell necrosis and viral egress was confirmed by treatment of infected cells by ER stress-inducing chemicals (MG132, dithiothreitol, and thapsigargin), which accelerated the egress and spreading of both the wild-type and the SAT(-) viruses. UV stress induction had no beneficial effect on PPV infection, underscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a coexpression experiment.IMPORTANCE SATp is encoded by an alternative open reading frame of the PPV genome. Earlier we showed that SATp of the attenuated PPV NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that slow spreading is a general feature of SAT(-) PPVs and is the consequence of prolonged cell integrity. PPV infection induced ER stress in infected cells regardless of the presence of SATp, as demonstrated by the morphological changes of the ER and expression of the stress response proteins Xbp1 and CHOP. However, the presence of SATp made the ER stress more severe

  8. Endoplasmic reticulum stress is involved in podocyte apoptosis induced by saturated fatty acid palmitate

    Institute of Scientific and Technical Information of China (English)

    TAO Jian-ling; WEN Yu-bing; SHI Bing-yang; ZHANG Hong; RUAN Xiong-zhong; LI Hang; LI Xue-mei; DONG Wen-ji; LI Xue-wang

    2012-01-01

    Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy.Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis,contributing to the onset of diabetes.We hypothesized that palmitate could induce podocyte apoptosis via ER stress,which initiates or aggravates proteinuria in diabetic nephropathy.Methods Podocyte apoptosis was detected by 4',6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain.The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78),indicators of ER-associated apoptosis C/EBP homologous protein (CHOP),and Bcl-2 were assayed by Western blotting and real-time PCR.GRP78 and synaptopodin were co-localized by immunofluorescence stain.Results Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours,13 hours,and 15 hours compared with 0 hour,P <0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control,P <0.001).Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP,and decreased that of Bcl-2.Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP,and decreased that of Bcl-2 compared to control (P <0.001),with the maximum concentration being 0.75 mmol/L.Palmitate 0.5 mmol/L loading for 3 hours,8 hours,and 12 hours significantly increased mRNA of GRP78 and CHOP,and decreased that of Bcl-2 compared to 0 hour (P <0.001),with the maximum effect at 3 hours.Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.Conclusion Palmitate could induce podocyte apoptosis via ER stress,suggesting podocyte apoptosis and consequent proteinuria caused

  9. Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells.

    Science.gov (United States)

    Moccia, Francesco; Berra-Romani, Roberto; Baruffi, Silvana; Spaggiari, Santina; Signorelli, Silvia; Castelli, Loretta; Magistretti, Jacopo; Taglietti, Vanni; Tanzi, Franco

    2002-01-01

    In non-excitable cells, many agonists increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing an inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the intracellular stores. Ca(2+) influx from the extracellular medium may then sustain the Ca(2+) signal. [Ca(2+)](i) recovers its resting level as a consequence of Ca(2+)-removing mechanisms, i.e. plasma-membrane Ca(2+)-ATPase (PMCA) pump, Na(+)/Ca(2+) exchanger (NCX) and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca(2+) transients, the Ca(2+) concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435-442]. It was therefore hypothesized that, in such a situation, intracellular Ca(2+) is not only picked up by the SERCA pump, but is also newly released through IP(3)-sensitive Ca(2+) channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca(2+) transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca(2+). We showed further that: (i) in order to avoid a rapid removal of Ca(2+) by the SERCA pump, continuous IP(3) production appears to be required throughout all of the decay phase of the Ca(2+) transient; and (ii) Ca(2+) picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435-442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the

  10. The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum.

    Science.gov (United States)

    Truchan, Hilary K; Cockburn, Chelsea L; Hebert, Kathryn S; Magunda, Forgivemore; Noh, Susan M; Carlyon, Jason A

    2016-01-01

    The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly

  11. Prolonged endoplasmic reticulum stress alters placental morphology and causes low birth weight

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, Takashige, E-mail: tkawakami@ph.bunri-u.ac.jp; Yoshimi, Masaki; Kadota, Yoshito; Inoue, Masahisa; Sato, Masao; Suzuki, Shinya

    2014-03-01

    The role of endoplasmic reticulum (ER) stress in pregnancy remains largely unknown. Pregnant mice were subcutaneously administered tunicamycin (Tun), an ER stressor, as a single dose [0, 50, and 100 μg Tun/kg/body weight (BW)] on gestation days (GDs) 8.5, 12.5, and 15.5. A high incidence (75%) of preterm delivery was observed only in the group treated with Tun 100 μg/kg BW at GD 15.5, indicating that pregnant mice during late gestation are more susceptible to ER stress on preterm delivery. We further examined whether prolonged in utero exposure to ER stress affects fetal development. Pregnant mice were subcutaneously administered a dose of 0, 20, 40, and 60 μg Tun/kg from GD 12.5 to 16.5. Tun treatment decreased the placental and fetal weights in a dose-dependent manner. Histological evaluation showed the formation of a cluster of spongiotrophoblast cells in the labyrinth zone of the placenta of Tun-treated mice. The glycogen content of the fetal liver and placenta from Tun-treated mice was lower than that from control mice. Tun treatment decreased mRNA expression of Slc2a1/glucose transporter 1 (GLUT1), which is a major transporter for glucose, but increased placental mRNA levels of Slc2a3/GLUT3. Moreover, maternal exposure to Tun resulted in a decrease in vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, and placental growth factor. These results suggest that excessive and exogenous ER stress may induce functional abnormalities in the placenta, at least in part, with altered GLUT and vascular-related gene expression, resulting in low infant birth weight. - Highlights: • Maternal exposure to excessive ER stress induced preterm birth and IUGR. • Prolonged excessive ER stress altered the formation of the placental labyrinth. • ER stress decreased GLUT1 mRNA expression in the placenta, but increased GLUT3. • ER stress-induced IUGR causes decreased glycogen and altered glucose transport.

  12. Cadmium-induced teratogenicity: Association with ROS-mediated endoplasmic reticulum stress in placenta

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhen; Wang, Hua; Xu, Zhong Mei; Ji, Yan-Li; Chen, Yuan-Hua; Zhang, Zhi-Hui; Zhang, Cheng; Meng, Xiu-Hong; Zhao, Mei; Xu, De-Xiang, E-mail: xudex@126.com

    2012-03-01

    The placenta is essential for sustaining the growth of the fetus. An increased endoplasmic reticulum (ER) stress has been associated with the impaired placental and fetal development. Cadmium (Cd) is a potent teratogen that caused fetal malformation and growth restriction. The present study investigated the effects of maternal Cd exposure on placental and fetal development. The pregnant mice were intraperitoneally injected with CdCl{sub 2} (4.5 mg/kg) on gestational day 9. As expected, maternal Cd exposure during early limb development significantly increased the incidences of forelimb ectrodactyly in fetuses. An obvious impairment in the labyrinth, a highly developed tissue of blood vessels, was observed in placenta of mice treated with CdCl{sub 2}. In addition, maternal Cd exposure markedly repressed cell proliferation and increased apoptosis in placenta. An additional experiment showed that maternal Cd exposure significantly upregulated the expression of GRP78, an ER chaperone. Moreover, maternal Cd exposure induced the phosphorylation of placental eIF2α, a downstream molecule of PERK signaling. In addition, maternal Cd exposure significantly increased the level of placental CHOP, another target of PERK signaling, indicating that the unfolded protein response (UPR) signaling was activated in placenta of mice treated with CdCl{sub 2}. Interestingly, alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, significantly alleviated Cd-induced placental ER stress and UPR. Taken together, these results suggest that reactive oxygen species (ROS)-mediated ER stress might be involved in Cd-induced impairment on placental and fetal development. Antioxidants may be used as pharmacological agents to protect against Cd-induced fetal malformation and growth restriction. -- Highlights: ► Cd induces fetal malformation and growth restriction. ► Cd induced placental ER stress and UPR. ► PBN alleviates Cd-induced ER stress and UPR in placenta. ► ROS-mediated ER

  13. Mechanical strain downregulates C/EBPβ in MSC and decreases endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Maya Styner

    Full Text Available Exercise prevents marrow mesenchymal stem cell (MSC adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPARγ expression and adipogenesis in MSC. We considered whether C/EBPβ might be mechanoresponsive as it is upstream to PPARγ, and also is known to upregulate endoplasmic reticulum (ER stress. MSC (C3H10T1/2 pluripotent cells as well as mouse marrow-derived MSC were cultured in adipogenic media and a daily mechanical strain regimen was applied. We demonstrate herein that mechanical strain represses C/EBPβ mRNA (0.6-fold ±0.07, p<0.05 and protein (0.4-fold ±0.1, p<0.01 in MSC. SiRNA silencing of β-catenin prevented mechanical repression of C/EBPβ. C/EBPβ overexpression did not override strain's inhibition of adipogenesis, which suggests that mechanical control of C/EBPβ is not the primary site at which adipogenesis is regulated. Mechanical inhibition of C/EBPβ, however, might be critical for further processes that regulate MSC health. Indeed, overexpression of C/EBPβ in MSC induced ER stress evidenced by a dose-dependent increase in the pro-apoptotic CHOP (protein 4-fold ±0.5, p<0.05 and a threshold reduction in the chaperone BiP (protein 0.6-fold ±0.1, p = 0.2; mRNA 0.3-fold ±0.1, p<0.01. ChIP-seq demonstrated a significant association between C/EBPβ and both CHOP and BiP genes. The strain regimen, in addition to decreasing C/EBPβ mRNA (0.5-fold ±0.09, p<0.05, expanded ER capacity as measured by an increase in BiP mRNA (2-fold ±0.2, p<0.05 and protein. Finally, ER stress induced by tunicamycin was ameliorated by mechanical strain as demonstrated by decreased C/EBPβ, increased BiP and decreased CHOP protein expression. Thus, C/EBPβ is a mechanically responsive transcription factor and its repression should counter increases in marrow fat as well as improve skeletal resistance to ER stress.

  14. Suggestibility and negative priming: two replication studies.

    Science.gov (United States)

    David, Daniel; Brown, Richard J

    2002-07-01

    Research suggests that inhibiting the effect of irrelevant stimuli on subsequent thought and action (cognitive inhibition) may be an important component of suggestibility. Two small correlation studies were conducted to address the relationship between different aspects of suggestibility and individual differences in cognitive inhibition, operationalized as the degree of negative priming generated by to-be-ignored stimuli in a semantic categorization task. The first study found significant positive correlations between negative priming, hypnotic suggestibility, and creative imagination; a significant negative correlation was obtained between negative priming and interrogative suggestibility, demonstrating the discriminant validity of the study results. The second study replicated the correlation between negative priming and hypnotic suggestibility, using a different suggestibility measurement procedure that assessed subjective experience and hypnotic involuntariness as well as objective responses to suggestions. These studies support the notion that the ability to engage in cognitive inhibition may be an important component of hypnotic responsivity and maybe of other forms of suggestibility.

  15. The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

    Directory of Open Access Journals (Sweden)

    A.J. Parodi

    1998-05-01

    Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

  16. Endoplasmic Reticulum Stress and the Related Signaling Networks in Severe Asthma

    OpenAIRE

    2014-01-01

    The endoplasmic reticulum (ER) is a specialized organelle that plays a central role in biosynthesis, correct protein folding, and posttranslational modifications of secretory and membrane proteins. Loss of homeostasis in ER functions triggers the ER stress response, resulting in activation of unfolded protein response (UPR), a hallmark of many inflammatory diseases. These pathways have been reported as critical players in the pathogenesis of various pulmonary disorders, including pulmonary fi...

  17. Six new chrysophycean stomatocysts ornamented with reticulum from the Great Xing'an Mountains, China

    Institute of Scientific and Technical Information of China (English)

    PANG Wanting; WANG Youfang; WANG Quanxi

    2012-01-01

    Six new chrysophycean stomatocysts ornamented with reticulum were illustrated based on SEM observation.They were described following the guidelines of the International Statospore Working Group (ISWG).All samples were collected from the Great Xing'an Mountains,China.Their taxonomic characteristics and habitats were described to provide new information on the biodiversity of chrysophycean stomatocysts.As is common with many morphotypes,their biological affinities remain unknown.

  18. Endoplasmic reticulum stress is induced in the human placenta during labour

    OpenAIRE

    Veerbeek, J.H.W.; Tissot Van Patot, M.C.; Burton, G.J.; Yung, H.W.

    2015-01-01

    This is the final published version. It originally appeared online at http://www.sciencedirect.com/science/article/pii/S0143400414008340#. Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for...

  19. Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

    OpenAIRE

    Sandvig, K.; Garred, O; van Deurs, B.

    1996-01-01

    Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. ...

  20. The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease

    Science.gov (United States)

    2016-07-01

    TECHNOLOGY? These studies identify a new target for the treatment of diabetic nephropathy . Studies with the NFAT inhibitor 11R- VIVIT will provide further...cells. In Aim 2, we will determine the role of CRT in mouse models of diabetic nephropathy . In year 2, we developed stably transduced HK-2 cells using...by western blot for fibronectin. 15. SUBJECT TERMS Diabetic nephropathy , calreticulin, TGF-beta, endoplasmic reticulum stress, fibrosis 16. SECURITY

  1. Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress

    Science.gov (United States)

    Li, Shengcun; Zhang, Lulu; Ni, Rui; Cao, Ting; Zheng, Dong; Xiong, Sidong; Greer, Peter A.; Fan, Guo-Chang; Peng, Tianqing

    2016-01-01

    Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4 weeks) were fed a high fat diet (HFD) or normal diet for 20 weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity. PMID:27523632

  2. Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Jian-wen Yang; Zhi-ping Hu

    2015-01-01

    Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cere-bral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the speciifc inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats in-tragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental ifndings indi-cate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.

  3. Evidence for Gamma-ray Emission from the Newly Discovered Dwarf Galaxy Reticulum 2

    CERN Document Server

    Geringer-Sameth, Alex; Koushiappas, Savvas M; Koposov, Sergey E; Belokurov, Vasily; Torrealba, Gabriel; Evans, N Wyn

    2015-01-01

    We present a search for gamma-ray emission from the direction of the newly discovered dwarf galaxy Reticulum 2. Using Fermi-LAT data, we detect a signal that exceeds expected backgrounds between ~2-10 GeV and is consistent with annihilation of dark matter for particle masses less than a few x 10^2 GeV. Modeling the background as a Poisson process based on Fermi-LAT diffuse models, and taking into account trials factors, we detect emission with p-value less than 9.8 x 10^-5 (>3.7 sigma). An alternative, model-independent treatment of background reduces the significance, raising the p-value to 9.7 x 10^-3 (2.3 sigma). Even in this case, however, Reticulum 2 has the most significant gamma-ray signal of any known dwarf galaxy. If Reticulum 2 has a dark matter halo that is similar to those inferred for other nearby dwarfs, the signal is consistent with the s-wave relic abundance cross section for annihilation.

  4. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    Science.gov (United States)

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  5. Changes in sarcoplasmic metabolite concentrations and pH associated with the catch contraction and relaxation of the anterior byssus retractor muscle of Mytilus edulis measured by phosphorus-31 nuclear magnetic resonance.

    Science.gov (United States)

    Ishii, N; Mitsumori, F; Takahashi, K

    1991-06-01

    The sarcoplasmic concentrations of phosphorus metabolites and pH (pHin) were measured in the anterior byssus retractor muscle (ABRM) of Mytilus edulis by 31P nuclear magnetic resonance spectroscopy. During an active contraction induced by 10(-3) acetylcholine, the concentration of arginine phosphate ([Arg-P]in) decreased from the resting value of 7.47 +/- 0.26 (mean +/- SE, n = 8) to 6.67 +/- 0.29 (n = 6) mumol g-1, and that of inorganic phosphate (Pi) consistently increased from 0.84 +/- 0.06 (n = 7) to 1.61 +/- 0.12 (n = 5) mumol g-1. In the 'catch' state following the active contraction, these concentrations were close to their resting levels, indicating that the catch is an inactive state. 5-hydroxytryptamine caused a rapid relaxation of the catch, which was associated with a slight decrease in [Arg-P]in and an increase in pHin by ca 0.2 units. The sarcoplasmic concentration of ATP (mean, 1.6 mumol g-1) did not change throughout the contraction-relaxation cycle.

  6. Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1.

    Science.gov (United States)

    Han, Sumin; Liu, Yu; Chang, Amy

    2007-09-01

    Cells have a variety of strategies for dealing with misfolded proteins. Heat shock response involves transcriptional induction of chaperones to promote and/or correct folding, and also activation of the ubiquitin/proteasome system to degrade defective proteins. In the secretory pathway, it is primarily luminal misfolded or unassembled proteins that trigger the unfolded protein response which, like heat shock, induces chaperones and components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. To understand cellular response to a misfolded polytopic membrane protein of the secretory pathway, we studied Pma1-D378S, a model ERAD substrate. Expression of misfolded Pma1 induces heat shock response in the absence of increased temperature. Overexpression of HSF1, the transcription factor that mediates heat shock response, increases degradation of Pma1-D378S without temperature upshift. Nevertheless, efficient Pma1-D378S degradation occurs in an hsf1 mutant that maintains basal transcription levels but cannot mediate transcriptional activation. Thus, heat shock protein induction enhances but is not necessary for ERAD. The Ssa group of cytoplasmic Hsp70 chaperones is required for ERAD of both Pma1-D378S and another transmembrane ERAD substrate, Ste6*. In the absence of Ssa chaperones, ubiquitination of both substrates is impaired, resulting in stabilization. We suggest a role for Hsp70 cytoplasmic chaperones in recognition by the endoplasmic reticulum-associated ubiquitination machinery.

  7. Sending Hidden Data via Google Suggest

    CERN Document Server

    Bialczak, Piotr; Szczypiorski, Krzysztof

    2011-01-01

    Google Suggest is a service incorporated within Google Web Search which was created to help user find the right search phrase by proposing the autocompleting popular phrases while typing. The paper presents a new network steganography method called StegSuggest which utilizes suggestions generated by Google Suggest as a hidden data carrier. The detailed description of the method's idea is backed up with the analysis of the network traffic generated by the Google Suggest to prove its feasibility. The traffic analysis was also performed to discover the occurrence of two TCP options: Window Scale and Timestamp which StegSuggest uses to operate. Estimation of method steganographic bandwidth proves that it is possible to insert 100 bits of steganogram into every suggestions list sent by Google Suggest service.

  8. Reinventing suggestion systems for continuous improvement

    NARCIS (Netherlands)

    Schuring, R.W.; Luijten, Harald

    2001-01-01

    This article reports an experiment to increase the effectiveness of a suggestion system by deliberately applying principles of the kaizen and performance management. Design rules for suggestion systems are derived from these theories. The suggestion system that resulted differs from traditional

  9. Reinventing suggestion systems for continuous improvement

    NARCIS (Netherlands)

    Schuring, Roel W.; Luijten, Harald

    2001-01-01

    This article reports an experiment to increase the effectiveness of a suggestion system by deliberately applying principles of the kaizen and performance management. Design rules for suggestion systems are derived from these theories. The suggestion system that resulted differs from traditional sugg

  10. An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids

    Directory of Open Access Journals (Sweden)

    Harriet Allison

    2014-10-01

    Full Text Available African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate ‘invariant glycoproteins’ (IGPs. IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism.

  11. An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids

    Science.gov (United States)

    Allison, Harriet; O’Reilly, Amanda J.; Sternberg, Jeremy; Field, Mark C.

    2014-01-01

    African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate ‘invariant glycoproteins’ (IGPs). IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism. PMID:26167471

  12. Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes.

    Science.gov (United States)

    Buton, Xavier; Hervé, Paulette; Kubelt, Janek; Tannert, Astrid; Burger, Koert N J; Fellmann, P; Müller, Peter; Herrmann, Andreas; Seigneuret, Michel; Devaux, Philippe F

    2002-10-29

    The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.

  13. The Possible Potentiating Role of Endoplasmic Reticulum Stress Response Inhibitors in Trans-Differentiation of white to Brown Adipocytes

    Directory of Open Access Journals (Sweden)

    Ali Mohammad Sharifi

    2012-01-01

    Full Text Available The brown adipose tissue (BAT is an organ with the specialised function of intracellular fat oxidation; in other words, brown fat points to a potential natural tool by which energy expenditure is being stimulated. Obesity is a serious illness which can lead to many medical complications such as cardiovascular disorders. The BAT production, therefore, could be a promising therapeutic strategy for managing obesity. While different approaches have been examined to generate brown adipocytes from various precursor cells, no study has proposed an efficient procedure for direct trans-differentiation of white to brown adipocytes. Bone morphogenic protein (BMP-7 is a possible potential agent by which most of the main factors involved in induction of brown adipocytogenesis such as early regulators of brown fat fate, positive regulatory domain containing 16 (PRDM16 and peroxisome proliferator-activated receptor gamma (PPARγ coactivator-1 alpha (PGC-1α are stimulated, but the rate of success was not so promising. It has been documented that mature white adipocytes exert endoplasmic reticulum stress response (ESR and consequently unfolded protein response (UPR becomes activated for the purpose of ESR recovery since the ESR exceeds the capacity of UPR to overcome the imposed stress, and in turn disables the cell to manage the protein synthesis cascade including those required for BMP-7 induction of brown adipogenesis. This was performed using three main ESR sensors: PKR-like endoplasmic reticulum kinase (PERK, inositol requiring enzyme-1 (IRE-1 and activating transcription factor 6 alpha (ATF-6α resulting in attenuation of protein translation by blocking the activation of transcriptional machinery of UPR genes and the cell behaviour would also be changed towards apoptosis.It may suggest and propose the hypothesis that pretreatment of the white adipocyte with an ESR inhibitor such as salubrinal by reducing ESR and turning on the protein synthesis machinery

  14. Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.

    Science.gov (United States)

    Hermanson, David; Addo, Sadiya N; Bajer, Anna A; Marchant, Jonathan S; Das, Sonia Goutam Kumar; Srinivasan, Balasubramanian; Al-Mousa, Fawaz; Michelangeli, Francesco; Thomas, David D; Lebien, Tucker W; Xing, Chengguo

    2009-09-01

    HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

  15. Effects of Weak Magnetic Field on Ca2+ Modulation of Skeletal Muscle Sarcoplamic Reticulum%弱磁场对骨骼肌肌质网钙调控作用的研究

    Institute of Scientific and Technical Information of China (English)

    刘仁臣; 吴永刚; 程和平; 谢国秋; 陆静; 夏子奂

    2011-01-01

    目的:探讨弱磁场对提取的骨骼肌肌质网系(SR)Ca2+转运、钙泵(Ca2 - Mg2+ -ATPase)及钙释放通道(RyR)活性的影响,从分子水平和细胞信号系统的角度来解释生物电磁效应.方法:利用动态光谱法检测0.4 mT弱磁场辐照过的SR Ca2+转运、Ca2 -ATPase活性,还原型辅酶(NADH)的氧化初速率和超氧(O2)产率,以及用同位素标记方法检测[3H] -Ryanodine与RyR的平衡结合度.结果:弱磁场辐照引起SR的Ca2+摄取功能和Ca2 -ATPase的活性明显下降,Ca2+释放和[3H] -Ryanodine平衡结合度上升,同时上调了NADH的氧化初速率和O2的产率.结论:提示0.4 mT弱磁场辐照30 min对SR Ca2 -ATPase活性有明显抑制,对RyR有一定的激活效果.%Objective: Discuss the effects of weak magnetic field on isolated sarcoplasmic reticulum (SR) Ca2+ modulation, calcium pump (Ca2+-Mg2+-ATPase) and Ca2 + release channel (Ryanodine Receptor, RyR) activity, so as to explain the mechanism of biological effects caused by electromagnetic fields in terms of molecular level and signal transduction system. Methods;The Ca + modulation and Ca + -ATPase activity, NADH oxidation initial rates and superoxide production of SR exposed to 0. 4 mT weak magnetic field ( MF) were investigated with dynamic Ca2 * dye spectrum method, [3 H ] -Ryanodine binding assay was investigated by isotope Labeling. Results: Weak MF exposure decreased SR Ca + uptake and Ca2 + -ATPase activity obviously, increased SR Ca2 + release and [3 H ] -Rryanodine binding, up-regulated the initial rates of NADH oxidation and the production of superoxide. Conclusion: It is indicated that 0. 4 mT weak magnetic field exposure for 30 min inhibited Ca2 + -ATPase activity and promoted the RyR channel function.

  16. Hypnotizability, not suggestion, influences false memory development.

    Science.gov (United States)

    Dasse, Michelle N; Elkins, Gary R; Weaver, Charles A

    2015-01-01

    Hypnotizability influences the development of false memories. In Experiment 1, participants heard a positive or negative suggestion regarding hypnosis and then listened to 8 Deese-Roediger-McDermott (DRM) false memory paradigm lists in a hypnotic state. Neither hypnosis nor prehypnotic suggestion affected memory. Highly hypnotizable participants were more accurate in recall and recognition. In Experiment 2, suggestions were delivered in the form of feedback. Participants heard a positive or negative suggestion about their performance prior to either the encoding or retrieval of 8 DRM lists. Neither accurate nor false memories were affected by the suggestion. Highly hypnotizable individuals recognized fewer critical lures if they received a negative suggestion about their performance. These results highlight the unusual role of hypnotizability in the creation of false memories.

  17. Calnexin deficiency and endoplasmic reticulum stress-induced apoptosis.

    Science.gov (United States)

    Zuppini, Anna; Groenendyk, Jody; Cormack, Lori A; Shore, Gordon; Opas, Michal; Bleackley, R Chris; Michalak, Marek

    2002-02-26

    In this study, we used calnexin-deficient cells to investigate the role of this protein in ER stress-induced apoptosis. We found that calnexin-deficient cells are relatively resistant to ER stress-induced apoptosis. However, caspase 3 and 8 cleavage and cytochrome c release were unchanged in these cells, indicating that ER to mitochondria "communication" during apoptotic stimulation is not affected in the absence of calnexin. The Bcl-2:Bax ratio was also not significantly changed in calnexin-deficient cells regardless of whether the ER stress was induced with thapsigargin or not. Ca(2+) homeostasis and ER morphology were unaffected by the lack of calnexin, but ER stress-induced Bap31 cleavage was significantly inhibited. Immunoprecipitation experiments revealed that Bap31 forms complexes with calnexin, which may play a role in apoptosis. The results suggest that calnexin may not play a role in the initiation of the ER stress but that the protein has an effect on later apoptotic events via its influence on Bap31 function.

  18. Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes

    Directory of Open Access Journals (Sweden)

    I. Golic

    2014-09-01

    Full Text Available Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1 and mitofusin 2 (MFN2 were increased, and mitochondrial fission as dynamin related protein 1 (DRP1 was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER. The level of uncoupling protein-1 (UCP1 was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes

  19. Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

    Directory of Open Access Journals (Sweden)

    Long-xing Xue

    2017-01-01

    Full Text Available Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum (ER stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein (CHOP and cleaved-caspase-3] and biomarkers of autophagy (Beclin-1 and light chain 3, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1 (IRE1, tumor necrosis factor receptor-associated factor 2 (TRAF2, apoptosis signal-regulating kinase 1 (ASK1, c-Jun N-terminal kinase (JNK and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

  20. Manganese induces endoplasmic reticulum (ER) stress and activates multiple caspases in nigral dopaminergic neuronal cells, SN4741.

    Science.gov (United States)

    Chun, H S; Lee, H; Son, J H

    2001-12-04

    Chronic exposure to manganese causes Parkinson's disease (PD)-like clinical symptoms (Neurotoxicology 5 (1984) 13; Arch. Neurol. 46 (1989) 1104; Neurology 56 (2001) 4). Occupational exposure to manganese is proposed as a risk factor in specific cases of idiopathic PD (Neurology 56 (2001) 8). We have investigated the mechanism of manganese neurotoxicity in nigral dopaminergic (DA) neurons using the DA cell line, SN4741 (J. Neurosci. 19 (1999) 10). Manganese treatment elicited endoplasmic reticulum (ER) stress responses, such as an increased level of the ER chaperone BiP, and simultaneously activated the ER resident caspase-12. Peak activation of other major initiator caspases-like activities, such as caspase-1, -8 and -9, ensued, resulting in activation of caspase-3-like activity during manganese-induced DA cell death. The neurotoxic cell death induced by manganese was significantly reduced in the Bcl-2-overexpressing DA cell lines. Our findings suggest that manganese-induced neurotoxicity is mediated in part by ER stress and considerably ameliorated by Bcl-2 overexpression in DA cells.