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Sample records for cytosolic calcium oscillations

  1. Circadian oscillations of cytosolic and chloroplastic free calcium in plants

    NASA Technical Reports Server (NTRS)

    Johnson, C. H.; Knight, M. R.; Kondo, T.; Masson, P.; Sedbrook, J.; Haley, A.; Trewavas, A.

    1995-01-01

    Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

  2. Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells

    PubMed Central

    de Menorval, Marie-Amelie; Andre, Franck M.; Silve, Aude; Dalmay, Claire; Français, Olivier; Le Pioufle, Bruno; Mir, Lluis M.

    2016-01-01

    Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate. PMID:27561994

  3. Fine tuning of cytosolic Ca 2+ oscillations

    PubMed Central

    Dupont, Geneviève; Combettes, Laurent

    2016-01-01

    Ca 2+ oscillations, a widespread mode of cell signaling, were reported in non-excitable cells for the first time more than 25 years ago. Their fundamental mechanism, based on the periodic Ca 2+ exchange between the endoplasmic reticulum and the cytoplasm, has been well characterized. However, how the kinetics of cytosolic Ca 2+ changes are related to the extent of a physiological response remains poorly understood. Here, we review data suggesting that the downstream targets of Ca 2+ are controlled not only by the frequency of Ca 2+ oscillations but also by the detailed characteristics of the oscillations, such as their duration, shape, or baseline level. Involvement of non-endoplasmic reticulum Ca 2+ stores, mainly mitochondria and the extracellular medium, participates in this fine tuning of Ca 2+ oscillations. The main characteristics of the Ca 2+ exchange fluxes with these compartments are also reviewed. PMID:27630768

  4. Sodium–calcium exchangers contribute to the regulation of cytosolic calcium levels in mouse taste cells

    PubMed Central

    Laskowski, Agnieszka I; Medler, Kathryn F

    2009-01-01

    Taste cells use multiple signalling mechanisms to generate unique calcium responses to distinct taste stimuli. Some taste stimuli activate G-protein coupled receptors (GPCRs) that cause calcium release from intracellular stores while other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). We recently demonstrated that a constitutive calcium influx exists in taste cells that is regulated by mitochondrial calcium transport and that the magnitude of this calcium influx correlates with the signalling mechanisms used by the taste cells. In this study, we used calcium imaging to determine that sodium–calcium exchangers (NCXs) also routinely contribute to the regulation of basal cytosolic calcium and that their relative role correlates with the signalling mechanisms used by the taste cells. RT-PCR analysis revealed that multiple NCXs and sodium–calcium–potassium exchangers (NCKXs) are expressed in taste cells. Thus, a dynamic relationship exists between calcium leak channels and calcium regulatory mechanisms in taste cells that functions to keep cytosolic calcium levels in the appropriate range for cell function. PMID:19581381

  5. Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana.

    PubMed

    Candeo, Alessia; Doccula, Fabrizio G; Valentini, Gianluca; Bassi, Andrea; Costa, Alex

    2017-03-31

    Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae, algal rhizoids). High resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that Light Sheet Fluorescence Microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosolic localised ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterised their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs.

  6. The Mechanical Environment Modulates Intracellular Calcium Oscillation Activities of Myofibroblasts

    PubMed Central

    Godbout, Charles; Follonier Castella, Lysianne; Smith, Eric A.; Talele, Nilesh; Chow, Melissa L.; Garonna, Adriano; Hinz, Boris

    2013-01-01

    Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair. PMID:23691248

  7. New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

    PubMed Central

    Vorndran, C; Minta, A; Poenie, M

    1995-01-01

    A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol. Images FIGURE 6 FIGURE 8 PMID:8580355

  8. Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

    PubMed

    McMahon, Shane M; Chang, Che-Wei; Jackson, Meyer B

    2016-03-01

    Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.

  9. Multiple cytosolic calcium buffers in posterior pituitary nerve terminals

    PubMed Central

    McMahon, Shane M.; Chang, Che-Wei

    2016-01-01

    Cytosolic Ca2+ buffers bind to a large fraction of Ca2+ as it enters a cell, shaping Ca2+ signals both spatially and temporally. In this way, cytosolic Ca2+ buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca2+ entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca2+ buffers in controlling pituitary Ca2+ signaling is poorly understood. Here, cytosolic Ca2+ buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca2+ indicator fluo-8 revealed stepwise increases in free Ca2+ after a series of brief depolarizing pulses in rapid succession. These Ca2+ increments grew larger as free Ca2+ rose to saturate the cytosolic buffers and reduce the availability of Ca2+ binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5–4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca2+ buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca2+ signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones. PMID:26880753

  10. Spatial Ca(2+) profiling: decrypting the universal cytosolic Ca(2+) oscillation.

    PubMed

    Samanta, Krishna; Parekh, Anant B

    2016-11-17

    Stimulation of cell-surface receptors that couple to phospholipase C to generate the second messenger inositol trisphosphate often evokes repetitive oscillations in cytosolic Ca(2+) . Signalling information is encoded in both the amplitude and frequency of the Ca(2+) spikes. Recent studies have revealed that the spatial profile of the oscillation also imparts signalling power; Ca(2+) microdomains near store-operated CRAC channels in the plasma membrane and inositol trisphosphate-gated channels in the endoplasmic reticulum both signal to distinct downstream targets. Spatial profiling therefore increases the transduction power of the universal oscillatory cytosolic Ca(2+) signal.

  11. Poliovirus protein 2BC increases cytosolic free calcium concentrations.

    PubMed Central

    Aldabe, R; Irurzun, A; Carrasco, L

    1997-01-01

    Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis. PMID:9223520

  12. Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes.

    PubMed Central

    Berlin, J R; Bassani, J W; Bers, D M

    1994-01-01

    Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires

  13. Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

    PubMed Central

    Li, Y X; Rinzel, J; Vergara, L; Stojilković, S S

    1995-01-01

    Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs. Images FIGURE 4 PMID:8519979

  14. Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in vivo.

    PubMed

    Tew, W P; Malis, C D; Howard, J E; Lehninger, A L

    1981-09-01

    Synthetic 3-phosphocitrate, an extremely potent inhibitor of calcium phosphate crystallization as determined in a nonbiological physical-chemical assay, has many similarities to a mitochondrial factor that inhibits crystallization of nondiffracting amorphous calcium phosphate. In order to determine whether phosphocitrate can prevent uptake and crystallization of calcium phosphate in mitochondria in vivo, it was administered intraperitoneally to animals given large daily doses of calcium gluconate or parathyroid hormone, a regimen that causes massive accumulation and crystallization of calcium phosphate in the mitochondria and cytosol of renal tubule cells in vivo. Administration of phosphocitrate greatly reduced the net uptake of Ca2+ by the kidneys and prevented the appearance of apatite-like crystalline structures within the mitochondrial matrix and cytosol of renal tubule cells. Phosphocitrate, which is a poor chelator of Ca2+, did not reduce the hypercalcemia induced by either agent. These in vivo observations therefore indicate that phosphocitrate acts primarily at the cellular level to prevent the extensive accumulation of calcium phosphate in kidney cells by inhibiting the mitochondrial accumulation or crystallization of calcium phosphate.

  15. Cytosolic calcium coordinates mitochondrial energy metabolism with presynaptic activity.

    PubMed

    Chouhan, Amit K; Ivannikov, Maxim V; Lu, Zhongmin; Sugimori, Mutsuyuki; Llinas, Rodolfo R; Macleod, Gregory T

    2012-01-25

    Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations that blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+ and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329±11 nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13 nM). In summary, we show that when MNs fire at endogenous rates, [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs.

  16. Cytosolic Calcium Coordinates Mitochondrial Energy Metabolism with Presynaptic Activity

    PubMed Central

    Chouhan, Amit K.; Ivannikov, Maxim V.; Lu, Zhongmin; Sugimori, Mutsuyuki; Llinas, Rodolfo R.; Macleod, Gregory T.

    2012-01-01

    Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pHm), inner membrane potential (Δψm), and NAD(P)H levels ([NAD(P)H]m) increase within seconds of nerve stimulation. The elevations of pHm, Δψm, and [NAD(P)H]m indicate an increased capacity for ATP production. Elevations in pHm were blocked by manipulations which blocked mitochondrial Ca2+ uptake, including replacement of extracellular Ca2+ with Sr2+, and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca2+ that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca2+ levels ([Ca2+]c) between MNs when each fires at its endogenous rate. The average [Ca2+]c level (329±11nM) was slightly above the average Ca2+ affinity of the mitochondria (281±13nM). In summary, we show that when MNs fire at endogenous rates [Ca2+]c is driven into a range where mitochondria rapidly acquire Ca2+. As we also show that Ca2+ stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca2+]c levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs. PMID:22279208

  17. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    SciTech Connect

    Bosche, Bert; Schäfer, Matthias; Graf, Rudolf; Härtel, Frauke V.; Schäfer, Ute; Noll, Thomas

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  18. Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors.

    PubMed

    Manzoor, Hamid; Chiltz, Annick; Madani, Siham; Vatsa, Parul; Schoefs, Benoît; Pugin, Alain; Garcia-Brugger, Angela

    2012-06-01

    Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca(2+) signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca(2+) signatures: both elicitors induced a biphasic cytosolic [Ca(2+)] elevation together with a single mitochondrial [Ca(2+)] elevation concomitant with the first cytosolic [Ca(2+)] peak. In addition, both elicitors induced a chloroplastic [Ca(2+)] elevation peaking later in comparison to cytosolic [Ca(2+)] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP(3) should play an important role in Ca(2+) signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca(2+)] variations. Our data also showed that, depending on [Ca(2+)] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles.

  19. Effect of Cyclosporin A and Angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts

    PubMed Central

    Supraja, Ajitkumar; Dinesh, Murugan Girija; Rajasekaran, Subbarayan; Balaji, Thodur Madapusi; Rao, Suresh Ranga

    2016-01-01

    Background: To evaluate the effect of Cyclosporin A (CsA) and angiotensin II (Ang II) on cytosolic calcium levels in cultured human gingival fibroblasts (HGFs). Materials and Methods: Healthy gingival samples from six volunteers were obtained, and primary HGFs were cultured. Cell viability and proliferation assay were performed to identify the ideal concentrations of CsA and Ang II. Cytosolic calcium levels in cultured gingival fibroblasts treated with CsA and Ang II were studied using colorimetric assay, confocal and fluorescence imaging. Statistical analyses were done using SPSS software and GraphPad Prism. Results: Higher levels of cytosolic levels were evident in cells treated with CsA and Ang II when compared to control group and was statistically significant (P < 0.05) in both colorimetric assay and confocal imaging. Fluorescent images of the cultured HGFs revealed the same. Conclusion: Thus calcium being a key player in major cellular functions, plays a major role in the pathogenesis of drug-induced gingival overgrowth. PMID:27857765

  20. Cytosolic free calcium-ion concentration in cleaving embryonic cells of Oryzias latipes measured with calcium-selective microelectrodes

    PubMed Central

    1985-01-01

    Calcium-selective microelectrodes were used to measure the free calcium- ion concentration ([Ca2+]i) in early-cleaving embryonic cells of the golden medaka, Oryzias latipes, a fresh water teleost fish. Embryos could be dechorionated as early as the four-cell stage using a three- step technique consisting of removal of some yolk to enlarge the perivitelline space, partial digestion of the chorion with pancreatin, and removal of the weakened chorion with forceps. Dechorionated embryos underwent cleavage at a normal rate. Intracellular cytosolic [Ca2+]i was monitored by impaling blastomeres first with a microelectrode filled with 5 M potassium acetate to measure membrane potential, and a few minutes later with a calcium-selective microelectrode. During nine rounds of cytokinesis from a total of six different embryos, cytosolic [Ca2+]i remained constant (with apparently random fluctuations of less than +/- 0.1 microM). During two successive cleavages in one embryo, however, [Ca2+]i rose transiently fourfold above the original resting level to 1.32 and 1.20 microM in synchrony with each period of cytokinesis and returned after each rise to submicromolar levels. Because a calcium-selective microelectrode can detect [Ca2+]i changes only in the immediate vicinity of its 2-microns tip, we interpreted these data to suggest that, although [Ca2+]i in most areas of the cytosol remains between 0.01 and 0.40 microM (mean of 0.14 microM), there may be small regions of the cell in which [Ca2+]i undergoes a substantial increase at the time of cleavage. Evidence also is presented to suggest that the membrane potential in these blastomeres undergoes a slow net hyperpolarization during early cleavage stages. PMID:3972904

  1. Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

    NASA Technical Reports Server (NTRS)

    Miyauchi, A.; Hruska, K. A.; Greenfield, E. M.; Duncan, R.; Alvarez, J.; Barattolo, R.; Colucci, S.; Zambonin-Zallone, A.; Teitelbaum, S. L.; Teti, A.

    1990-01-01

    The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

  2. Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures.

    PubMed Central

    Teti, A; Blair, H C; Schlesinger, P; Grano, M; Zambonin-Zallone, A; Kahn, A J; Teitelbaum, S L; Hruska, K A

    1989-01-01

    Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment. Images PMID:2547838

  3. Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels

    PubMed Central

    Gopal, Sandeep; Søgaard, Pernille; Multhaupt, Hinke A.B.; Pataki, Csilla; Okina, Elena; Xian, Xiaojie; Pedersen, Mikael E.; Stevens, Troy; Griesbeck, Oliver; Park, Pyong Woo; Pocock, Roger

    2015-01-01

    Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior. In fibroblasts, ligand interactions with heparan sulfate of syndecan-4 recruit cytoplasmic protein kinase C to target serine714 of TRPC7 with subsequent control of the cytoskeleton and the myofibroblast phenotype. In epidermal keratinocytes a syndecan–TRPC4 complex controls adhesion, adherens junction composition, and early differentiation in vivo and in vitro. In Caenorhabditis elegans, the TRPC orthologues TRP-1 and -2 genetically complement the loss of syndecan by suppressing neuronal guidance and locomotory defects related to increases in neuronal calcium levels. The widespread and conserved syndecan–TRPC axis therefore fine tunes cytoskeletal organization and cell behavior. PMID:26391658

  4. The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations.

    PubMed

    Hernández-SanMiguel, Esther; Vay, Laura; Santo-Domingo, Jaime; Lobatón, Carmen D; Moreno, Alfredo; Montero, Mayte; Alvarez, Javier

    2006-07-01

    There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.

  5. Activation of TRPV1-dependent calcium oscillation exacerbates seawater inhalation-induced acute lung injury

    PubMed Central

    LI, CONGCONG; BO, LIYAN; LIU, QINGQING; LIU, WEI; CHEN, XIANGJUN; XU, DUNQUAN; JIN, FAGUANG

    2016-01-01

    Calcium is an important second messenger and it is widely recognized that acute lung injury (ALI) is often caused by oscillations of cytosolic free Ca2+. Previous studies have indicated that the activation of transient receptor potential-vanilloid (TRPV) channels and subsequent Ca2+ entry initiates an acute calcium-dependent permeability increase during ALI. However, whether seawater exposure induces such an effect through the activation of TRPV channels remains unknown. In the current study, the effect of calcium, a component of seawater, on the inflammatory reactions that occur during seawater drowning-induced ALI, was examined. The results demonstrated that a high concentration of calcium ions in seawater increased lung tissue myeloperoxidase activity and the secretion of inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β and IL-6. Further study demonstrated that the seawater challenge elevated cytosolic Ca2+ concentration, indicated by [Ca2+]c, by inducing calcium influx from the extracellular medium via TRPV1 channels. The elevated [Ca2+c] may have resulted in the increased release of TNF-α and IL-1β via increased phosphorylation of nuclear factor-κB (NF-κB). It was concluded that a high concentration of calcium in seawater exacerbated lung injury, and TRPV1 channels were notable mediators of the calcium increase initiated by the seawater challenge. Calcium influx through TRPV1 may have led to greater phosphorylation of NF-κB and increased release of TNF-α and IL-1β. PMID:26796050

  6. Effects of antihypertensive therapy on platelet cytosolic calcium responses to low density lipoprotein cholesterol.

    PubMed

    Sowers, J R; Raman, B B; Afonso, L C; Bedford-Rice, K; Standley, P R

    1996-03-01

    This study examines the effects of antihypertensive therapy on platelet cytosolic calcium [Ca2+]i responses to low-density lipoprotein cholesterol (LDL) and vasopressin (AVP) in 15 patients (50-80 years) participating in the Hypertension Optimal Treatment International Study. All patients (diastolic blood pressure (DBP) > or = 100 mm Hg and < or = 115 mm Hg) were treated with the calcium antagonist felodipine (10 mg p.o.) with or without addition of enalapril (up to 20 mg daily as needed) to lower diastolic pressures to < 85 mm Hg. This antihypertensive therapy lowered DBP (104 +/- 0.8 to 78 +/- 1.6 mm Hg, P < 0.0001), but had no effect on basal [Ca2+]i or AVP-stimulated [Ca2+]i responses. Basal platelet [Ca2+]i following antihypertensive therapy (49 +/- 3.4 ng/ml) were not different from those prior to therapy (52 +/- 4.7 ng/ml). Additionally, [Ca2+]i responses to AVP following therapy (554 +/- 74 units) were not different from those prior to treatment (595 +/- 49 units). Following antihypertensive therapy, [Ca2+]i responses to 200 micrograms/ml of LDL were decreased fourfold (P < 0.05). These results suggest that antihypertensive therapy with a calcium channel blocker may potentially impact the atherogenic process by reducing the platelet [Ca2+]i rise, and potentially the aggregatory response, to LDL.

  7. Separation between cytosolic calcium and secretion in chromaffin cells superfused with calcium ramps.

    PubMed Central

    Michelena, P; García-Pérez, L E; Artalejo, A R; García, A G

    1993-01-01

    This paper describes experiments in which cytosolic Ca2+ concentrations ([Ca2+]i) and catecholamine release were measured in two populations of chromaffin cells stimulated with a solution enriched in K+ (100 mM). Once depolarized, external Ca2+ or Ba2+ ions were offered to cells either as a single 2.5 mM step or as a ramp that linearly increased the concentration from 0 to 2.5 mM over a 10-min period. A clear separation between the changes of the [Ca2+]i and the time course of secretion was observed. Specifically, secretion and [Ca2+]i rose in parallel when a Ca2+ step was used to reach a peak in a few seconds; however, while secretion declined to the basal level, [Ca2+]i remained elevated at a plateau of 400 nM. With a Ca2+ ramp, only a transient small peak of secretion was observed, yet the [Ca2+]i remained elevated throughout the 10-min stimulation period. The separation between secretion and [Ca2+]i was observed even when voltage-dependent Ca2+ channels were expected to remain open (mild depolarization in the presence of 1 microM Bay K 8644). By using Ba2+ steps or ramps, sustained noninactivating secretory responses were obtained. The results suggest that the rate and extent of secretion are not a simple function of the [Ca2+]i at a given time; they are compatible with the following conclusions: (i) A steep extracellular-to-cytosolic Ca2+ gradient is required to produce a sharp increase in the [Ca2+]i at exocytotic sites capable of evoking a fast but transient secretory response. (ii) As a result of Cai(2+)-dependent inactivation of Ca2+ channels, those high [Ca2+]i are possible only at early times after cell depolarization. (iii) The Cai(2+)-dependent supply of storage granules to the secretory machinery cooperates with the supply of Ca2+ through Ca2+ channels to regulate the rate and extent of secretion. PMID:8475070

  8. Cytosolic calcium and pH signaling in plants under salinity stress.

    PubMed

    Kader, Md Abdul; Lindberg, Sylvia

    2010-03-01

    Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH(cyt), either individually, or in coordination with changes in cytosolic Ca(2+) concentration, [Ca(2+)](cyt), evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca(2+) and protons. The variety and complexity of Ca(2+) and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca(2+)](cyt) concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca(2+)](cyt) and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca(2+)](cyt) and pH(cyt) at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.

  9. Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

    NASA Astrophysics Data System (ADS)

    Neef, Maren; Ecke, Margret; Hampp, Rüdiger

    2015-07-01

    In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca^{2+}_{ {cyt}}) concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8 g and μ g during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca^{2+}_{ {cyt}} level during the hypergravity phases of a parabola but a significant increase of the Ca^{2+}_{ {cyt}} concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca^{2+}_{ {cyt}} response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca^{2+}_{ {cyt}} changes entirely. We thus conclude that the significant increase in Ca^{2+}_{ {cyt}} under microgravity is largely due to extracellular Ca2+ sources.

  10. The regulation of cytosolic calcium in rat brain synaptosomes by sodium-dependent calcium efflux.

    PubMed Central

    Nachshen, D A; Sanchez-Armass, S; Weinstein, A M

    1986-01-01

    1. When pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain are incubated in a low-Na (24-36 mM) medium, they take up 45Ca in a time-dependent manner. In a medium containing 1 mM-Ca, this Na-dependent 45Ca uptake amounts to approximately 10 nmol/mg protein at 1 min, and to approximately 40 nmol/mg protein at 20 min. The Na-dependent Ca uptake is not reduced when the synaptosomes are loaded with concentrations of quin 2 as high as 2 mM. 2. The increase in 45Ca uptake is paralleled by an increase in the free cytosolic Ca concentration [Ca]i, as monitored with the fluorescent Ca indicators quin 2 or fura 2. [Ca]i increases from the value of approximately 200 to approximately 500 nM within 3-5 min, and thereafter, remains at this elevated level. 3. When synaptosomes that have been loaded with 45Ca (for 1 min, in a low-Na medium) are diluted into an Na-containing medium, there is a rapid efflux of the Ca load. After correcting for Ca that is taken up during the efflux period, calculations show that the total Ca in the synaptosomes returns to the control level within 1 min. Measurements of total chemical Ca parallel the measurements made with radiotracer Ca, and confirm that the Ca loaded into the nerve terminals during a 5 min incubation in a low-Na medium is extruded from the nerve terminals within 1 min in a normal-Na medium. 4. The efflux of Ca from the synaptosomes is paralleled by a drop of [Ca]i to its basal level, also within 1 min. 5. The mitochondrial uncoupler, carbonyl cyanide p-trifluoromethyloxy-phenyl-hydrazone (FCCP, 1 microM), has no effect on either Na-dependent Ca uptake or efflux in synaptosomes. FCCP causes a slight (100-200 nM) increase in [Ca]i in synaptosomes resuspended in either a Na or a low-Na medium. This indicates that little of the Ca that is taken up by the synaptosomes in a low-Na medium is sequestered by the mitochondria. 6. These results suggest that Na-dependent Ca efflux (probably Na-Ca exchange) plays an

  11. Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

    PubMed Central

    Kribben, A; Wieder, E D; Wetzels, J F; Yu, L; Gengaro, P E; Burke, T J; Schrier, R W

    1994-01-01

    The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases. Images PMID:8182125

  12. Calcium sensing receptors and calcium oscillations: calcium as a first messenger.

    PubMed

    Breitwieser, Gerda E

    2006-01-01

    Calcium sensing receptors (CaR) are unique among G-protein-coupled receptors (GPCRs) since both the first (extracellular) and second (intracellular) messengers are Ca(2+). CaR serves to translate small fluctuations in extracellular Ca(2+) into intracellular Ca(2+) oscillations. In many cells and tissues, CaR also acts as a coincidence detector, sensing both changes in extracellular Ca(2+) plus the presence of various allosteric activators including amino acids, polyamines, and/or peptides. CaR oscillations are uniquely shaped by the activating agonist, that is, Ca(2+) triggers sinusoidal oscillations while Ca(2+) plus phenylalanine trigger transient oscillations of lower frequency. The distinct oscillation patterns generated by Ca(2+)versus Ca(2+) plus phenylalanine are the results of activation of distinct signal transduction pathways. CaR is a member of Family C GPCRs, having a large extracellular agonist binding domain, and functioning as a disulfide-linked dimer. The CaR dimer likely can be driven to distinct active conformations by various Ca(2+) plus modulator combinations, which can drive preferential coupling to divergent signaling pathways. Such plasticity with respect to both agonist and signaling outcomes allows CaR to uniquely contribute to the physiology of organs and tissues where it is expressed. This chapter will examine the structural features of CaR, which contribute to its unique properties, the nature of CaR-induced intracellular Ca(2+) signals and the potential role(s) for CaR in development and differentiation.

  13. Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

    PubMed

    Zhang, Xuanbo; Wang, Lihong; Zhou, Anhua; Zhou, Qing; Huang, Xiaohua

    2016-04-01

    The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots.

  14. Impaired presynaptic cytosolic and mitochondrial calcium dynamics in aged compared to young adult hippocampal CA1 synapses ameliorated by calcium chelation.

    PubMed

    Tonkikh, A A; Carlen, P L

    2009-04-10

    Impaired regulation of presynaptic intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. We studied presynaptic cytosolic and mitochondrial calcium (Ca) dynamics using axonally loaded Calcium Green-AM and Rhod-2 AM fluorescence respectively in young (2-3 months) and aged (23-26 months) CA3 to CA1 Schaffer collateral excitatory synapses in hippocampal brain slices from Fisher 344 rats. After a tetanus (100 Hz, 200 ms), the presynaptic cytosolic Ca peaked at approximately 10 s in the young and approximately 12 s in the aged synapses. Administration of the membrane permeant Ca chelator, bis (O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), significantly attenuated the Ca response in the aged slices, but not in the young slices. The presynaptic mitochondrial Ca signal was much slower, peaking at approximately 90 s in both young and aged synapses, returning to baseline by 300 s. BAPTA-AM significantly attenuated the mitochondrial calcium signal only in the young synapses. Uncoupling mitochondrial respiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP) application evoked a massive intracellular cytosolic Ca increase and a significant drop of mitochondrial Ca, especially in aged slices wherein the cytosolic Ca signal disappeared after approximately 150 s of washout and the mitochondrial Ca signal disappeared after 25 s of washout. These signals were preserved in aged slices by BAPTA-AM. Five minutes of oxygen glucose deprivation (OGD) was associated with a significant increase in cytosolic Ca in both young and aged synapses, which was irreversible in the aged synapses. These responses were significantly attenuated by BAPTA-AM in both the young and aged synapses. These results support the hypothesis that increasing intracellular calcium neuronal buffering in aged rats ameliorates age-related impaired presynaptic Ca regulation.

  15. Nitric oxide triggers specific and dose-dependent cytosolic calcium transients in Arabidopsis.

    PubMed

    Aboul-Soud, Mourad A M; Aboul-Enein, Ahmed M; Loake, Gary J

    2009-03-01

    Calcium (Ca(2+)) transients have been shown to take place in response to diverse developmental and physiological cues. Also, it is involved in biotic and abiotic stress signaling. Nitric oxide (NO) is an important signaling molecule that plays a crucial role in plant growth and development, starting from germination to flowering, ripening of fruit and senescence of organs. Moreover, it plays a pivotal role in several biotic and abiotic stress signaling processes. In the present work, the ability of NO to trigger increases in cytosolic calcium concentration ([Ca(2+)](cyt)) was investigated. For this purpose, transgenic Arabidopsis seedlings constitutively expressing the luminescent Ca(2+)-sensitive protein apoaequorin (35S::APOAEQUORIN) was employed. In chemiluminescence and in vivo Ca(2+) imaging assays, the NO-donor sodium nitroprusside (SNP) triggered a strong, instantaneous, reproducible, and dose-dependent rise in [Ca(2+)](cyt). Moreover, the observed rise in [Ca(2+)](cyt) was shown to be NO-specific and not associated with decomposition products of SNP, as the NO-scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (C-PTIO) significantly blunted the observed NO-mediated spike in [Ca(2+)](cyt). Interestingly, preincubation of 35S::APOAEQUORIN Arabidopsis seedlings with the plasma membrane channel blocker lanthanum chloride resulted in partial concentration-dependent blocking of the NO-specific Ca(2+) transient. This observation indicates that, in addition to the mobilization of [Ca(2+)](cyt), as an external source in response to NO treatment, there also exists an appreciable contribution of an as yet unidentified internal pool.

  16. Cytosolic Calcium Concentration Changes in Neuronal Cells Under Clinorotation and in Parabolic Flight Missions

    NASA Astrophysics Data System (ADS)

    Hauslage, Jens; Abbrecht, Medea; Hanke, Lars; Hemmersbach, Ruth; Koch, Claudia; Hanke, Wolfgang; Kohn, Florian P. M.

    2016-12-01

    All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. To study the effects especially of microgravity on biological systems, a variety of platforms are available, from drop towers to the ISS. Due to the costs of these platforms and their limited availability, as an alternative, numerous simulators have been developed for so called "simulated" microgravity. A classical systems is a clinostat, basically rotating a sample around one axis, and by integration of the gravity vector for 360° arguing that thus the effects of gravity are depleted. Indeed, a variety of studies has shown that taking out the direction of gravity from a biological system often results in consequences similar to the exposure of the system to real microgravity. Nevertheless, the opposite has been shown, too, and as a consequence the relevance of clinostats in microgravity research is still under discussion. To get some more insight into this problem we have constructed a small fluorescence clinostat and have studied the effects of clinorotation on the cytosolic calcium concentration of neuroglioma cells. The results have been compared to experiments with identical cells in real microgravity, utilizing parabolic flight missions. Our results show that in case of a cell suspension used in a small florescence clinostat within a tube diameter of 2mm, the effects of clinorotation are comparable to those under real microgravity, both showing a significant increase in intracellular calcium concentration.

  17. Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes.

    PubMed Central

    Giovannelli, A; Grassi, F; Mattei, E; Mileo, A M; Eusebi, F; Giovanelli, A

    1991-01-01

    Electrophysiological, biochemical, and Ca2+ imaging studies of cultured mouse myotubes were used to investigate whether the neurotransmitter acetylcholine causes an increase in intracellular Ca2+ concentration ([Ca2+]i) through activation of a second messenger system. Bath applications of acetylcholine to myotubes (i) elicited a significant membrane current even in a Na(+)-free Ca2+ medium, when the current was carried mainly by calcium ions; (ii) caused a rapid and transient cytosolic accumulation of inositol 1,4,5-trisphosphate; (iii) evoked a conspicuous alpha-bungarotoxin-sensitive long-lasting [Ca2+]i enhancement even in the presence of Cd2+; and (iv) transiently increased [Ca2+]i when cells were equilibrated in a Ca(2+)-free atropine-containing medium. We propose that, in addition to opening ion channels, the nicotinic action of acetylcholine on the muscle cell membrane increases [Ca2+]i through activation of the inositol 1,4,5-trisphosphate second messenger system and mobilization of Ca2+ from intracellular stores. Images PMID:1946425

  18. Selective use of a reserved mechanism for inducing calcium oscillations.

    PubMed

    Ma, Chun Yan; Chen, Chun Ying; Cui, Zong Jie

    2004-12-01

    Concentration-dependent transformation of hormone- and neurotransmitter-induced calcium oscillation is a common phenomenon in diverse types of cells especially of the secretory type. The rodent submandibular acinar cells are an exception to this rule, which show elevated plateau increase in intracellular calcium under all stimulatory concentrations of both norepinephrine and acetylcholine. However, under depolarized state this cell type could also show a variation of periodic calcium changes. This reserved mechanism of calcium oscillation is jump-started by depolarization only with muscarinic cholinergic stimulation, but not with adrenergic stimulation. This latter effect is attributable to alpha receptor activation, not due to simultaneous activation of alpha and beta receptors, with beta receptor activation only serving to enhance the magnitude. These data suggest that this reserved mechanism for inducing calcium oscillation can be selectively used only by specific receptor-signaling pathways, and may therefore partly explain the long-known differences between secretion induced by sympathetic and parasympathetic stimulation in the submandibular gland.

  19. Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla.

    PubMed

    Vinet, Raúl; Cortés, Magdalena P; Alvarez, Rocío; Delpiano, Marco A

    2014-09-01

    We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca(2+)]i ) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca(2+)]i changes in BAMCECs the intracellular fluorescent probe, fluo-3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca(2+)]i , which was depressed by B1650 (0.1 µM), a B2-bradykinin receptor antagonist (D-Arg-[Hyp(3), Thi(5,8) , D-Phe(7)]-Bradykinin). Similarly, increase in [Ca(2+)]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1-histamine receptor antagonist. [Ca(2+)]i increase induced by both agonists was unaffected in the absence of extracellular Ca(2+) or presence of antagonists of voltage operated Ca(2+) channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca(2+)]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca(2+)]i response induced by bradykinin (3 µM), but did not affect the [Ca(2+)]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin- and H1 histamine-receptors in BAMCECs. Liberation of Ca(2+) induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca(2+) -ATPase (SERCA) thapsigargin-insensitive, histamine activates a SER containing a SERCA thapsigargin-sensitive. We suggest that the increase in [Ca(2+)]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

  20. Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

    PubMed

    Horváth, Balázs; Hegyi, Bence; Kistamás, Kornél; Váczi, Krisztina; Bányász, Tamás; Magyar, János; Szentandrássy, Norbert; Nánási, Péter P

    2015-07-01

    This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

  1. Effects of parathyroid hormone on cytosolic free calcium concentration in individual rabbit connecting tubules.

    PubMed Central

    Bourdeau, J E; Lau, K

    1989-01-01

    PTH stimulates active Ca reabsorption in isolated perfused rabbit kidney connecting tubules (CNTs). The existence of PTH-sensitive adenylate cyclase and the reproduction of increased epithelial Ca transport by dibutyryl-cAMP suggest that cAMP is the mediator. Accordingly, we studied the effects of PTH and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) on cytosolic free calcium concentration [( Ca2+]i) in individual rabbit CNTs. [Ca2+]i was estimated by continuous epifluorescence microscopy of single fura-2-loaded tubules during dual wave-length excitation. In nonperfused controls at 37 degrees C, [Ca2+]i decreased with time. In contrast to vehicle controls, synthetic bovine (1-34) PTH (0.1 nM) increased [Ca2+]i within 4 min, produced a maximal effect in 7.2 min, and sustained its effect for at least 2 min after washout. 8-Br-cAMP (1 mM) mimicked the effect of PTH, but with an earlier onset of action. To test the hypothesis that lumen Ca is the predominant source of the rise in [Ca2+]i, we studied singly perfused CNTs. In the absence of bath and lumen Ca, PTH elicited no rise in [Ca2+]i, implying that intracellular Ca stores are not the major source. In contrast, there was a rise when Ca was replenished in both media. In the continuous presence of bath Ca, lumen Ca was estimated to contribute 65% of the total rise in [Ca2+]i in response to PTH when it was first deleted and then replenished. However, when the sequence of lumen Ca manipulation was reversed, the contributions by lumen and bath Ca were found to be essentially equal. We conclude (a) at a physiologic concentration, PTH increases [Ca2+]i in rabbit CNTs, (b) 8-Br-cAMP mimics this action, implicating cAMP as a second messenger, and (c) the PTH-stimulated rise in [Ca2+]i depends importantly on both bath and tubular luminal fluid Ca. PMID:2536396

  2. Estrogen evokes a rapid effect on intracellular calcium in neurons characterized by calcium oscillations in the arcuate nucleus.

    PubMed

    Fricke, Oliver; Kow, Lee-Ming; Bogun, Magda; Pfaff, Donald W

    2007-06-01

    Rapid estrogen effects became an interesting topic to explain estrogen effects not associated with the classical nuclear pathway. The rapid estrogen effect on intracellular calcium oscillations was characterized in neurons of the arcuate nucleus. Ratiometric calcium imaging (fura-2AM) was used to measure intracellular calcium in brain slices of female Swiss Webster mice (median of age 27 days p.n.). Calcium oscillations were dependent on intracellular calcium and also on calcium influx from the extracellular space. The perfusion of slices with calcium-free solution inhibited spontaneous calcium oscillations. The metabotropic glutamate receptor agonist t-ACPD (5 microM) and low concentrated ryanodine (100 nM) induced intracellular calcium release when slices were perfused with calcium-free solution. 17beta-estradiol (10 nM) also induced intracellular calcium release in calcium-free ACSF. This effect was inhibited by the preceding administration of thapsigargin (2 microM) indicating the association of the rapid estrogen effect with intracellular calcium stores. The administration of the non-selective phospholipase C-inhibitor ET-18 (30 microM), but not U73122 (10 microM), and the inhibition of protein kinase A by H-89 (0.25 microM) suppressed the rapid estrogen effect. Analyses indicated a qualitative, but not quantitatively significant effect of 17beta-estradiol on calcium oscillations.

  3. Effects of Ca2+ channel antagonists on chromaffin cell death and cytosolic Ca2+ oscillations induced by veratridine.

    PubMed

    Maroto, R; De la Fuente, M T; Artalejo, A R; Abad, F; López, M G; García-Sancho, J; García, A G

    1994-08-03

    Exposure of bovine chromaffin cells to 30 microM veratridine for 24 h led to 70-80% cell death as reflected by phase contrast microscopy, trypan blue exclusion, lactate dehydrogenase (LDH) release and cell catecholamine contents. Na+ deprivation, Ca2+ deletion or tetrodotoxin (5 microM) prevented the veratridine-induced cell damage. Nimodipine and verapamil, but not omega-conotoxin GVIA afforded 20-30% protection. Flunarizine protected the cells by 80% and R56865 by 60%. Stimulation of fura-2-loaded single bovine chromaffin cells with 30 microM of 1,1-dimethyl-4-phenylpiperazinium (DMPP) or 59 mM K+ caused fast increases in cytosolic Ca2+ concentrations, ([Ca2+]i). The [Ca2+]i rose from 0.1 to peaks of 1.9 microM, which quickly declined to near basal levels with a t1/2 of around 30 s. In spite of sustained stimulation with these two depolarizing agents, the [Ca2+]i remained low and did not undergo oscillations. In contrast, veratridine (30 microM) caused large and frequent oscillatory changes in the [Ca2+]i which were long-lasting and did not disappear even 30 min after washing out the toxin. The [Ca2+]i oscillations were reversibly suppressed by Na+ or Ca2+ removal and by 5 microM tetrodotoxin. Selective L-type Ca2+ channel blockers (10 microM nimodipine or verapamil) or N-type Ca2+ channel blockers (1 microM omega-conotoxin GVIA) did not affect the [Ca2+]i oscillations. In contrast, flunarizine or R56865 (10 microM each) suppressed the oscillations of [Ca2+]i. The results demonstrate that bovine chromaffin cells have the necessary machinery to develop prolonged and repetitive [Ca2+]i oscillations in the presence of veratridine; however, 'physiological' depolarizing stimuli did not cause oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. The human two-pore channel 1 is modulated by cytosolic and luminal calcium

    PubMed Central

    Lagostena, Laura; Festa, Margherita; Pusch, Michael; Carpaneto, Armando

    2017-01-01

    Two-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low μM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling. PMID:28252105

  5. Apparent cytosolic calcium gradients in T-lymphocytes due to fura-2 accumulation in mitochondria.

    PubMed

    Quintana, Ariel; Hoth, Markus

    2004-08-01

    Fura-2 is the most common dye to measure cytosolic Ca2+ concentrations ([Ca2+]i). To facilitate simultaneous imaging of many cells while preserving their cytosolic environment, fura-2 is often loaded into the cytosol in its membrane-permeant ester form. It has been reported that small amounts of fura-2 accumulate in intracellular compartments, an effect that is usually neglected. We show that either focal or non-focal stimulation methods induce large [Ca2+]i gradients in T-lymphocytes during both, Ca2+ release and Ca2+ influx across the plasma membrane. Interfering with mitochondrial Ca2+ homeostasis and by labeling mitochondria with MitoTracker, we demonstrate that [Ca2+]i gradients co-localize with mitochondria and are attributable to mitochondrial fura-2 sequestration. Gradients could not be avoided by different loading protocols, compromising measurements of "real" [Ca2+]i gradients following T-cell stimulation. They were observed in human blood and lamina propria lymphocytes, Jurkat T-cells, mast cells, but not to the same extent in HEK-293 cells. Finally, we show that T-lymphocytes can be efficiently loaded with the membrane-impermeant fura-2 salt by electroporation and by osmotic lysis of pinocytic vesicles, which result in the loss of [Ca2+]i gradients. These methods are therefore suitable to study localized Ca2+ signals in large populations of T-cells while preserving their cytosolic integrity.

  6. Perturbation of cytosolic calcium by 2-aminoethoxydiphenyl borate and caffeine affects zebrafish myofibril alignment.

    PubMed

    Wu, Hsin-Ju; Fong, Tsorng-Harn; Chen, Shen-Liang; Wei, Jen-Cheng; Wang, I-Jong; Wen, Chi-Chung; Chang, Chao-Yuan; Chen, Xing-Guang; Chen, Wei-Yu; Chen, Hui-Min; Horng, Juin-Lin; Wang, Yun-Hsin; Chen, Yau-Hung

    2015-03-01

    The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.

  7. Low-frequency calcium oscillations accompany deoxyhemoglobin oscillations in rat somatosensory cortex

    PubMed Central

    Du, Congwu; Volkow, Nora D.; Koretsky, Alan P.; Pan, Yingtian

    2014-01-01

    Spontaneous low-frequency oscillations (LFOs) of blood-oxygen-level-dependent (BOLD) signals are used to map brain functional connectivity with functional MRI, but their source is not well understood. Here we used optical imaging to assess whether LFOs from vascular signals covary with oscillatory intracellular calcium (Ca2+i) and with local field potentials in the rat’s somatosensory cortex. We observed that the frequency of Ca2+i oscillations in tissue (∼0.07 Hz) was similar to the LFOs of deoxyhemoglobin (HbR) and oxyhemoglobin (HbO2) in both large blood vessels and capillaries. The HbR and HbO2 fluctuations within tissue correlated with Ca2+i oscillations with a lag time of ∼5–6 s. The Ca2+i and hemoglobin oscillations were insensitive to hypercapnia. In contrast, cerebral-blood-flow velocity (CBFv) in arteries and veins fluctuated at a higher frequency (∼0.12 Hz) and was sensitive to hypercapnia. However, in parenchymal tissue, CBFv oscillated with peaks at both ∼0.06 Hz and ∼0.12 Hz. Although the higher-frequency CBFv oscillation (∼0.12 Hz) was decreased by hypercapnia, its lower-frequency component (∼0.06 Hz) was not. The sensitivity of the higher CBFV oscillations to hypercapnia, which triggers blood vessel vasodilation, suggests its dependence on vascular effects that are distinct from the LFOs detected in HbR, HbO2, Ca2+i, and the lower-frequency tissue CBFv, which were insensitive to hypercapnia. Hemodynamic LFOs correlated both with Ca2+i and neuronal firing (local field potentials), indicating that they directly reflect neuronal activity (perhaps also glial). These findings show that HbR fluctuations (basis of BOLD oscillations) are linked to oscillatory cellular activity and detectable throughout the vascular tree (arteries, capillaries, and veins). PMID:25313035

  8. Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture.

    PubMed

    Nguyen, Hieu T H; Umemura, Kenji; Kawano, Tomonori

    2016-08-01

    Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca(2+) concentration ([Ca(2+)]c). The IAA-induced [Ca(2+)]c elevation was inhibited by Ca(2+) channel blockers and a Ca(2+) chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca(2+)]c elevation.

  9. Cyclopiazonic acid disturbs the regulation of cytosolic calcium when repetitive action potentials are evoked in Dionaea traps.

    PubMed

    Trebacz, Kazimierz; Busch, Marion B; Hejnowicz, Zygmunt; Sievers, Andreas

    1996-04-01

    Evoking of action potentials (APs) in the trap of Dionaea muscipula Ellis at intervals shorter than 20 s caused a gradual decrease in the amplitude of the APs. At longer intervals the amplitude was constant. The calcium ionophore A23187 (1 μM) caused a considerable decrease of AP amplitude. Pretreatment of a segment of the Dionaea trap with cyclopiazonic acid (CPA), which is a specific inhibitor of the Ca(2+)-ATPase in the sarcoplasmic seticulum of animal cells and in ER vesicles isolated from plant cells, only slightly affected the amplitude when APs were evoked every 10 min; however, it caused a considerable decrease in the amplitude when the stimulation was repeated every 2 min. Assuming that APs increase the concentration of cytosolic Ca(2+) and the amplitude of AP depends on the gradient of Ca(2+) across the plasma membrane, the effect of CPA on the AP amplitude indicates that CPA inhibits the sequestration of Ca(2+) in Dionaea cells.

  10. By Releasing ADP, Acanthamoeba castellanii Causes an Increase in the Cytosolic Free Calcium Concentration and Apoptosis in Wish Cells

    PubMed Central

    Mattana, A.; Tozzi, M. G.; Costa, M.; Delogu, G.; Fiori, P. L.; Cappuccinelli, P.

    2001-01-01

    The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here that Acanthamoeba castellanii trophozoites constitutively release ADP in the medium. Cell-free supernatants prepared from A. castellanii, by interaction with specific P2y2 purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca2+]i, extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P2y2 inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-living A. castellanii may have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis. PMID:11349088

  11. The control of brain mitochondrial energization by cytosolic calcium: the mitochondrial gas pedal.

    PubMed

    Gellerich, Frank Norbert; Gizatullina, Zemfira; Gainutdinov, Timur; Muth, Katharina; Seppet, Enn; Orynbayeva, Zulfiya; Vielhaber, Stefan

    2013-03-01

    This review focuses on problems of the intracellular regulation of mitochondrial function in the brain via the (i) supply of mitochondria with ADP by means of ADP shuttles and channels and (ii) the Ca(2+) control of mitochondrial substrate supply. The permeability of the mitochondrial outer membrane for adenine nucleotides is low. Therefore rate dependent concentration gradients exist between the mitochondrial intermembrane space and the cytosol. The existence of dynamic ADP gradients is an important precondition for the functioning of ADP shuttles, for example CrP-shuttle. Cr at mM concentrations instead of ADP diffuses from the cytosol through the porin pores into the intermembrane space. The CrP-shuttle isoenzymes work in different directions which requires different metabolite concentrations mainly caused by dynamic ADP compartmentation. The ADP shuttle mechanisms alone cannot explain the load dependent changes in mitochondrial energization, and a complete model of mitochondrial regulation have to account the Ca(2+) -dependent substrate supply too. According to the old paradigmatic view, Ca(2+) (cyt) taken up by the mitochondrial Ca(2+) uniporter activates dehydrogenases within the matrix. However, recently it was found that Ca(2+) (cyt) at low nM concentrations exclusively activates the state 3 respiration via aralar, the mitochondrial glutamate/aspartate carrier. At higher Ca(2+) (cyt) (> 500 nM), brain mitochondria take up Ca(2+) for activation of substrate oxidation rates. Since brain mitochondrial pyruvate oxidation is only slightly influenced by Ca(2+) (cyt) , it was proposed that the cytosolic formation of pyruvate from its precursors is tightly controlled by the Ca(2+) dependent malate/aspartate shuttle. At low (50-100 nM) Ca(2+) (cyt) the pyruvate formation is suppressed, providing a substrate limitation control in neurons. This so called "gas pedal" mechanism explains why the energy metabolism of neurons in the nucleus suprachiasmaticus could be down

  12. Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

    PubMed Central

    Hou, Yanyan; Arai, Satoshi; Takei, Yoshiaki; Murata, Atsushi; Takeoka, Shinji; Suzuki, Madoka

    2016-01-01

    Abstract Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)–biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors. PMID:27877882

  13. Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum.

    PubMed

    Hou, Yanyan; Arai, Satoshi; Takei, Yoshiaki; Murata, Atsushi; Takeoka, Shinji; Suzuki, Madoka

    2016-01-01

    Ca(2+) distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca(2+) sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca(2+) sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca(2+) sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca(2+) concentration, ER-localized N3-fura-2 successfully sensed the Ca(2+) level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca(2+) level changes with the specifically targeted Ca(2+) sensors.

  14. Mitochondrial Matrix Ca2+ Accumulation Regulates Cytosolic NAD+/NADH Metabolism, Protein Acetylation, and Sirtuin Expression

    PubMed Central

    Marcu, Raluca; Wiczer, Brian M.; Neeley, Christopher K.

    2014-01-01

    Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD+/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades. PMID:24865966

  15. Speract induces calcium oscillations in the sperm tail.

    PubMed

    Wood, Chris D; Darszon, Alberto; Whitaker, Michael

    2003-04-14

    Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

  16. Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

    PubMed Central

    Baud, L; Goetzl, E J; Koo, C H

    1987-01-01

    The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane. PMID:3477571

  17. Copper elicits an increase in cytosolic free calcium in cultured tobacco cells.

    PubMed

    Inoue, Hiroki; Kudo, Tomoko; Kamada, Hiroshi; Kimura, Makoto; Yamaguchi, Isamu; Hamamoto, Hiroshi

    2005-12-01

    At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.

  18. Evaluation of Cisplatin Neurotoxicity in Cultured Rat Dorsal Root Ganglia via Cytosolic Calcium Accumulation

    PubMed Central

    Erol, Kevser; Yiğitaslan, Semra; Ünel, Çiğdem; Kaygısız, Bilgin; Yıldırım, Engin

    2016-01-01

    Background: Calcium homeostasis is considered to be important in antineoplastic as well as in neurotoxic adverse effects of cisplatin. Aims: This study aimed to investigate the role of Ca2+ in cisplatin neurotoxicity in cultured rat dorsal root ganglia (DRG) cells. Study Design: Cell culture study. Methods: DRG cells prepared from 1-day old Sprague-Dawley rats were used to determine the role of Ca2+ in the cisplatin (10–600 μM) neurotoxicity. The cells were incubated with cisplatin plus nimodipine (1–3 μM), dizocilpine (MK-801) (1–3 μM) or thapsigargin (100–300 nM). Toxicity of cisplatinon DRG cells was determined by the MTT assay. Results: The neurotoxicity of cisplatin was significant when used in high concentrations (100–600 μM). Nimodipine (1 μM) but not MK-801 or thapsigargin prevented the neurotoxic effects of 200 μM of cisplatin. Conclusion: Voltage-dependent calcium channels may play a role in cisplatin neurotoxicity. PMID:27403382

  19. Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress.

    PubMed Central

    McAinsh, M. R.; Clayton, H.; Mansfield, T. A.; Hetherington, A. M.

    1996-01-01

    We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture. PMID:12226345

  20. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors.

    PubMed

    Nakata, Masanori; Yada, Toshihiko

    2008-01-10

    Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.

  1. Noise induced calcium oscillations in a cell exposed to electromagnetic fields.

    PubMed

    Zhang, Yuhong; Zhao, Yongli; Chen, Yafei; Yuan, Changqing; Zhan, Yong

    2015-01-01

    The effects of noise on the calcium oscillations in a cell exposed to electromagnetic fields are described by a dynamic model. Noise is a very important factor to be considered in the dynamic research on the calcium oscillations in a cell exposed to electromagnetic fields. Some meaningful results have been obtained here based on the discussion. The results show that the pattern of intracellular calcium oscillations exposure to electromagnetic fields can be influenced by noise. Furthermore, the intracellular calcium oscillations exposure to electromagnetic fields can also be induced by noise. And the work has also studied the relationships between the voltage sensitive calcium channel's open probability and electromagnetic field. The result can provide new insights into constructive roles and potential applications of selecting appropriate electromagnetic field frequency during the research of biological effect of electromagnetic field.

  2. Fura-2 measurement of cytosolic calcium in HgCl/sub 2/-treated rabbit renal turbular cells

    SciTech Connect

    Trump, B.F.; Smith, M.W.

    1986-05-01

    This abstract reports the effect of HgCl/sub 2/ on cytosolic ionized calcium (Ca/sup 2 +/)/sub c/, measured by the fluorescent chelator Fura-2, in trypsinized rabbit renal tubular cells at 37/sup 0/C in Hanks salt solution, pH 7.2, containing 1.37 mM CaCl/sub 2/. Viability measured fluorometrically with propidium iodide correlated well with that determined using trypan blue. HgCl/sub 2/ (1-10 ..mu..M) induced rapid and dose-dependent increases up to 5-fold normal (Ca/sup 2 +/)/sub c/. After 1-3 min the rate of increase slowed or stopped. At higher doses of HgCl/sub 2/ (20-100 ..mu..M) an unexpected pattern of (Ca/sup 2 +/)/sub c/ changes occurred. After an initial 5-6-fold increase by 1 min, (Ca/sup 2 +/)/sub c/ decreased in the next 2-3 min to 2-3-fold normal levels. This change was followed by a second increase of (Ca/sup 2 +/)/sub c/ at a much slower rate which did appear to be dose-related. Calcium channel blockers and calmodulin inhibitors had little or no effect. Inhibitors of mitochondrial function, antimycin and 2,4-dinitrophenol, interfered with the fluorescent assay; KCN totally inhibited HgCl/sub 2/-induced (Ca/sup 2 +/)/sub c/ changes while hypoxia had no apparent effect. The -SH group binding compound N-ethyl maleimide increased (Ca/sup 2 +/)/sub c/ 4-5 fold; addition of 25 ..mu..M Hg caused faster peaking and recovery of (Ca/sup 2 +/)/sub c/. The mechanism of Ca/sup 2 +/ buffering triggered by higher HgCl/sub 2/ concentrations is as yet unknown.

  3. A diffusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells.

    PubMed

    Navazio, Lorella; Moscatiello, Roberto; Genre, Andrea; Novero, Mara; Baldan, Barbara; Bonfante, Paola; Mariani, Paola

    2007-06-01

    The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca(2+) indicator aequorin to detect intracellular Ca(2+) changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca(2+) were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca(2+)-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca(2+) transient were constitutively released in the medium, and the induced Ca(2+) signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca(2+) response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

  4. Hormone-induced calcium oscillations depend on cross-coupling with inositol 1,4,5-trisphosphate oscillations.

    PubMed

    Gaspers, Lawrence D; Bartlett, Paula J; Politi, Antonio; Burnett, Paul; Metzger, Walson; Johnston, Jane; Joseph, Suresh K; Höfer, Thomas; Thomas, Andrew P

    2014-11-20

    Receptor-mediated oscillations in cytosolic Ca(2+) concentration ([Ca(2+)]i) could originate either directly from an autonomous Ca(2+) feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca(2+) feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca(2+) oscillations or could just be a secondary response to the [Ca(2+)]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca(2+)]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca(2+)]i rise and the speed of Ca(2+) wave propagation and, at high levels, abolished [Ca(2+)]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca(2+)]i oscillations and waves.

  5. Magnesium Sensitizes Slow Vacuolar Channels to Physiological Cytosolic Calcium and Inhibits Fast Vacuolar Channels in Fava Bean Guard Cell Vacuoles.

    PubMed

    Pei; Ward; Schroeder

    1999-11-01

    Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 µM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening.

  6. Ryanodine receptors are involved in nuclear calcium oscillation in primary pancreatic {beta}-cells

    SciTech Connect

    Zheng, Ji; Chen, Zheng; Yin, Wenxuan; Miao, Lin; Zhou, Zhansong; Ji, Guangju

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Black-Right-Pointing-Pointer We showed that the pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. Black-Right-Pointing-Pointer Our results demonstrate that ryanodine-sensitive Ca{sup 2+} stores exist and have function in the pancreatic {beta}-cell nucleus. -- Abstract: Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca{sup 2+} oscillation in pancreatic {beta}-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca{sup 2+} oscillation we designed and conducted experiments in intact primary pancreatic {beta}-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca{sup 2+} indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca{sup 2+} oscillation. The pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. The reduction of Ca{sup 2+} oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca{sup 2+} amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic {beta}-cells.

  7. Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration

    PubMed Central

    1991-01-01

    Transient increases in cytosolic free calcium concentration, [Ca2+]i, appear to be required for the migration of human neutrophils on poly-D- lysine-coated glass in the presence of dilute serum (Marks, P. W., and F. R. Maxfield. 1990. J. Cell Biol. 110:43-52). In contrast, no requirement for [Ca2+]i transients exists when neutrophils migrate on albumin-coated glass in the absence of serum. To determine the mechanism that necessitates [Ca2+]i transients on poly-D-lysine in the presence of serum, migration was examined on substrates consisting of purified adhesive glycoproteins. In the absence of external Ca2+, a treatment which causes the cessation of [Ca2+]i transients, migration on fibronectin (fn) and vitronectin (vn) was significantly inhibited. Migration was also inhibited in Ca2(+)-buffered cells on these substrates, indicating that this effect was the result of an alteration of [Ca2+]i. In the absence of external Ca2+, the inhibition of migration on fn or vn was more pronounced when soluble fn or vn was added to cells migrating on these substrates. This effect of soluble adhesive glycoprotein was specific: in the absence of external Ca2+, soluble fn did not affect the migration of cells on vn, and soluble vn did not affect the migration on fn. No additional inhibition of migration was observed in Ca2(+)-buffered cells with the addition of soluble adhesive glycoprotein. These data indicate that [Ca2+]i transients are involved in continued migration of human neutrophils on fn or vn, proteins which are part of the extracellular matrix that neutrophils encounter in vivo. PMID:1702443

  8. Spatiotemporal properties of high-speed calcium oscillations in the pedunculopontine nucleus

    PubMed Central

    Hyde, James; Kezunovic, Nebojsa; Urbano, Francisco J.

    2013-01-01

    The pedunculopontine nucleus (PPN) is a component of the reticular activating system (RAS), and is involved in the activated states of waking and rapid eye movement (REM) sleep. Gamma oscillations (approximately 30–80 Hz) are evident in all PPN neurons and are mediated by high-threshold voltage-dependent N- and P/Q-type calcium channels. We tested the hypothesis that high-speed calcium imaging would reveal calcium-mediated oscillations in dendritic compartments in synchrony with patch-clamp recorded oscillations during depolarizing current ramps. Patch-clamped 8- to 16-day-old rat PPN neurons (n = 67 out of 121) were filled with Fura 2, Bis Fura, or OGB1/CHR. This study also characterized a novel ratiometric technique using Oregon Green BAPTA-1 (OGB1) with coinjections of a new long-stokes-shift dye, Chromeo 494 (CHR). Fluorescent calcium transients were blocked with the nonspecific calcium channel blocker cadmium, or by the combination of ω-agatoxin-IVA, a specific P/Q-type calcium channel blocker, and ω-conotoxin-GVIA, a specific N-type calcium channel blocker. The calcium transients were evident in different dendrites (suggesting channels are present throughout the dendritic tree) along the sampled length without interruption (suggesting channels are evenly distributed), and appeared to represent a summation of oscillations present in the soma. We confirm that PPN calcium channel-mediated oscillations are due to P/Q- and N-type channels, and reveal that these channels are distributed along the dendrites of PPN cells. PMID:23990242

  9. Calcium spikes, waves and oscillations in a large, patterned epithelial tissue.

    PubMed

    Balaji, Ramya; Bielmeier, Christina; Harz, Hartmann; Bates, Jack; Stadler, Cornelia; Hildebrand, Alexander; Classen, Anne-Kathrin

    2017-02-20

    While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet.

  10. Calcium spikes, waves and oscillations in a large, patterned epithelial tissue

    PubMed Central

    Balaji, Ramya; Bielmeier, Christina; Harz, Hartmann; Bates, Jack; Stadler, Cornelia; Hildebrand, Alexander; Classen, Anne-Kathrin

    2017-01-01

    While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet. PMID:28218282

  11. Calcium oscillations in interstitial cells of the rabbit urethra

    PubMed Central

    Johnston, L; Sergeant, GP; Hollywood, MA; Thornbury, KD; McHale, NG

    2005-01-01

    Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 μm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release. PMID:15760947

  12. Calcium oscillations in interstitial cells of the rabbit urethra.

    PubMed

    Johnston, L; Sergeant, G P; Hollywood, M A; Thornbury, K D; McHale, N G

    2005-06-01

    Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with D-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 microm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release.

  13. A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

    PubMed Central

    Giri, Lopamudra; Patel, Anilkumar K.; Karunarathne, W.K. Ajith; Kalyanaraman, Vani; Venkatesh, K.V.; Gautam, N.

    2014-01-01

    G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of Gβγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the Gβγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential Gβγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component. PMID:24988358

  14. Calcium dynamics during NMDA-induced membrane potential oscillations in lamprey spinal neurons--contribution of L-type calcium channels (CaV1.3).

    PubMed

    Wang, Di; Grillner, Sten; Wallén, Peter

    2013-05-15

      NMDA receptor-dependent, intrinsic membrane potential oscillations are an important element in the operation of the lamprey locomotor network. They involve a cyclic influx of calcium, leading to an activation of calcium-activated potassium (KCa) channels that in turn contributes to the termination of the depolarized plateau and membrane repolarization. In this study, we have investigated the calcium dynamics in different regions of lamprey spinal neurons during membrane potential oscillations, using confocal calcium imaging in combination with intracellular recordings. Calcium fluctuations were observed in both soma and dendrites, timed to the oscillations. The calcium level increased sharply at the onset of membrane depolarization, to reach its maximum by the end of the plateau. The calcium peak in distal dendrites typically occurred earlier than in the soma during the oscillatory cycle. The L-type calcium channel blocker nimodipine increased the duration of the depolarized plateau phase in most cells tested, whereas the agonist Bay K 8644 decreased plateau duration. Bay K 8644 increased the amplitude of calcium fluctuations, particularly in distal dendrites, whereas nimodipine caused a decrease, suggesting that L-type low-voltage-activated calcium channels are mainly localized in these regions. Our results thus indicate that dendritic CaV1.3-like calcium channels are activated during NMDA-mediated membrane potential oscillations. This calcium influx activates KCa channels involved in plateau termination.

  15. Homocysteine and cytosolic GSH depletion induce apoptosis and oxidative toxicity through cytosolic calcium overload in the hippocampus of aged mice: involvement of TRPM2 and TRPV1 channels.

    PubMed

    Övey, I S; Naziroğlu, M

    2015-01-22

    Oxidative stress and apoptosis were induced in neuronal cultures by inhibition of glutathione (GSH) biosynthesis with d,l-buthionine-S,R-sulfoximine (BSO). Transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) cation channels are gated by oxidative stress. The oxidant effects of homocysteine (Hcy) may induce activation of TRPV1 and TRPM2 channels in aged mice as a model of Alzheimer's disease (AD). We tested the effects of Hcy, BSO and GSH on oxidative stress, apoptosis and Ca2+ and influx via TRPM2 and TRPV1 channels in the hippocampus of mice. Native mice hippocampal neurons were divided into five groups as follows; control, Hcy, BSO, Hcy+BSO and Hcy+BSO+GSH groups. The neurons in TRPM2 and TRPV1 experiments were stimulated by hydrogen peroxide and capsaicin, respectively. BSO and Hcy incubations increased intracellular free Ca2+ concentrations, reactive oxygen species, apoptosis, mitochondrial depolarization, and levels of caspase 3 and 9. All of these increases were reduced by GSH treatments. Treatment with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA) as potent inhibitors of TRPM2, capsazepine as a potent inhibitor of TRPV1, verapamil+diltiazem (V+D) as inhibitors of the voltage-gated Ca2+ channels (VGCC) and MK-801 as a N-methyl-d-aspartate (NMDA) channel antagonist indicated that GSH depletion and Hcy elevation activated Ca2+ entry into the neurons through TRPM2, TRPV1, VGCC and NMDA channels. Inhibitor roles of 2-APB and capsazepine on the Ca2+ entry higher than in V+D and MK-801 antagonists. In conclusion, these findings support the idea that GSH depletion and Hcy elevation can have damaging effects on hippocampal neurons by perturbing calcium homeostasis, mainly through TRPM2 and TRPV1 channels. GSH treatment can partially reverse these effects.

  16. Spontaneous Calcium Oscillations Regulate Human Cardiac Progenitor Cell Growth

    PubMed Central

    Ferreira-Martins, João; Rondon-Clavo, Carlos; Tugal, Derin; Korn, Justin A; Rizzi, Roberto; Padin-Iruegas, Maria Elena; Ottolenghi, Sergio; De Angelis, Antonella; Urbanek, Konrad; Iwata, Noriko; D’Amario, Domenico; Hosoda, Toru; Leri, Annarosa; Kajstura, Jan; Anversa, Piero; Rota, Marcello

    2009-01-01

    Rationale The adult heart possesses a pool of progenitor cells stored in myocardial niches but the mechanisms involved in the activation of this cell compartment are currently unknown. Objective Ca2+ promotes cell growth raising the possibility that changes in intracellular Ca2+ initiate division of c-kit-positive human cardiac progenitor cells (hCPCs) and determine their fate. Methods and Results Ca2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca2+. These findings were confirmed in the heart of transgenic mice in which EGFP was under the control of the c-kit-promoter. Ca2+ oscillations in hCPCs were regulated by the release of Ca2+ from the ER through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the re-uptake of Ca2+ by the sarco/endoplasmic reticulum Ca2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs while ryanodine receptors were not detected. Although Na+-Ca2+ exchanger, store-operated Ca2+-channels and plasma membrane Ca2+-pump were present and functional in hCPCs, they had no direct effects on Ca2+ oscillations. Conversely, Ca2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and BrdUrd incorporation. Induction of Ca2+ oscillations in hCPCs prior to their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny. Conclusion IP3R-mediated Ca2+ mobilization control hCPC growth and their regenerative potential. PMID:19745162

  17. Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers.

    PubMed

    Stary, Creed M; Hogan, Michael C

    2016-05-15

    The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: <3 mmHg) or AMP kinase activation had no effect on HSP72 mRNA levels. These results suggest that the intermittent cytosolic Ca(2+) transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

  18. Lack of changes in cytosolic ionized calcium in primary cultures of rat kidney cortical cells exposed to cytotoxic concentrations of gentamicin.

    PubMed

    Swann, J D; Ulrich, R; Acosta, D

    1990-10-01

    Gentamicin nephrotoxicity in vivo has a delayed onset. Our assessment of gentamicin-induced cell death in vitro, by measuring the release of cytosolic lactate dehydrogenase (LDH), indicated a prolonged onset as well. A recent study, which showed that gentamicin caused an abrupt increase in the concentration of cytosolic free calcium ([Ca2+]i) in a trypsin-harvested kidney cell line, suggested that immediate changes in calcium homeostasis may initiate the pathogenesis of gentamicin nephrotoxicity. To study the immediate effect of gentamicin on [Ca2+]i, gentamicin was perfused for 1 hr over primary monolayer cultures of renal cortical epithelial cells, and suspensions of trypsin-harvested renal cells (from primary cultures and a cell line) were treated with gentamicin for 30 min. [Ca2+]i was determined using the fluorescent probe fura-2. Positive controls (ionomycin and mercury) reliably increased [Ca2+]i in each experimental model, but no increase in [Ca2+]i was observed with gentamicin. Because enzyme release data indicated that significant cytotoxicity did not occur until 48 hr of exposure to 2 mM gentamicin, primary cultures were exposed to gentamicin (1-2 mM) for 24-48 hr and [Ca2+]i was measured. No gentamicin-induced increase in [Ca2+]i was observed in these longer exposures, whether or not significant LDH release occurred. These results do not support a role for elevated [Ca2+]i in the cytotoxicity of gentamicin in cultured kidney cells, either immediately after exposure or following prolonged exposures.

  19. Increased phase synchronization of spontaneous calcium oscillations in epileptic human versus normal rat astrocyte cultures

    NASA Astrophysics Data System (ADS)

    Balázsi, Gábor; Cornell-Bell, Ann H.; Moss, Frank

    2003-06-01

    Stochastic synchronization analysis is applied to intracellular calcium oscillations in astrocyte cultures prepared from epileptic human temporal lobe. The same methods are applied to astrocyte cultures prepared from normal rat hippocampus. Our results indicate that phase-repulsive coupling in epileptic human astrocyte cultures is stronger, leading to an increased synchronization in epileptic human compared to normal rat astrocyte cultures.

  20. Inhibition of neuronal calcium oscillations by cell surface APP phosphorylated on T668.

    PubMed

    Santos, Susana Ferrao; Tasiaux, Bernadette; Sindic, Christian; Octave, Jean-Noël

    2011-12-01

    Adenoviral expression of human APP (hAPP), but not of hAPP deleted from its C-terminal intracellular domain, in rat cortical neurons abolishes spontaneous synchronous calcium oscillations. The intracellular domain of APP695 contains several residues that can be phosphorylated. Contrary to non-neuronal cells, a very high phosphorylation of APP on T668 is observed in neurons, which is mediated by JNK, GSK3 and Cdk5 protein kinases. JNK activity, modulated by GSK3, enhances the traffic of phosphorylated APP to nerve terminals, contrary to Cdk5. Here we show that inhibition of GSK3 and JNK restores calcium oscillations in an hAPP expressing neuronal network, whereas inhibition of Cdk5 does not. Expression of mutant hAPPT668A does not inhibit calcium oscillations, and the proportion of hAPPT668A at the plasma membrane is reduced by more than 50%. Altogether, these results indicate that the intracellular domain of APP is needed to inhibit neuronal calcium oscillations because GSK3/JNK phosphorylation of T668 controls APP trafficking at the plasma membrane.

  1. Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

    PubMed

    Morton, Andrew; Marland, Jamie R K; Cousin, Michael A

    2015-08-01

    Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

  2. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

    PubMed

    Rey, Osvaldo; Young, Steven H; Jacamo, Rodrigo; Moyer, Mary P; Rozengurt, Enrique

    2010-10-01

    The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

  3. Oscillatory NAD(P)H Waves and Calcium Oscillations in Neutrophils? A Modeling Study of Feasibility

    PubMed Central

    Slaby, Oliver; Lebiedz, Dirk

    2009-01-01

    Abstract The group of Howard Petty has claimed exotic metabolic wave phenomena together with mutually phase-coupled NAD(P)H- and calcium-oscillations in human neutrophils. At least parts of these phenomena are highly doubtful due to extensive failure of reproducibility by several other groups and hints that unreliable data from the Petty lab are involved in publications concerning circular calcium waves. The aim of our theoretical spatiotemporal modeling approach is to propose a possible and plausible biochemical mechanism which would, in principle, be able to explain metabolic oscillations and wave phenomena in neutrophils. Our modeling suggests the possibility of a calcium-controlled glucose influx as a driving force of metabolic oscillations and a potential role of polarized cell geometry and differential enzyme distribution for various NAD(P)H wave phenomena. The modeling results are supposed to stimulate further controversial discussions of such phenomena and potential mechanisms and experimental efforts to finally clarify the existence and biochemical basis of any kind of temporal and spatiotemporal patterns of calcium signals and metabolic dynamics in human neutrophils. Independent of Petty's observations, they present a general feasibility study of such phenomena in cells. PMID:19167293

  4. Melatonin attenuates the mitochondrial translocation of mitochondrial fission proteins and Bax, cytosolic calcium overload and cell death in methamphetamine-induced toxicity in neuroblastoma SH-SY5Y cells.

    PubMed

    Parameyong, Arisa; Govitrapong, Piyarat; Chetsawang, Banthit

    2015-09-01

    Methamphetamine (METH) is an addictive drug that can cause toxicity and degeneration in the brain. Several pieces of evidence have demonstrated that METH toxicity results in increases in oxidative stress that regulate an intracellular signaling cascade that leads to cell death. Recently, several studies have emphasized that the overload of cytosolic calcium levels and mitochondrial fission into a small mitochondrial structure is involved in cell death processes. In the present study, we aimed to investigate the effects of METH toxicity on cytosolic calcium overload and mitochondrial fission in neuroblastoma SH-SY5Y cells. Additionally, the protective effect of melatonin against METH-induced toxicity was also investigated. The results of the present study demonstrated that METH significantly decreases cell viability and increases the levels of mitochondrial fission (Fis1 and Drp1) proteins and pro-apoptotic protein, Bax in isolated mitochondria. The levels of Drp1 in the cytosol of METH-treated cells had no significant differences compared to the control untreated cells. METH also significantly increased the cytosolic calcium levels. Melatonin reversed the toxic effects of METH by restoring cell viability and inhibiting the increase in mitochondrial Fis1 levels and the mitochondrial translocation of Drp1 and Bax. Additionally, melatonin was able to reduce the METH-induced increase in cytosolic calcium levels and fragmented mitochondria into small globular structures in SH-SY5Y cells. The results of the present study demonstrate the potential abilities of melatonin to maintain the homeostasis of mitochondrial dynamics and cytosolic calcium levels in METH-induced toxicity in neuronal cells.

  5. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

    PubMed

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2015-04-24

    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

  6. Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus-response coupling of insulin release.

    PubMed Central

    Shepherd, R. M.; Hashmi, M. N.; Kane, C.; Squires, P. E.; Dunne, M. J.

    1996-01-01

    1. Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 microM), phenotolamine (200-500 microM) and idazoxan (200-500 microM) on the intracellular free Ca2+ concentration ([Ca2+]i) in mouse isolated islets of Langerhans. 2. The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]i when Ca2+ was removed from outside of the islets and diazoxide (100-250 microM) attenuated the responses. 3. Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+]i. 4. Efaroxan- and phentolamine-evoked increases in [Ca2+]i were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+]i under any experimental condition tested. 5. The putative endogenous ligand of imidazoline receptors, agmatine (1 microM-1 mM), blocked KATP channels in isolated patches of beta-cell membrane, but effects upon [Ca2+]i could not be further investigated since agmatine disrupts fura-2 fluorescence. 6. In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+]i in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic beta-cells. PMID:8922740

  7. Nonlinear Time Series Analysis of Nodulation Factor Induced Calcium Oscillations: Evidence for Deterministic Chaos?

    PubMed Central

    Hazledine, Saul; Sun, Jongho; Wysham, Derin; Downie, J. Allan; Oldroyd, Giles E. D.; Morris, Richard J.

    2009-01-01

    Legume plants form beneficial symbiotic interactions with nitrogen fixing bacteria (called rhizobia), with the rhizobia being accommodated in unique structures on the roots of the host plant. The legume/rhizobial symbiosis is responsible for a significant proportion of the global biologically available nitrogen. The initiation of this symbiosis is governed by a characteristic calcium oscillation within the plant root hair cells and this signal is activated by the rhizobia. Recent analyses on calcium time series data have suggested that stochastic effects have a large role to play in defining the nature of the oscillations. The use of multiple nonlinear time series techniques, however, suggests an alternative interpretation, namely deterministic chaos. We provide an extensive, nonlinear time series analysis on the nature of this calcium oscillation response. We build up evidence through a series of techniques that test for determinism, quantify linear and nonlinear components, and measure the local divergence of the system. Chaos is common in nature and it seems plausible that properties of chaotic dynamics might be exploited by biological systems to control processes within the cell. Systems possessing chaotic control mechanisms are more robust in the sense that the enhanced flexibility allows more rapid response to environmental changes with less energetic costs. The desired behaviour could be most efficiently targeted in this manner, supporting some intriguing speculations about nonlinear mechanisms in biological signaling. PMID:19675679

  8. Three types of ependymal cells with intracellular calcium oscillation are characterized by distinct cilia beating properties.

    PubMed

    Liu, Tongyu; Jin, Xingjian; Prasad, Rahul M; Sari, Youssef; Nauli, Surya M

    2014-09-01

    Ependymal cells are multiciliated epithelial cells that line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia has been associated with various neurological deficits. For the first time, we report three distinct ependymal cell types, I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells have specific localizations within the third ventricle of the mouse brain. Furthermore, neither ependymal cell types nor their localizations are altered by aging. Our high-speed fluorescence imaging analysis reveals that these ependymal cells have an intracellular pacing calcium oscillation property. Our study further shows that alcohol can significantly repress the amplitude of calcium oscillation and the frequency of ciliary beating, resulting in an overall decrease in volume replacement by the cilia. Furthermore, the pharmacological agent cilostazol could differentially increase cilia beating frequency in type II, but not in type I or type III, ependymal cells. In summary, we provide the first evidence of three distinct types of ependymal cells with calcium oscillation properties.

  9. Sources of variability in cytosolic calcium transients triggered by stimulation of homogeneous uro-epithelial cell monolayers

    PubMed Central

    Appleby, Peter A.; Shabir, Saqib; Southgate, Jennifer; Walker, Dawn

    2015-01-01

    Epithelial tissue structure is the emergent outcome of the interactions between large numbers of individual cells. Experimental cell biology offers an important tool to unravel these complex interactions, but current methods of analysis tend to be limited to mean field approaches or representation by selected subsets of cells. This may result in bias towards cells that respond in a particular way and/or neglect local, context-specific cell responses. Here, an automated algorithm was applied to examine in detail the individual calcium transients evoked in genetically homogeneous, but asynchronous populations of cultured non-immortalized normal human urothelial cells when subjected to either the global application of an external agonist or a localized scratch wound. The recorded calcium transients were classified automatically according to a set of defined metrics and distinct sub-populations of cells that responded in qualitatively different ways were observed. The nature of this variability in the homogeneous cell population was apportioned to two sources: intrinsic variation in individual cell responses and extrinsic variability due to context-specific factors of the environment, such as spatial heterogeneity. Statistically significant variation in the features of the calcium transients evoked by scratch wounding according to proximity to the wound edge was identified. The manifestation of distinct sub-populations of cells is considered central to the coordination of population-level response resulting in wound closure. PMID:25694543

  10. Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

    SciTech Connect

    Kumar, Binod; Kumar, Amit; Ghosh, Subhalakshmi; Pandey, Badri N.; Mishra, Kaushala P.; Hazra, Banasri

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. Black-Right-Pointing-Pointer Activated PC-PLC induced a sustained-release of Ca{sup 2+} from endoplasmic reticulum. Black-Right-Pointing-Pointer The elevated cytosolic Ca{sup +2} led to the calpain-caspase12 dependent apoptosis. Black-Right-Pointing-Pointer D7-Induced Ca{sup +2} also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca{sup 2+}]{sub c} leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca{sup 2+}]{sub c} and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca{sup 2+} mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca{sup 2+}]{sub c} which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca{sup 2+}-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca{sup 2+}]{sub c} was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca{sup 2+} uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.

  11. Calcium-Sensing Receptor Regulates Cytosolic [Ca2+] and Plays a Major Role in the Development of Pulmonary Hypertension

    PubMed Central

    Smith, Kimberly A.; Ayon, Ramon J.; Tang, Haiyang; Makino, Ayako; Yuan, Jason X.-J.

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation and migration, both contributing to the development of pulmonary vascular remodeling. PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated, and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers (e.g., nifedipine), which are used to treat PAH patients but are only effective in 15–20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced

  12. Effects of endothelin on the mechanical activity and cytosolic calcium level of various types of smooth muscle.

    PubMed Central

    Sakata, K.; Ozaki, H.; Kwon, S. C.; Karaki, H.

    1989-01-01

    1. Effects of porcine/human endothelin (endothelin-1), a novel vasoconstrictor peptide, on various smooth muscles were examined. 2. In rat aorta, endothelin (1 pM-30nM) induced contraction in a concentration-dependent manner. Removal of endothelium shifted the concentration-response curve to the left. When added during the sustained contraction induced by 0.1 microM noradrenaline, endothelin (1 nM) induced a relaxation that was inhibited by removing endothelium or by methylene blue. 3. In rat aorta without endothelium, endothelin (1-30 nM) increased cytosolic Ca2+ level [( Ca2+]cyt) followed by contraction. Endothelin induced less contraction than high K+ at a given [Ca2+] cyt when the concentration of endothelin was lower (1-3nm) and/or during the early phase of the contraction (less than 10 min). In contrast, endothelin induced a greater contraction than KCl after prolonged exposure to high concentrations (greater than 10 nM). 4. The increase in [Ca2+]cyt due to endothelin was strongly inhibited by 10 microM verapamil or 0.3 microM nicardipine although muscle contraction was only partially inhibited. 5.In Ca2+ -free solution, endothelin (30 nM) induced a transient increase in [Ca2+] cyt and a slow increase in muscle tension. After a prolonged incubation in Ca2+-free solution, endothelin (30 nM) still induced a slow increase in tension without changing [Ca2+]cyt. This contraction was inhibited by 1 microM sodium nitropusside or 10 microM forskolin. 6. In canine trachea and guinea-pig uterus, endothelin (30 nM) induced sustained contraction with an increase in [Ca2+]cyt. In the absence of external Ca2+, endothelin (30 nM) induced a sustained contraction in canine trachea without changing [Ca2+]cyt. In guinea-pig vas deferens, taenia caeci and ileal longitudinal muscle, endothelin induced small increases in [Ca2+]cyt and tension. 7. In permeabilized smooth muscles, endothelin (30 nM) did not change the muscle tone. 8. These results suggest that endothelin acts on

  13. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  14. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

    PubMed Central

    Jing, Da; Baik, Andrew D.; Lu, X. Lucas; Zhou, Bin; Lai, Xiaohan; Wang, Liyun; Luo, Erping; Guo, X. Edward

    2014-01-01

    Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. PMID:24347610

  15. Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity.

    PubMed

    Eisner, Verónica; Cupo, Ryan R; Gao, Erhe; Csordás, György; Slovinsky, William S; Paillard, Melanie; Cheng, Lan; Ibetti, Jessica; Chen, S R Wayne; Chuprun, J Kurt; Hoek, Jan B; Koch, Walter J; Hajnóczky, György

    2017-01-31

    Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

  16. BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

    PubMed Central

    Wakle-Prabagaran, Monali; Lorca, Ramón A.; Ma, Xiaofeng; Stamnes, Susan J.; Amazu, Chinwendu; Hsiao, Jordy J.; Hyrc, Krzysztof L.; Wright, Michael E.; England, Sarah K.

    2016-01-01

    The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCa and both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCa in an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy. PMID:27044074

  17. Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

    PubMed

    Testard, Ambroise; Da Silva, Daniel; Ormancey, Mélanie; Pichereaux, Carole; Pouzet, Cécile; Jauneau, Alain; Grat, Sabine; Robe, Eugénie; Brière, Christian; Cotelle, Valérie; Mazars, Christian; Thuleau, Patrice

    2016-10-01

    Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

  18. Phototropins Function in High-Intensity Blue Light-Induced Hypocotyl Phototropism in Arabidopsis by Altering Cytosolic Calcium1[C][W][OA

    PubMed Central

    Zhao, Xiang; Wang, Yan-Liang; Qiao, Xin-Rong; Wang, Jin; Wang, Lin-Dan; Xu, Chang-Shui; Zhang, Xiao

    2013-01-01

    Phototropins (phot1 and phot2), the blue light receptors in plants, regulate hypocotyl phototropism in a fluence-dependent manner. Especially under high fluence rates of blue light (HBL), the redundant function mediated by both phot1 and phot2 drastically restricts the understanding of the roles of phot2. Here, systematic analysis of phototropin-related mutants and overexpression transgenic lines revealed that HBL specifically induced a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis (Arabidopsis thaliana) hypocotyls and that the increase in [Ca2+]cyt was primarily attributed to phot2. Pharmacological and genetic experiments illustrated that HBL-induced Ca2+ increases were modulated differently by phot1 and phot2. Phot2 mediated the HBL-induced increase in [Ca2+]cyt mainly by an inner store-dependent Ca2+-release pathway, not by activating plasma membrane Ca2+ channels. Further analysis showed that the increase in [Ca2+]cyt was possibly responsible for HBL-induced hypocotyl phototropism. An inhibitor of auxin efflux carrier exhibited significant inhibitions of both phototropism and increases in [Ca2+]cyt, which indicates that polar auxin transport is possibly involved in HBL-induced responses. Moreover, PHYTOCHROME KINASE SUBSTRATE1 (PKS1), the phototropin-related signaling element identified, interacted physically with phototropins, auxin efflux carrier PIN-FORMED1 and calcium-binding protein CALMODULIN4, in vitro and in vivo, respectively, and HBL-induced phototropism was impaired in pks multiple mutants, indicating the role of the PKS family in HBL-induced phototropism. Together, these results provide new insights into the functions of phototropins and highlight a potential integration point through which Ca2+ signaling-related HBL modulates hypocotyl phototropic responses. PMID:23674105

  19. Modulation of the Kinetics of Inositol 1,4,5-Trisphosphate-Induced [Ca2+]i Oscillations by Calcium Entry in Pituitary Gonadotrophs

    PubMed Central

    Kukuljan, Manuel; Vergara, Leoncio; Stojilkovic, Stanko S.

    1997-01-01

    Inositol 1,4,5-trisphosphate (InsP3) binds to its receptor channels and causes liberation of Ca2+ from intracellular stores, frequently in an oscillatory manner. In addition to InsP3, the activation and inactivation properties of these intracellular channels are controlled by Ca2+. We studied the influence of Ca2+ entry on the kinetics of InsP3-triggered oscillations in cytosolic calcium ([Ca2+]i) in gonadotrophs stimulated with gonadotropin-releasing hormone, an agonist that activates InsP3 production. The natural expression of voltage-gated Ca2+ channels (VGCC) in these cells was employed to manipulate Ca2+ entry by voltage clamping the cells at different membrane potentials (Vm). Under physiological conditions, the frequency of the GnRH-induced oscillations increased with time, while the amplitude decreased, until both reached stable values. However, in cells with Vm held at -50 mV or lower, both parameters progressively decreased until the signal was abolished. These effects were reverted by a depolarization of the membrane positive to -45 mV in both agonist- and InsP3-stimulated gonadotrophs. Depolarization also led to an increase in the fraction of time during which the [Ca2+]i remained elevated; this effect originated from both an increase in the mean duration of spikes and a decrease in the interval between spikes. The frequency and amplitude of spiking depended on the activity of VGCC, but displayed different temporal courses and voltage relationships. The depolarization-driven recovery of the frequency was instantaneous, whereas the recovery of the amplitude of spiking was more gradual. The midpoints of the Vm sensitivity curve for amplitude and duration of spiking (-15 mV) were close to the value observed for L-type Ca2+ current and for depolarization-induced increase in [Ca2+]i, whereas this parameter was much lower (-35 mV) for interval between spikes and frequency of oscillations. These observations are compatible with at least two distinct effects of

  20. Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

    PubMed

    Granqvist, Emma; Sun, Jongho; Op den Camp, Rik; Pujic, Petar; Hill, Lionel; Normand, Philippe; Morris, Richard J; Downie, J Allan; Geurts, Rene; Oldroyd, Giles E D

    2015-08-01

    Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.

  1. 9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes.

    PubMed

    Burt, Rees; Graves, Bridget M; Gao, Ming; Li, Chaunfu; Williams, David L; Fregoso, Santiago P; Hoover, Donald B; Li, Ying; Wright, Gary L; Wondergem, Robert

    2013-09-01

    It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca(2+)-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

  2. Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils [published erratum appears in J Cell Biol 1990 Mar;110(3):861

    PubMed Central

    1990-01-01

    Human neutrophils exhibit multiple increases in cytosolic free calcium concentration [( Ca2+]i) spontaneously and in response to the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Jaconi, M. E. E., R. W. Rivest, W. Schlegel, C. B. Wollheim, D. Pittet, and P. D. Lew. 1988. J. Biol. Chem. 263:10557-10560). The function of these repetitive increases in [Ca2+]i, as well as the role of Ca2+ in human neutrophil migration, remain unresolved. We have used microspectrofluorometry to measure [Ca2+]i in single fura-2-loaded human neutrophils as they moved on poly-D-lysine-coated glass in the presence of serum. To investigate the role of Ca2+ in human neutrophil migration, we examined cells in the presence and absence of extracellular Ca2+, as well as intracellular Ca2(+)-buffered and Ca2(+)- depleted cells. In the presence of extracellular Ca2+, multiple increases and decreases in [Ca2+]i were frequently observed, and at least one such transient increase in [Ca2+]i occurred in every moving cell during chemokinesis, chemotaxis, and phagocytosis. In addition, neutrophils that extended pseudopodia and assumed a polarized morphology after plating onto a surface were always observed to exhibit [Ca2+]i transients even in the absence of chemoattractant. In contrast, a [Ca2+]i transient was observed in only one of the nonpolarized stationary cells that were examined (n = 15). Although some cells exhibited relatively periodic increases and decreases in [Ca2+]i, resembling the regular oscillations that have been observed in some cell types, many others exhibited increases and decreases in [Ca2+]i that varied in their timing, magnitude, and duration. Buffering of [Ca2+]i or removal of extracellular Ca2+ damped out or blocked transient increases in [Ca2+]i and reduced or inhibited the migration of neutrophils. Under these conditions, polarized cells were often observed to make repeated attempts at migration, but they remained anchored at their rear. These data suggest

  3. Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

    PubMed Central

    Perez, H D; Elfman, F; Marder, S; Lobo, E; Ives, H E

    1989-01-01

    We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane. PMID:2723068

  4. Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes

    PubMed Central

    De Pittà, Maurizio; Goldberg, Mati; Volman, Vladislav; Berry, Hugues

    2009-01-01

    Recent years have witnessed an increasing interest in neuron–glia communication. This interest stems from the realization that glia participate in cognitive functions and information processing and are involved in many brain disorders and neurodegenerative diseases. An important process in neuron–glia communications is astrocyte encoding of synaptic information transfer—the modulation of intracellular calcium (Ca2 + ) dynamics in astrocytes in response to synaptic activity. Here, we derive and investigate a concise mathematical model for glutamate-induced astrocytic intracellular Ca2 +  dynamics that captures the essential biochemical features of the regulatory pathway of inositol 1,4,5-trisphosphate (IP3). Starting from the well-known two-variable (intracellular Ca2 +  and inactive IP3 receptors) Li–Rinzel model for calcium-induced calcium release, we incorporate the regulation of IP3 production and phosphorylation. Doing so, we extend it to a three-variable model (which we refer to as the ChI model) that could account for Ca2 +  oscillations with endogenous IP3 metabolism. This ChI model is then further extended into the G-ChI model to include regulation of IP3 production by external glutamate signals. Compared with previous similar models, our three-variable models include a more realistic description of IP3 production and degradation pathways, lumping together their essential nonlinearities within a concise formulation. Using bifurcation analysis and time simulations, we demonstrate the existence of new putative dynamical features. The cross-couplings between IP3 and Ca2 +  pathways endow the system with self-consistent oscillatory properties and favor mixed frequency–amplitude encoding modes over pure amplitude–modulation ones. These and additional results of our model are in general agreement with available experimental data and may have important implications for the role of astrocytes in the synaptic transfer of information

  5. Calcium

    MedlinePlus

    ... You'll also find calcium in broccoli and dark green, leafy vegetables (especially collard and turnip greens, ... can enjoy good sources of calcium such as dark green, leafy vegetables, broccoli, chickpeas, and calcium-fortified ...

  6. Amyloid β Peptide Enhances RANKL-Induced Osteoclast Activation through NF-κB, ERK, and Calcium Oscillation Signaling

    PubMed Central

    Li, Shangfu; Yang, Bu; Teguh, Dian; Zhou, Lin; Xu, Jiake; Rong, Limin

    2016-01-01

    Osteoporosis and Alzheimer’s disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aβ), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aβ on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aβ exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aβ enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aβ enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aβ may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis. PMID:27735865

  7. Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

    PubMed Central

    Wayman, Christopher P; McFadzean, Ian; Gibson, Alan; Tucker, John F

    1997-01-01

    At a holding potential of −40 mV, carbachol (50 μM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (IDOC). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (Ioscil) superimposed on IDOC.Carbachol-induced Ioscil (amplitude 97±11 pA; frequency 0.26±0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution.In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; Ioscil could be restored by re-admission of calcium. The frequency, but not the amplitude, of Ioscil increased with increasing concentrations of extracellular calcium (0.5–10 mM).Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml−1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), prevented the activation of Ioscil by carbachol. Caffeine (10 mM) activated both ICl(Ca) and IDOC and prevented the induction of Ioscil by carbachol. Caffeine and CPA also abolished Ioscil in the presence of carbachol, as did both a low (3 μM) and a high (30 μM) concentration of ryanodine.Carbachol-induced Ioscil was abolished by the general calcium entry blocker SKF 96365 (10 μM) and by Cd2+ (100 μM), but was unaffected by La3+ (400 μM). As found previously, IDOC was also blocked by

  8. Calcium- and potassium-permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production.

    PubMed

    Rodrigo-Moreno, Ana; Andrés-Colás, Nuria; Poschenrieder, Charlotte; Gunsé, Benet; Peñarrubia, Lola; Shabala, Sergey

    2013-04-01

    Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH(•) ), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper-transport activity: Col-0, high-affinity copper transporter COPT1-overexpressing (C1(OE) ) seedlings, and T-DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm) stimulated a dose-dependent Gd(3+) and verapamil sensitive net Ca(2+) influx in the root apex but not in mature zone. C1(OE) also showed a fivefold higher Cu-induced K(+) efflux at the root tip level compared with Col-0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1(OE) seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd(3+) . Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH(•) -sensitive Ca(2+) and K(+) transport systems.

  9. Calcium oscillations index the extent of calcium loading and predict functional recovery during reperfusion in rat myocardium.

    PubMed Central

    Weiss, R G; Gerstenblith, G; Lakatta, E G

    1990-01-01

    Delayed recovery of contractile function after myocardial ischemia may be due to prolonged recovery of high-energy phosphates, persistent acidosis, increased inorganic phosphate, and/or calcium loading. To examine these potential mechanisms, metabolic parameters measured by 31P nuclear magnetic resonance spectroscopy, and spontaneous diastolic myofilament motion caused by sarcoplasmic reticulum-myofilament calcium cycling indexed by the scattered light intensity fluctuations (SLIF) it produces in laser beam reflected from the heart, were studied in isolated atrioventricularly blocked rat hearts (n = 10) after 65 min of ischemia at 30 degrees C. All metabolic parameters recovered to their full extent 5 min after reperfusion. Developed pressure evidenced a small recovery but then fell abruptly. This was accompanied by an increase in end diastolic pressure to 37 +/- 5 mm Hg and a fourfold increase in SLIF, to 252 +/- 58% of baseline. In another series of hearts initial reperfusion with calcium of 0.08 mM prevented the SLIF rise and resulted in improved developed pressure (74 +/- 3% vs. 39 +/- 13% of control), and lower cell calcium (5.9 +/- 3 vs. 10.3 +/- 1.4 mumol/g dry wt). Thus, during reperfusion, delayed contractile recovery is not associated with delayed recovery of pH, inorganic phosphate, or high-energy phosphates and can be attributed, in part, to an adverse effect of calcium loading which can be indexed by increased SLIF occurring at that time. PMID:2312726

  10. Modulation of gamma oscillations in the pedunculopontine nucleus by neuronal calcium sensor protein-1: relevance to schizophrenia and bipolar disorder

    PubMed Central

    D'Onofrio, Stasia; Kezunovic, Nebojsa; Hyde, James R.; Luster, Brennon; Messias, Erick; Urbano, Francisco J.

    2014-01-01

    Reduced levels of gamma-band activity are present in schizophrenia and bipolar disorder patients. In the same disorders, increased neuronal calcium sensor protein-1 (NCS-1) expression was reported in a series of postmortem studies. These disorders are also characterized by sleep dysregulation, suggesting a role for the reticular activating system (RAS). The discovery of gamma-band activity in the pedunculopontine nucleus (PPN), the cholinergic arm of the RAS, revealed that such activity was mediated by high-threshold calcium channels that are regulated by NCS-1. We hypothesized that NCS-1 normally regulates gamma-band oscillations through these calcium channels and that excessive levels of NCS-1, such as would be expected with overexpression, decrease gamma-band activity. We found that PPN neurons in rat brain slices manifested gamma-band oscillations that were increased by low levels of NCS-1 but suppressed by high levels of NCS-1. Our results suggest that NCS-1 overexpression may be responsible for the decrease in gamma-band activity present in at least some schizophrenia and bipolar disorder patients. PMID:25376789

  11. Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

    NASA Astrophysics Data System (ADS)

    Kim, Suhwan; Jung, Unsang; Baek, Juyoung; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-01-01

    Recently, mouse neuroblastoma cells have been considered as an attractive model for the study of human neurological and prion diseases, and they have been intensively used as a model system in different areas. For example, the differentiation of neuro2a (N2A) cells, receptor-mediated ion current, and glutamate-induced physiological responses have been actively investigated with these cells. These mouse neuroblastoma N2A cells are of interest because they grow faster than other cells of neural origin and have a number of other advantages. The calcium oscillations and neural spikes of mouse neuroblastoma N2A cells in epileptic conditions are evaluated. Based on our observations of neural spikes in these cells with our proposed imaging modality, we reported that they can be an important model in epileptic activity studies. We concluded that mouse neuroblastoma N2A cells produce epileptic spikes in vitro in the same way as those produced by neurons or astrocytes. This evidence suggests that increased levels of neurotransmitter release due to the enhancement of free calcium from 4-aminopyridine causes the mouse neuroblastoma N2A cells to produce epileptic spikes and calcium oscillations.

  12. Mouse neuroblastoma cell based model and the effect of epileptic events on calcium oscillations and neural spikes

    NASA Astrophysics Data System (ADS)

    Kim, Suhwan; Baek, Juyeong; Jung, Unsang; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-05-01

    Recently, Mouse neuroblastoma cells are considered as an attractive model for the study of human neurological and prion diseases, and intensively used as a model system in different areas. Among those areas, differentiation of neuro2a (N2A) cells, receptor mediated ion current, and glutamate induced physiological response are actively investigated. The reason for the interest to mouse neuroblastoma N2A cells is that they have a fast growing rate than other cells in neural origin with a few another advantages. This study evaluated the calcium oscillations and neural spikes recording of mouse neuroblastoma N2A cells in an epileptic condition. Based on our observation of neural spikes in mouse N2A cell with our proposed imaging modality, we report that mouse neuroblastoma N2A cells can be an important model related to epileptic activity studies. It is concluded that the mouse neuroblastoma N2A cells produce the epileptic spikes in vitro in the same way as produced by the neurons or the astrocytes. This evidence advocates the increased and strong level of neurotransmitters release by enhancement in free calcium using the 4-aminopyridine which causes the mouse neuroblastoma N2A cells to produce the epileptic spikes and calcium oscillation.

  13. Investigation of the effects of distance from sources on apoptosis, oxidative stress and cytosolic calcium accumulation via TRPV1 channels induced by mobile phones and Wi-Fi in breast cancer cells.

    PubMed

    Çiğ, Bilal; Nazıroğlu, Mustafa

    2015-10-01

    TRPV1 is a Ca2+ permeable channel and gated by noxious heat, oxidative stress and capsaicin (CAP). Some reports have indicated that non-ionized electromagnetic radiation (EMR)-induces heat and oxidative stress effects. We aimed to investigate the effects of distance from sources on calcium signaling, cytosolic ROS production, cell viability, apoptosis, plus caspase-3 and -9 values induced by mobile phones and Wi-Fi in breast cancer cells MCF-7 human breast cancer cell lines were divided into A, B, C and D groups as control, 900, 1800 and 2450 MHz groups, respectively. Cells in Group A were used as control and were kept in cell culture conditions without EMR exposure. Groups B, C and D were exposed to the EMR frequencies at different distances (0 cm, 1 cm, 5 cm, 10 cm, 20 cm and 25 cm) for 1h before CAP stimulation. The cytosolic ROS production, Ca2+ concentrations, apoptosis, caspase-3 and caspase-9 values were higher in groups B, C and D than in A group at 0 cm, 1 cm and 5 cm distances although cell viability (MTT) values were increased by the distances. There was no statistically significant difference in the values between control, 20 and 25 cm. Wi-Fi and mobile phone EMR placed within 10 cm of the cells induced excessive oxidative responses and apoptosis via TRPV1-induced cytosolic Ca2+ accumulation in the cancer cells. Using cell phones and Wi-Fi sources which are farther away than 10 cm may provide useful protection against oxidative stress, apoptosis and overload of intracellular Ca2+. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

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

    NASA Astrophysics Data System (ADS)

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

    1999-07-01

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

  15. Glutamate Receptor-Like Channel3.3 Is Involved in Mediating Glutathione-Triggered Cytosolic Calcium Transients, Transcriptional Changes, and Innate Immunity Responses in Arabidopsis1[W][OA

    PubMed Central

    Li, Feng; Wang, Jing; Ma, Chunli; Zhao, Yongxiu; Wang, Yingchun; Hasi, Agula; Qi, Zhi

    2013-01-01

    The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related. PMID:23656893

  16. Optogenetic Restoration of Disrupted Slow Oscillations Halts Amyloid Deposition and Restores Calcium Homeostasis in an Animal Model of Alzheimer’s Disease

    PubMed Central

    Kastanenka, Ksenia V.; Hou, Steven S.; Shakerdge, Naomi; Logan, Robert; Feng, Danielle; Wegmann, Susanne; Chopra, Vanita; Hawkes, Jonathan M.; Chen, Xiqun; Bacskai, Brian J.

    2017-01-01

    Slow oscillations are important for consolidation of memory during sleep, and Alzheimer’s disease (AD) patients experience memory disturbances. Thus, we examined slow oscillation activity in an animal model of AD. APP mice exhibit aberrant slow oscillation activity. Aberrant inhibitory activity within the cortical circuit was responsible for slow oscillation dysfunction, since topical application of GABA restored slow oscillations in APP mice. In addition, light activation of channelrhodopsin-2 (ChR2) expressed in excitatory cortical neurons restored slow oscillations by synchronizing neuronal activity. Driving slow oscillation activity with ChR2 halted amyloid plaque deposition and prevented calcium overload associated with this pathology. Thus, targeting slow oscillatory activity in AD patients might prevent neurodegenerative phenotypes and slow disease progression. PMID:28114405

  17. Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

    PubMed

    Caporarello, N; Salmeri, M; Scalia, M; Motta, C; Parrino, C; Frittitta, L; Olivieri, M; Toscano, M A; Anfuso, C D; Lupo, G

    2015-12-21

    Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin β-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

  18. Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs

    PubMed Central

    NAKAI, Michiko; ITO, Junya; SUZUKI, Shun-ichi; FUCHIMOTO, Dai-ichiro; SEMBON, Shoichiro; SUZUKI, Misae; NOGUCHI, Junko; KANEKO, Hiroyuki; ONISHI, Akira; KASHIWAZAKI, Naomi; KIKUCHI, Kazuhiro

    2016-01-01

    In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation. PMID:27725347

  19. Front-surface fluorometry with fura-2 and effects of nitroglycerin on cytosolic calcium concentrations and on tension in the coronary artery of the pig.

    PubMed Central

    Abe, S.; Kanaide, H.; Nakamura, M.

    1990-01-01

    1. By use of front-surface fluorometry and fura-2-loaded strips of the coronary artery of the pig, the effects of nitroglycerin (NG) on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension development were measured simultaneously. 2. Both high K+ depolarization and histamine increased [Ca2+]i and tension in a concentration-dependent manner. However, the tension development in relation to the [Ca2+]i increase ([Ca2+]i-tension relation) observed with histamine was much greater than that observed with K+ depolarization. 3. NG reduced in a concentration-dependent manner both [Ca2+]i and tension, irrespective of whether the vascular strips were in a resting state or during exposure to high K+ or to histamine stimulation. However, the extent of reduction in tension (relaxation) was greater than that expected from the reduction in [Ca2+]i based on the [Ca2+]i-tension relationship observed with K(+)-depolarization. 4. In the absence of extracellular Ca2+, NG depleted stored Ca2+ and also inhibited Ca2+ release from histamine-sensitive stores, but had no effect on the caffeine-sensitive stores. NG inhibited the caffeine-induced tension development with no change in [Ca2+]i. 5. We suggest that NG relaxes the coronary artery of the pig by reducing [Ca2+]i and also by directly controlling contractile elements through second messengers not related to changes in [Ca2+]i. PMID:2127551

  20. Ent-7α-acetoxytrachyloban-18-oic acid and ent-7α-hydroxytrachyloban-18-oic acid from Xylopia langsdorfiana A. St-Hil. & Tul. modulate K(+) and Ca(2+) channels to reduce cytosolic calcium concentration on guinea pig ileum.

    PubMed

    Santos, Rosimeire F; Martins, Italo R R; Travassos, Rafael A; Tavares, Josean F; Silva, Marcelo S; Paredes-Gamero, Edgar J; Ferreira, Alice T; Nouailhetas, Viviane L A; Aboulafia, Jeannine; Rigoni, Vera L S; da Silva, Bagnólia A

    2012-03-05

    In this study we investigated the mechanism underlying the spasmolytic action of ent-7α-acetoxytrachyloban-18-oic acid (trachylobane-360) and ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318), diterpenes obtained from Xylopia langsdorfiana, on guinea pig ileum. Both compounds inhibited histamine-induced cumulative contractions (slope=3.5±0.9 and 4.4±0.7) that suggests a noncompetitive antagonism to histaminergic receptors. CaCl(2)-induced contractions were nonparallelly and concentration-dependently reduced by both diterpenes, indicating blockade of calcium influx through voltage-dependent calcium channels (Ca(v)). The Ca(v) participation was confirmed since both trachylobanes equipotently relaxed ileum pre-contracted with S-(-)-Bay K8644 (EC(50)=3.5±0.7×10-(5) and 1.1±0.2×10-(5)M) and KCl (EC(50)=5.5±0.3×10-(5) and 1.4±0.2×10-(5)M). K(+) channels participation was confirmed since diterpene-induced relaxation curves were significantly shifted to right in the presence of 5mM tetraethylammonium (TEA(+)) (EC(50)=0.5±0.04×10-(4) and 2.0±0.5×10-(5)M). ATP-sensitive K(+) channel (K(ATP)), voltage activated K(+) channels (K(V)), small conductance calcium-activated K(+) channels (SK(Ca)) or big conductance calcium-activated K(+) channels (BK(Ca)) did not seem to participate of trachylobane-360 spasmolytic action. However trachylobane-318 modulated positively K(ATP), K(V) and SK(Ca) (EC(50)=1.1±0.3×10-(5), 0.7±0.2×10-(5) and 0.7±0.2×10-(5)M), but not BK(Ca). A fluorescence analysis technique confirmed the decrease of cytosolic calcium concentration ([Ca(2+)](c)) induced by both trachylobanes in ileal myocytes. In conclusion, trachylobane-360 and trachylobane-318 induced spasmolytic activity by K(+) channel positive modulation and Ca(2+) channel blockade, which results in [Ca(2+)](c) reduction at cellular level leading to smooth muscle relaxation.

  1. Cytosolic Calcium, hydrogen peroxide, and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: Parabolic flight data

    NASA Astrophysics Data System (ADS)

    Hampp, Ruediger; Hausmann, Niklas; Neef, Maren; Fengler, Svenja

    Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide and cytosolic Ca2+ were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion, for RNA; acid/base for NADPH, NADP) at typical stages of a parabola (1g before pull up; end of pull up (1.8 g), end of microgravity (µg, 20 sec), and end of pull out (1.8 g)). Cells exhibited an increase of both Ca2+ and hydrogen peroxide with the onset of µg, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating a Ca2+-dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca2+- and ROS(reactive oxygen species)-related gene products. The same material was also used for the analysis of phosphopeptides by 2D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of reactive oxygen species. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS.

  2. Cytosolic calcium, hydrogen peroxide and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: parabolic flight data.

    PubMed

    Hausmann, N; Fengler, S; Hennig, A; Franz-Wachtel, M; Hampp, R; Neef, M

    2014-01-01

    Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular, short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide (H2 O2 ) and cytosolic Ca(2+) were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion for RNA; acid/base for NADPH, NADP) at typical stages of a parabola [1 g before pull up; end of pull up (1.8 g), end of microgravity (20 s) and end of pull out (1.8 g)]. Cells exhibited an increase in both Ca(2+) and H2 O2 with the onset of microgravity, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating Ca(2+) -dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca(2+) - and ROS-related gene products. The same material was also used for analysis of phosphopeptides with 2-D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of ROS. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS.

  3. Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery.

    PubMed

    Tesarik, J; Sousa, M; Mendoza, C

    1995-06-01

    Temporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady-state period of sperm-induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal-induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm-induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca(2+)-induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator.

  4. Calcium

    MedlinePlus

    ... milligrams) of calcium each day. Get it from: Dairy products. Low-fat milk, yogurt, cheese, and cottage ... lactase that helps digest the sugar (lactose) in dairy products, and may have gas, bloating, cramps, or ...

  5. Low levels of serum ionized magnesium are found in patients early after stroke which result in rapid elevation in cytosolic free calcium and spasm in cerebral vascular muscle cells.

    PubMed

    Altura, B T; Memon, Z I; Zhang, A; Cheng, T P; Silverman, R; Cracco, R Q; Altura, B M

    1997-07-11

    Ninety-eight patients admitted to the emergency rooms of three urban hospitals with a diagnosis of either ischemic stroke or hemorrhagic stroke exhibited early and significant deficits in serum ionized Mg2+ (IMg2+), but not total Mg, as measured with a unique Mg2+-sensitive ion-selective electrode. Twenty-five percent of these stroke patients exhibited >65% reductions in the mean serum IMg2+ found in normal healthy human volunteers or patients admitted for minor bruises, cuts or deep lacerations. The stroke patients also demonstrated significant elevation in the serum ionized Ca2+ (ICa2+)/IMg2+ ratio, a sign of increased vascular tone and cerebrovasospasm. Exposure of primary cultured canine cerebral vascular smooth muscle cells to the low concentrations of IMg2+ found in the stroke patients, e.g. 0.30-0.48 mM, resulted in rapid and marked elevations in cytosolic free calcium ions ([Ca2+]i) as measured with the fluorescent probe, fura-2, and digital image analysis. Coincident with the rise in [Ca2+]i, many of the cerebral vascular cells went into spasm. Reintroduction of normal extracellular Mg2+ ion concentrations failed to either lower the [Ca2+]i overload or reverse the rounding-up of the cerebral vascular cells. These results suggest that changes in Mg2+ metabolism play important roles in stroke syndromes and in the etiology of cerebrovasospasm associated with cerebral hemorrhage.

  6. Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

    PubMed

    Barman, Ananya; Tamuli, Ranjan

    2015-04-01

    Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

  7. Mutation of a NCKX Eliminates Glial Microdomain Calcium Oscillations and Enhances Seizure Susceptibility

    PubMed Central

    Melom, Jan E.; Littleton, J. Troy

    2013-01-01

    Glia exhibit spontaneous and activity-dependent fluctuations in intracellular Ca2+, yet it is unclear whether glial Ca2+ oscillations are required during neuronal signaling. Somatic glial Ca2+ waves are primarily mediated by the release of intracellular Ca2+ stores, and their relative importance in normal brain physiology has been disputed. Recently, near-membrane microdomain Ca2+ transients were identified in fine astrocytic processes and found to arise via an intracellular store-independent process. Here, we describe the identification of rapid, near-membrane Ca2+ oscillations in Drosophila cortex glia of the CNS. In a screen for temperature-sensitive conditional seizure mutants, we identified a glial-specific Na+/Ca2+, K+ exchanger (zydeco) that is required for microdomain Ca2+ oscillatory activity. We found that zydeco mutant animals exhibit increased susceptibility to seizures in response to a variety of environmental stimuli, and that zydeco is required acutely in cortex glia to regulate seizure susceptibility. We also found that glial expression of calmodulin is required for stress-induced seizures in zydeco mutants, suggesting a Ca2+/calmodulin-dependent glial signaling pathway underlies glial–neuronal communication. These studies demonstrate that microdomain glial Ca2+ oscillations require NCKX-mediated plasma membrane Ca2+ flux, and that acute dysregulation of glial Ca2+ signaling triggers seizures. PMID:23325253

  8. Effect of extracellular calcium, pH and borate on growth oscillations in Lilium formosanum pollen tubes.

    PubMed

    Holdaway-Clarke, Terena L; Weddle, Nicole M; Kim, SaRa; Robi, Amsale; Parris, Colleen; Kunkel, Joseph G; Hepler, Peter K

    2003-01-01

    Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.

  9. Ab initio oscillator strengths and transition probabilities in aluminum-like calcium, Ca VIII

    SciTech Connect

    Karpuskiene, R. Bogdanovich, P.

    2009-07-15

    An ab initio study of aluminum-like calcium is presented. The calculations are performed within the configuration interaction method in the basis of transformed radial orbitals with a variable parameter. Relativistic effects are accounted for within the Breit-Pauli approximation. Energy spectra, transition characteristics and lifetimes of excited levels of configurations 3s{sup 2}3p, 3s3p{sup 2}, 3s{sup 2}3d, 3p{sup 3}, 3s3p3d, 3p{sup 2}3d, 3s{sup 2}4s, 3s{sup 2}4p, 3s{sup 2}4d, 3s{sup 2}4f, 3s3p4s, and 3s3p4p are obtained. The results are compared with available experimental and theoretical data.

  10. The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.

    PubMed

    Carrithers, Lisette M; Hulseberg, Paul; Sandor, Matyas; Carrithers, Michael D

    2011-12-01

    Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.

  11. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

    PubMed

    Caporarello, Nunzia; Salmeri, Mario; Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

  12. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells

    PubMed Central

    Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion. PMID:27631977

  13. Alendronate affects calcium dynamics in cardiomyocytes in vitro.

    PubMed

    Kemeny-Suss, Naomi; Kasneci, Amanda; Rivas, Daniel; Afilalo, Jonathan; Komarova, Svetlana V; Chalifour, Lorraine E; Duque, Gustavo

    2009-01-01

    Therapy with bisphosphonates, including alendronate (ALN), is considered a safe and effective treatment for osteoporosis. However, recent studies have reported an unexpected increase in serious atrial fibrillation (AF) in patients treated with bisphosphonates. The mechanism that explains this side effect remains unknown. Since AF is associated with an altered sarcoendoplasmic reticulum calcium load, we studied how ALN affects cardiomyocyte calcium homeostasis and protein isoprenylation in vitro. Acute and long-term (48h) treatment of atrial and ventricular cardiomyocytes with ALN (10(-8)-10(-6)M) was performed. Changes in calcium dynamics were determined by both fluorescence measurement of cytosolic free Ca(2+) concentration and western blot analysis of calcium-regulating proteins. Finally, effect of ALN on protein farnesylation was also identified. In both atrial and ventricular cardiomyocytes, ALN treatment delayed and diminished calcium responses to caffeine. Only in atrial cells, long-term exposure to ALN-induced transitory calcium oscillations and led to the development of oscillatory component in calcium responses to caffeine. Changes in calcium dynamics were accompanied by changes in expression of proteins controlling sarcoendoplasmic reticulum calcium. In contrast, ALN minimally affected protein isoprenylation in these cells. In summary, treatment of atrial cardiomyocytes with ALN-induced abnormalities in calcium dynamics consistent with induction of a self-stimulatory, pacemaker-like behavior, which may contribute to the development of cardiac side effects associated with these drugs.

  14. Role of a T-type calcium current in supporting a depolarizing potential, damped oscillations, and phasic firing in vasopressinergic guinea pig supraoptic neurons.

    PubMed

    Erickson, K R; Ronnekleiv, O K; Kelly, M J

    1993-05-01

    Guinea pig magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) were studied using the in vitro slice preparation. Intracellular recordings were made with biocytin-filled electrodes, permitting immunocytochemical identification of the recorded cells as arginine vasopressin- (AVP) versus oxytocin- (OT) containing. Only AVP cells displaying a depolarizing potential (DP) fired phasically. The DP was associated with a transient inward current measured in voltage clamp, which exhibited a number of properties of the T-type calcium current: activation threshold of -64 mV, time course of up to 250 ms, blockade by nickel and augmentation by barium chloride. This current has not been reported previously in SON neurons. The T-type current (IT) was always associated with a damped oscillation of the membrane following the offset from hyperpolarizing steps. In all cells tested, an apamin-sensitive afterhyperpolarization (AHP) was observed, similar to the calcium-dependent potassium current (IK, Ca) described in rat SON and other CNS regions. Therefore, as with other CNS regions displaying damped oscillations, guinea pig SON cells possess both an IT and an IK, Ca. We have previously described an Ih activating at hyperpolarized potentials in these cells, which depolarizes the membrane to a range in which the IT and IK, Ca can interactively support oscillations. In summary, the IT and associated depolarizing potential appears to be a requisite feature for phasic firing in AVP cells of guinea pig SON.

  15. Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

    PubMed

    Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

  16. High-power continuous-wave tunable 544- and 272-nm beams based on a diode-oscillator fiber-amplifier for calcium spectroscopy

    NASA Astrophysics Data System (ADS)

    Ko, Kwang-Hoon; Kim, Yonghee; Park, Hyunmin; Cha, Yong-Ho; Kim, Taek-Soo; Lee, Lim; Lim, Gwon; Han, Jaemin; Ko, Kwang-Hee; Jeong, Do-Young

    2015-08-01

    Continuous-wave single-frequency tunable 544- and 272-nm beams have been demonstrated by the second- and fourth-harmonic conversions of a 1088-nm fundamental beam from a diode-oscillator fiber-amplifier. The single-pass second-harmonic generation with a MgO-doped periodically poled stoichiometric LiTaO3 crystal and the external-cavity frequency-doubling technique with a bulk BBO crystal were employed to achieve an approximately 6-W 544-nm beam and a 1.5-W 272-nm beam, respectively. We characterized the second- and fourth-harmonic generations and discussed their applications to calcium spectroscopy.

  17. Extrinsic periodic information interpolates between monostable and bistable states in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Lin, Ling; Duan, Wei-Long

    2015-06-01

    Extrinsic periodic information including physiological cyclical and circadian replacement would affect inevitably a real cell, in this paper we investigate the effect of extrinsic periodic information on intracellular calcium dynamics by means of second-order algorithm for stochastic simulation colored noises. By simulating time evolutions and stationary probability distribution of intracellular Ca2+ concentrations, the results show: (i) intracellular calcium oscillation between cytosol and calcium store shows synchronous and anti-synchronous oscillation as intensity and frequency of extrinsic periodic information vary; (ii) extrinsic periodic information interpolates stability from bistable state → monostable state → bistable state → monostable state as frequency of extrinsic periodic information increases; (iii) extrinsic periodic information interpolates stability from monostable state → bistable state as intensity of extrinsic periodic information increases.

  18. Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

    PubMed

    Rizaner, Nahit; Onkal, Rustem; Fraser, Scott P; Pristerá, Alessandro; Okuse, Kenji; Djamgoz, Mustafa B A

    2016-10-01

    The possible association of intracellular Ca(2+) with metastasis in human cancer cells is poorly understood. We have studied Ca(2+) signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca(2+) was measured using a membrane-permeant fluorescent Ca(2+)-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca(2+) oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca(2+) level could be induced by applying a high-K(+) solution. Various parameters of the oscillations depended on extracellular Ca(2+) and voltage-gated Na(+) channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na(+) channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca(2+) oscillations. It is concluded that the functional voltage-gated Na(+) channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca(2+) activity. Possible mechanisms and consequences of the Ca(2+) oscillations are discussed.

  19. Cytosolic free Ca2+ oscillations induced by diadenosine 5',5"'-P1,P3-triphosphate and diadenosine 5',5"'-P1,P4-tetraphosphate in single rat hepatocytes are indistinguishable from those induced by ADP and ATP respectively.

    PubMed

    Green, A K; Cobbold, P H; Dixon, C J

    1995-09-01

    Diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.

  20. [Electrophysiology and calcium signalling in human bronchial smooth muscle].

    PubMed

    Marthan, R; Hyvelin, J M; Roux, E; Savineau, J P

    1999-01-01

    Recently, cells isolated from airways have been used to characterize precisely the electrophysiological properties of this smooth muscle and to describe the changes in cytosolic calcium concentration ([Ca2+]i) occurring upon agonist stimulation. Although most studies have produced consistent results in terms of types of ion channel and pathways of calcium signalling implicated in the mechanical activity of airways, there are differences according to (i) the site along the bronchial tree (trachea vs. bronchi); (ii) the proliferating status of the cells (freshly isolated vs. cultured) and (iii) the species (human vs. animals). With regard to the electrophysiological properties of airway smooth muscle, the contribution to [Ca2+]i rise of Ca2+ influx through L-type voltage-dependent calcium channels depends on the balance between depolarization related to non-specific cation channel and/or chloride channel activation and hyperpolarization related to activation of a variety of potassium channels. Most of the above-mentioned channels appear to be controlled, directly or indirectly, by agonists in human bronchial smooth muscle. With regard to calcium signalling, the pattern of agonist-induced [Ca2+]i responses, the so-called [Ca2+]i oscillations, has been observed recently in freshly isolated airway smooth muscle cells. The role and the calcium sources involved in these oscillations in human bronchial smooth muscle are currently being investigated.

  1. Metabotropic glutamate receptor 5 modulates calcium oscillation and innate immune response induced by lipopolysaccharide in microglial cell.

    PubMed

    Liu, F; Zhou, R; Yan, H; Yin, H; Wu, X; Tan, Y; Li, L

    2014-12-05

    Microglia, the primary immune cells in the brain, have been implicated as the predominant cells governing inflammation-mediated neuronal damage. In response to immunological challenges such as lipopolysaccharide (LPS), microglia are activated and subsequently inflammatory process is initiated as evidenced by the release of pro-inflammatory chemokines and cytokines. Here we show that Group I metabotropic glutamate receptor 5 (mGluR5) is involved in LPS-induced microglia activation. LPS triggered a similar pattern of [Ca2+]i oscillation in N9, Toll-like receptor 4 (TLR4)-mutant EOC 20, TLR4-wild-type and TLR4-deficient primary mouse microglia, suggesting that LPS-induced [Ca2+]i oscillation is independent of TLR4. The characteristics of [Ca2+]i oscillation induced by LPS are consistent with those observed in mGluR5 activation. In addition, mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) abolished LPS-induced [Ca2+]i oscillation. Immunocytochemistry demonstrated that LPS colocalizes with mGluR5 in microglia and the direct binding of LPS and mGluR5 was further validated by antibody-based fluorescence resonance energy transfer (FRET) technology. Activation of mGluR5 using a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) significantly expanded LPS-induced nuclear factor-kappa B (NF-κB) activity and CHPG alone increased NF-κB activity as well. But, mGluR5 antagonist MTEP attenuated the actions of LPS, CHPG and the additive effect of LPS and CHPG in microglia. LPS induced tumor necrosis factor-α (TNF-α) secretion in N9 microglia, but not in TLR4-mutant EOC 20 and TLR4-deficient primary mouse microglia. CHPG reduced LPS-caused TNF-α production, but MTEP increased LPS-induced TNF-α production and blocked the effect of CHPG in N9 microglia. These data demonstrate that mGluR5 and TLR4 are two critical receptors that mediate microglia activation in response to LPS, suggesting that mGluR5 may represent a novel target for modulating

  2. Aphanomyces euteiches Cell Wall Fractions Containing Novel Glucan-Chitosaccharides Induce Defense Genes and Nuclear Calcium Oscillations in the Plant Host Medicago truncatula

    PubMed Central

    Nars, Amaury; Lafitte, Claude; Chabaud, Mireille; Drouillard, Sophie; Mélida, Hugo; Danoun, Saïda; Le Costaouëc, Tinaig; Rey, Thomas; Benedetti, Julie; Bulone, Vincent; Barker, David George; Bono, Jean-Jacques; Dumas, Bernard; Jacquet, Christophe; Heux, Laurent; Fliegmann, Judith; Bottin, Arnaud

    2013-01-01

    N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes. PMID:24086432

  3. Calcium in plant defence-signalling pathways.

    PubMed

    Lecourieux, David; Ranjeva, Raoul; Pugin, Alain

    2006-01-01

    In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

  4. Monthly Strontium/Calcium oscillations in symbiotic coral aragonite: Biological effects limiting the precision of the paleotemperature proxy

    USGS Publications Warehouse

    Meibom, A.; Stage, M.; Wooden, J.; Constantz, B.R.; Dunbar, R.B.; Owen, A.; Grumet, N.; Bacon, C.R.; Chamberlain, C.P.

    2003-01-01

    In thermodynamic equilibrium with sea water the Sr/Ca ratio of aragonite varies predictably with temperature and the Sr/Ca ratio in coral have thus become a frequently used proxy for past Sea Surface Temperature (SST). However, biological effects can offset the Sr/Ca ratio from its equilibrium value. We report high spatial resolution ion microprobe analyses of well defined skeletal elements in the reef-building coral Porites lutea that reveal distinct monthly oscillations in the Sr/Ca ratio, with an amplitude in excess of ten percent. The extreme Sr/Ca variations, which we propose result from metabolic changes synchronous with the lunar cycle, introduce variability in Sr/Ca measurements based on conventional sampling techniques well beyond the analytical precision. These variations can limit the accuracy of Sr/Ca paleothermometry by conventional sampling techniques to about 2??C. Our results may help explain the notorious difficulties involved in obtaining an accurate and consistent calibration of the Sr/Ca vs. SST relationship.

  5. Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid.

    PubMed

    Gonugunta, Vijay K; Srivastava, Nupur; Puli, Mallikarjuna R; Raghavendra, Agepati S

    2008-11-01

    Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.

  6. When Isolated at Full Receptivity, in Vitro Fertilized Wheat (Triticum aestivum, L.) Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin

    PubMed Central

    Pónya, Zsolt; Corsi, Ilaria; Hoffmann, Richárd; Kovács, Melinda; Dobosy, Anikó; Kovács, Attila Zoltán; Cresti, Mauro; Barnabás, Beáta

    2014-01-01

    During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation. PMID:25535074

  7. Calcium Activation of Mougeotia Potassium Channels 1

    PubMed Central

    Lew, Roger R.; Serlin, Bruce S.; Schauf, Charles L.; Stockton, Marsha E.

    1990-01-01

    Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca2+] as low as 20 micromolar. However, external [Ca2+] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID:16667356

  8. CYTOSOL

    EPA Pesticide Factsheets

    Technical product bulletin: this surface washing agent for oil spill cleanups extracts and recovers weathered petroleum by flotation, remaining residual hydrocarbons are biodegraded. Suitable for sensitive/ high impact sites including fisheries, rip rap.

  9. Intracellular click reaction with a fluorescent chemical Ca2+ indicator to prolong its cytosolic retention.

    PubMed

    Takei, Yoshiaki; Murata, Atsushi; Yamagishi, Kento; Arai, Satoshi; Nakamura, Hideki; Inoue, Takafumi; Takeoka, Shinji

    2013-08-25

    The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca(2+) indicator in the cytosol. Specifically, a novel clickable Ca(2+) indicator "N3-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca(2+) oscillation was observed for an extended period of time.

  10. In vivo effects of calcium entry blockers on human parathyroid adenoma cells with special reference to calcium sensing ability and the hormone secretion.

    PubMed

    Horikawa, Y; Nakajima, H; Iizuka, K; Imagawa, A; Tomita, K; Shiba, E; Takai, S; Miyagawa, J; Kuwajima, M; Namba, M; Hanafusa, T; Matsuzawa, Y

    1999-01-01

    We evaluated the effects of calcium-entry blockers on parathyroid hormone (PTH) secretion by human parathyroid adenoma cells in vitro. Nifedipine and bamidipine inhibited PTH secretion, while diltiazem had no significant effect. Cytosolic calcium concentrations were measured by use of the calcium-sensitive fluorescent dye fluo-3 with confocal laser scanning microscopy. Nifedipine increased the cytosolic concentration of calcium, whereas diltiazem decreased it. Results suggest that, in parathyroid adenoma cells, regulation of PTH secretion with respect to intracellular calcium concentration would be maintained despite differing response of intracellular calcium concentration following exposure to calcium-entry blockers.

  11. Life cycle of cytosolic prions.

    PubMed

    Hofmann, Julia; Vorberg, Ina

    2013-01-01

    Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.

  12. Life cycle of cytosolic prions

    PubMed Central

    Hofmann, Julia; Vorberg, Ina

    2013-01-01

    Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions. PMID:24021964

  13. Inositol trisphosphate and calcium signalling

    NASA Astrophysics Data System (ADS)

    Berridge, Michael J.

    1993-01-01

    Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

  14. Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1.

    PubMed

    Xu, Ningyong; Cioffi, Donna L; Alexeyev, Mikhail; Rich, Thomas C; Stevens, Troy

    2015-02-15

    Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.

  15. Calcium signals activated by ghrelin and D-Lys(3)-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells.

    PubMed

    Erriquez, Jessica; Bernascone, Silvia; Ciarletta, Monica; Filigheddu, Nicoletta; Graziani, Andrea; Distasi, Carla

    2009-09-01

    Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys(3)-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca(2+)](i) followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca(2+) entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys(3)-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca(2+) activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys(3)-GHRP-6 ghrelin antagonist features ghrelin independent activities.

  16. Calcium signalling and calcium channels: evolution and general principles.

    PubMed

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-09-15

    Calcium as a divalent cation was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules handling calcium. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, neurotransmitters, second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms.

  17. Similarities between catalase and cytosolic epoxide hydrolase.

    PubMed

    Guenthner, T M; Qato, M; Whalen, R; Glomb, S

    1989-01-01

    Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

  18. Calcium supplements

    MedlinePlus

    ... TYPES OF CALCIUM SUPPLEMENTS Forms of calcium include: Calcium carbonate: Over-the-counter (OTC) antacid products, such as Tums and Rolaids, contain calcium carbonate. These sources of calcium do not cost much. ...

  19. Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells

    PubMed Central

    Felle, Hubert; Brummer, Benno; Bertl, Adam; Parish, Roger W.

    1986-01-01

    Microelectrodes were used to measure simultaneously the effects of indole-3-acetic acid (IAA) on membrane potential and cytosolic pH of corn coleoptile cells. IAA caused an initial depolarization followed by hyperpolarization, the latter displaying rhythmic oscillations. The extent of the changes in membrane potential was dependent on IAA concentration, and hyperpolarization, but not depolarization, could be detected with concentrations of IAA as low as 10 nM. Membrane hyperpolarization was preceded by a decrease in cytosolic pH. The decrease commenced ≈5 min after adding IAA and continued for 15-20 min before reaching a new steady state ≈0.1 pH unit lower than the original pH. The decrease in pH was readily detectable after treatment with 0.1 μM IAA. Fusicoccin and acetate, which, like IAA, induce elongation growth, caused a similar drop in cytosolic pH and subsequent membrane hyperpolarization, the decrease in pH commencing within seconds. The addition of fusicoccin to IAA-treated cells resulted in a further cytosolic acidification and membrane hyperpolarization. The two substances probably change cytosolic pH via different mechanisms. The results imply that one of the primary effects of auxins in coleoptiles is to lower cytosolic pH. PMID:16593782

  20. Biochemical Oscillations and Cellular Rhythms

    NASA Astrophysics Data System (ADS)

    Goldbeter, Albert; Berridge, Foreword by M. J.

    1997-04-01

    1. Introduction; Part I. Glycolytic Oscillations: 2. Oscillatory enzymes: simple periodic behaviour in an allosteric model for glycolytic oscillations; Part II. From Simple to Complex Oscillatory Behaviour; 3. Birhythmicity: coexistence between two stable rhythms; 4. From simple periodic behaviour to complex oscillations, including bursting and chaos; Part III. Oscillations Of Cyclic Amo In Dictyostelium Cells: 5. Models for the periodic synthesis and relay of camp signals in Dictyostelium discoideum amoebae; 6. Complex oscillations and chaos in the camp signalling system of Dictyostelium; 7. The onset of camp oscillations in Dictyostelium as a model for the ontogenesis of biological rhythms; Part IV. Pulsatile Signalling In Intercellular Communication: 8. Function of the rhythm of intercellular communication in Dictyostelium. Link with pulsatile hormone secretion; Part V. Calcium Oscillations: 9. Oscillations and waves of intracellular calcium; Part VI. The Mitotic Oscillator: 10. Modelling the mitotic oscillator driving the cell division cycle; Part VII. Circadian Rhythms: 11. Towards a model for circadian oscillations in the Drosophila period protein (PER); 12. Conclusions and perspectives; References.

  1. Calcium-Mediated Abiotic Stress Signaling in Roots

    PubMed Central

    Wilkins, Katie A.; Matthus, Elsa; Swarbreck, Stéphanie M.; Davies, Julia M.

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. PMID:27621742

  2. The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

    PubMed Central

    Miller, Evan W.; Slak Rupnik, Marjan

    2013-01-01

    Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. PMID:24324777

  3. A model of propagating calcium-induced calcium release mediated by calcium diffusion

    PubMed Central

    1989-01-01

    The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading. PMID:2738577

  4. Cardiac alternans and intracellular calcium cycling

    PubMed Central

    Edwards, Joshua N.; Blatter, Lothar A.

    2014-01-01

    Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans), and Ca2+ transient amplitude (Ca2+ alternans). The cause of alternans is multifactorial, however alternans always originate from disturbances of the bi-directional coupling between membrane voltage (Vm) and intracellular calcium ([Ca2+]i). Bi-directional coupling refers to the fact that in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca2+]i that is required for contraction (a process referred to as excitation-contraction coupling), the changes of [Ca2+]i on the other hand control Vm because important membrane currents are Ca2+-dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca2+ signaling. Here we review how two key factors of cardiac cellular Ca2+ cycling - the release of Ca2+ from internal stores and the capability of clearing the cytosol from Ca2+ after each beat - determine the conditions under which alternans occurs. The contributions from key Ca2+ handling proteins - surface membrane channels, ion pumps and transporters, and internal Ca2+ release channels - are discussed. PMID:25040398

  5. Disease causing mutations of calcium channels.

    PubMed

    Lorenzon, Nancy M; Beam, Kurt G

    2008-01-01

    Calcium ions play an important role in the electrical excitability of nerve and muscle, as well as serving as a critical second messenger for diverse cellular functions. As a result, mutations of genes encoding calcium channels may have subtle affects on channel function yet strongly perturb cellular behavior. This review discusses the effects of calcium channel mutations on channel function, the pathological consequences for cellular physiology, and possible links between altered channel function and disease. Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca(2+) ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca(2+) stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores.

  6. Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

    PubMed Central

    Patel, Sandip; Docampo, Roberto

    2010-01-01

    Changes in cytosolic calcium concentration are crucial for a variety of cellular processes in all cells. It has long been appreciated that calcium is stored and released from intracellular calcium stores such as the endoplasmic reticulum. However, emerging evidence indicates that calcium is also dynamically regulated by a seemingly disparate collection of acidic organelles. Here, we review the defining features of these acidic calcium stores and highlight recent progress in understanding the mechanisms of uptake and release of calcium from these stores. We also examine the nature of calcium buffering within the stores and summarize the physiological and patho-physiological significance of these ubiquitous organelles in calcium signaling. PMID:20303271

  7. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.

    PubMed

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K

    2005-07-01

    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  8. Cytosolic selection systems to study protein stability.

    PubMed

    Malik, Ajamaluddin; Mueller-Schickert, Antje; Bardwell, James C A

    2014-12-01

    Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.

  9. Calcium Dyshomeostasis in Tubular Aggregate Myopathy

    PubMed Central

    Lee, Jong-Mok; Noguchi, Satoru

    2016-01-01

    Calcium is a crucial mediator of cell signaling in skeletal muscles for basic cellular functions and specific functions, including contraction, fiber-type differentiation and energy production. The sarcoplasmic reticulum (SR) is an organelle that provides a large supply of intracellular Ca2+ in myofibers. Upon excitation, it releases Ca2+ into the cytosol, inducing contraction of myofibrils. During relaxation, it takes up cytosolic Ca2+ to terminate the contraction. During exercise, Ca2+ is cycled between the cytosol and the SR through a system by which the Ca2+ pool in the SR is restored by uptake of extracellular Ca2+ via a specific channel on the plasma membrane. This channel is called the store-operated Ca2+ channel or the Ca2+ release-activated Ca2+ channel. It is activated by depletion of the Ca2+ store in the SR by coordination of two main molecules: stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (ORAI1). Recently, myopathies with a dominant mutation in these genes have been reported and the pathogenic mechanism of such diseases have been proposed. This review overviews the calcium signaling in skeletal muscles and role of store-operated Ca2+ entry in calcium homeostasis. Finally, we discuss the phenotypes and the pathomechanism of myopathies caused by mutations in the STIM1 and ORAI1 genes. PMID:27879676

  10. Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism.

    PubMed

    Gorodeski, G I

    2000-11-01

    Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of L-[(3)H]arginine to L-[(3)H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

  11. Cytosolic phospholipase A2: physiological function and role in disease

    PubMed Central

    Leslie, Christina C.

    2015-01-01

    The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)α, -β, -γ, -δ, -ε, and -ζ. They contain a Ser-Asp catalytic dyad and all except cPLA2γ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2α, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2α catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2α is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2α is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2α activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2α KO mice. However, humans recently identified with cPLA2α deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2α, indicating that much remains to be learned about this interesting enzyme. PMID:25838312

  12. Cytosolic phospholipase A₂: physiological function and role in disease.

    PubMed

    Leslie, Christina C

    2015-08-01

    The group IV phospholipase A2 (PLA2) family is comprised of six intracellular enzymes (GIVA, -B, -C, -D, -E, and -F) commonly referred to as cytosolic PLA2 (cPLA2)α, -β, -γ, -δ, -ε, and -ζ. They contain a Ser-Asp catalytic dyad and all except cPLA2γ have a C2 domain, but differences in their catalytic activities and subcellular localization suggest unique regulation and function. With the exception of cPLA2α, the focus of this review, little is known about the in vivo function of group IV enzymes. cPLA2α catalyzes the hydrolysis of phospholipids to arachidonic acid and lysophospholipids that are precursors of numerous bioactive lipids. The regulation of cPLA2α is complex, involving transcriptional and posttranslational processes, particularly increases in calcium and phosphorylation. cPLA2α is a highly conserved widely expressed enzyme that promotes lipid mediator production in human and rodent cells from a variety of tissues. The diverse bioactive lipids produced as a result of cPLA2α activation regulate normal physiological processes and disease pathogenesis in many organ systems, as shown using cPLA2α KO mice. However, humans recently identified with cPLA2α deficiency exhibit more pronounced effects on health than observed in mice lacking cPLA2α, indicating that much remains to be learned about this interesting enzyme.

  13. Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

    PubMed

    Takaoka, Yousuke; Shigenaga, Miyuki; Imai, Masaki; Nukadzuka, Yuuki; Ishimaru, Yasuhiro; Saito, Kei; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ueda, Minoru

    2016-01-01

    In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

  14. Neurodynamic oscillators

    NASA Technical Reports Server (NTRS)

    Espinosa, Ismael; Gonzalez, Hortensia; Quiza, Jorge; Gonazalez, J. Jesus; Arroyo, Ruben; Lara, Ritaluz

    1995-01-01

    Oscillation of electrical activity has been found in many nervous systems, from invertebrates to vertebrates including man. There exists experimental evidence of very simple circuits with the capability of oscillation. Neurons with intrinsic oscillation have been found and also neural circuits where oscillation is a property of the network. These two types of oscillations coexist in many instances. It is nowadays hypothesized that behind synchronization and oscillation there is a system of coupled oscillators responsible for activities that range from locomotion and feature binding in vision to control of sleep and circadian rhythms. The huge knowledge that has been acquired on oscillators from the times of Lord Rayleigh has made the simulation of neural oscillators a very active endeavor. This has been enhanced with more recent physiological findings about small neural circuits by means of intracellular and extracellular recordings as well as imaging methods. The future of this interdisciplinary field looks very promising; some researchers are going into quantum mechanics with the idea of trying to provide a quantum description of the brain. In this work we describe some simulations using neuron models by means of which we form simple neural networks that have the capability of oscillation. We analyze the oscillatory activity with root locus method, cross-correlation histograms, and phase planes. In the more complicated neural network models there is the possibility of chaotic oscillatory activity and we study that by means of Lyapunov exponents. The companion paper shows an example of that kind.

  15. Increased oxidative-modifications of cytosolic proteins in 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-exposed rat liver.

    PubMed

    Upreti, Vijay V; Moon, Kwan-Hoon; Yu, Li-Rong; Lee, Insong J; Eddington, Natalie D; Ye, Xiaoying; Veenstra, Timothy D; Song, Byoung-Joon

    2011-01-01

    It is well established that 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA-exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2-DE analysis showed markedly increased levels of biotin-N-methylimide-labeled oxidized cytosolic proteins in MDMA-exposed rats compared to vehicle-treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti-oxidant defensive enzymes, a calcium-binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c-Jun N-terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti-apoptotic Bcl-2 protein, their activation may promote apoptosis in MDMA-exposed tissues. Our results show for the first time that MDMA induces oxidative-modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.

  16. Do calcium buffers always slow down the propagation of calcium waves?

    PubMed

    Tsai, Je-Chiang

    2013-12-01

    Calcium buffers are large proteins that act as binding sites for free cytosolic calcium. Since a large fraction of cytosolic calcium is bound to calcium buffers, calcium waves are widely observed under the condition that free cytosolic calcium is heavily buffered. In addition, all physiological buffered excitable systems contain multiple buffers with different affinities. It is thus important to understand the properties of waves in excitable systems with the inclusion of buffers. There is an ongoing controversy about whether or not the addition of calcium buffers into the system always slows down the propagation of calcium waves. To solve this controversy, we incorporate the buffering effect into the generic excitable system, the FitzHugh-Nagumo model, to get the buffered FitzHugh-Nagumo model, and then to study the effect of the added buffer with large diffusivity on traveling waves of such a model in one spatial dimension. We can find a critical dissociation constant (K = K(a)) characterized by system excitability parameter a such that calcium buffers can be classified into two types: weak buffers (K ∈ (K(a), ∞)) and strong buffers (K ∈ (0, K(a))). We analytically show that the addition of weak buffers or strong buffers but with its total concentration b(0)(1) below some critical total concentration b(0,c)(1) into the system can generate a traveling wave of the resulting system which propagates faster than that of the origin system, provided that the diffusivity D1 of the added buffers is sufficiently large. Further, the magnitude of the wave speed of traveling waves of the resulting system is proportional to √D1 as D1 --> ∞. In contrast, the addition of strong buffers with the total concentration b(0)(1) > b(0,c)(1) into the system may not be able to support the formation of a biologically acceptable wave provided that the diffusivity D1 of the added buffers is sufficiently large.

  17. Privileged crosstalk between TRPV1 channels and mitochondrial calcium shuttling machinery controls nociception.

    PubMed

    Nita, Iulia I; Caspi, Yaki; Gudes, Sagi; Fishman, Dimitri; Lev, Shaya; Hersfinkel, Michal; Sekler, Israel; Binshtok, Alexander M

    2016-12-01

    The nociceptive noxious heat-activated receptor - TRPV1, conducts calcium and sodium, thus producing a depolarizing receptor potential, leading to activation of nociceptive neurons. TRPV1-mediated calcium and sodium influx is negatively modulated by calcium, via calcium-dependent desensitization of TRPV1 channels. A mitochondrial Ca(2+) uniporter - MCU, controls mitochondrial Ca(2+) entry while a sodium/calcium transporter - NCLX shapes calcium and sodium transients by mediating sodium entry into and removing calcium from the mitochondria. The functional interplay between TRPV1, MCU and NCLX, in controlling the cytosolic and mitochondrial calcium and sodium transients and subsequently the nociceptive excitability, is poorly understood. Here, we used cytosolic and mitochondrial fluorescent calcium and sodium imaging together with electrophysiological recordings of TRPV1-induced currents in HEK293T cells and nociceptor-like dissociated rat dorsal root ganglion neurons, while modulating NCLX or MCU expression using specific small interfering RNA (siNCLX). We show that the propagation of the TRPV1-induced cytosolic calcium and sodium fluxes into mitochondria is dependent on coordinated activity of NCLX and MCU. Thus, knocking-down of NCLX triggers down regulation of MCU dependent mitochondrial Ca(2+) uptake. This in turn decreases rate and amplitude of TRPV1-mediated cytosolic calcium, which inhibits capsaicin-induced inward current and neuronal firing. TRPV1-mediated currents were fully rescued by intracellular inclusion of the fast calcium chelator BAPTA. Finally, NCLX controls capsaicin-induced cell death, by supporting massive mitochondrial Ca(2+) shuttling. Altogether, our results suggest that NCLX, by regulating cytosolic and mitochondrial ionic transients, modulates calcium-dependent desensitization of TRPV1 channels, thereby, controlling nociceptive signaling.

  18. Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement.

    PubMed

    Basset, Olivier; Boittin, François-Xavier; Dorchies, Olivier M; Chatton, Jean-Yves; van Breemen, Cornelis; Ruegg, Urs T

    2004-11-05

    In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.

  19. Calcium - urine

    MedlinePlus

    ... into the urine, which causes calcium kidney stones Sarcoidosis Taking too much calcium Too much production of ... Milk-alkali syndrome Proximal renal tubular acidosis Rickets Sarcoidosis Vitamin D Review Date 5/3/2015 Updated ...

  20. Interactions of cytosolic sulfotransferases with xenobiotics.

    PubMed

    James, Margaret O; Ambadapadi, Sriram

    2013-11-01

    Cytosolic sulfotransferases are a superfamily of enzymes that catalyze the transfer of the sulfonic group from 3'-phosphoadenosine-5'-phosphosulfate to hydroxy or amine groups in substrate molecules. The human cytosolic sulfotransferases that have been most studied, namely SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1, are expressed in different tissues of the body, including liver, intestine, adrenal, brain and skin. These sulfotransferases play important roles in the sulfonation of endogenous molecules such as steroid hormones and neurotransmitters, and in the elimination of xenobiotic molecules such as drugs, environmental chemicals and natural products. There is often overlapping substrate selectivity among the sulfotransferases, although one isoform may exhibit greater enzyme efficiency than other isoforms. Similarly, inhibitors or enhancers of one isoform often affect other isoforms, but typically with different potency. This means that if the activity of one form of sulfotransferase is altered (either inhibited or enhanced) by the presence of a xenobiotic, the sulfonation of endogenous and xenobiotic substrates for other isoforms may well be affected. There are more examples of inhibitors than enhancers of sulfonation. Modulators of sulfotransferase enzymes include natural products ingested as part of the human diet as well as environmental chemicals and drugs. This review will discuss recent work on such interactions.

  1. Calcium in Plants

    PubMed Central

    WHITE, PHILIP J.; BROADLEY, MARTIN R.

    2003-01-01

    Calcium is an essential plant nutrient. It is required for various structural roles in the cell wall and membranes, it is a counter‐cation for inorganic and organic anions in the vacuole, and the cytosolic Ca2+ concentration ([Ca2+]cyt) is an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges. This article provides an overview of the nutritional requirements of different plants for Ca, and how this impacts on natural flora and the Ca content of crops. It also reviews recent work on (a) the mechanisms of Ca2+ transport across cellular membranes, (b) understanding the origins and specificity of [Ca2+]cyt signals and (c) characterizing the cellular [Ca2+]cyt‐sensors (such as calmodulin, calcineurin B‐like proteins and calcium‐dependent protein kinases) that allow plant cells to respond appropriately to [Ca2+]cyt signals. PMID:12933363

  2. Cytosolic delivery of materials with endosome-disrupting colloids

    DOEpatents

    Helms, Brett A.; Bayles, Andrea R.

    2016-03-15

    A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

  3. Store-operated calcium entry is essential for glial calcium signalling in CNS white matter.

    PubMed

    Papanikolaou, M; Lewis, A; Butt, A M

    2017-02-28

    'Calcium signalling' is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca(2+) stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca(2+)]o) resulted in a marked depletion of glial cytosolic calcium ([Ca(2+)]i), which recovered rapidly on restoration of [Ca(2+)]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in white matter glia.

  4. Calcium signalling and calcium channels: Evolution and general principles

    PubMed Central

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-01-01

    Calcium as a divalent ion was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, by neurotransmitters, by second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms. PMID:24291103

  5. Protein Aggregation Profile of the Bacterial Cytosol

    PubMed Central

    de Groot, Natalia S.; Ventura, Salvador

    2010-01-01

    Background Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings Here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance Overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms. PMID:20195530

  6. Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2.

    PubMed

    Chen, Min; Wang, Yanru; Hou, Tingting; Zhang, Huiliang; Qu, Aijuan; Wang, Xianhua

    2011-10-01

    Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

  7. Cytosolic [Ca2+] signaling pathway in macula densa cells.

    PubMed

    Peti-Peterdi, J; Bell, P D

    1999-09-01

    Previous micropuncture studies suggested that macula densa (MD) cells might detect variations in luminal sodium chloride concentration ([NaCl]l) through changes in cytosolic calcium ([Ca2+]c). To test this hypothesis, MD [Ca2+]c was measured with fluorescence microscopy using fura 2 in the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney. Tubules were bathed and perfused with a Ringer solution, [NaCl]l was varied and isosmotically replaced with N-methyl-D-glucamine cyclamate. Control [Ca2+]c, during perfusion with 25 mM NaCl and 150 mM NaCl in the bath, averaged 101. 6 +/- 8.2 nM (n = 21). Increasing [NaCl]l to 150 mM elevated [Ca2+]c by 39.1 +/- 5.2 nM (n = 21, P < 0.01). This effect was concentration dependent between zero and 60 mM [NaCl]l. The presence of either luminal furosemide or basolateral nifedipine or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a potent Cl- channel blocker, significantly reduced resting [Ca2+]c and abolished the increase in [Ca2+]c in response to increased [NaCl]l. Nifedipine failed to produce a similar inhibitory effect when added exclusively to the luminal perfusate. Also, 100 nM BAY K 8644, a voltage-gated Ca2+ channel agonist, added to the bathing solution increased [Ca2+]c by 33.2 +/- 8.1 nM (n = 5, P < 0.05). These observations suggest that MD cells may detect variations in [NaCl]l through a signaling pathway that includes Na+-2Cl--K+ cotransport, basolateral membrane depolarization via Cl- channels, and Ca2+ entry through voltage-gated Ca2+ channels.

  8. Decoding of calcium signal through calmodulin: calmodulin-binding proteins in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many abiotic and biotic stimuli such as heat, cold, drought, salt, light, wind, touch, wounding, symbionts and pathogens as well as growth, developmental and hormonal cues can quickly induce cytosolic calcium increases. Calmodulin, the most thoroughly studied calcium sensor, mediates interpretation...

  9. Power oscillator

    DOEpatents

    Gitsevich, Aleksandr

    2001-01-01

    An oscillator includes an amplifier having an input and an output, and an impedance transformation network connected between the input of the amplifier and the output of the amplifier, wherein the impedance transformation network is configured to provide suitable positive feedback from the output of the amplifier to the input of the amplifier to initiate and sustain an oscillating condition, and wherein the impedance transformation network is configured to protect the input of the amplifier from a destructive feedback signal. One example of the oscillator is a single active element device capable of providing over 70 watts of power at over 70% efficiency. Various control circuits may be employed to match the driving frequency of the oscillator to a plurality of tuning states of the lamp.

  10. Calcium pumps in the central nervous system.

    PubMed

    Mata, Ana M; Sepúlveda, M Rosario

    2005-09-01

    Two families of Ca2+ transport ATPases are involved in the maintenance of Ca2+ homeostasis in the nervous system, the plasma membrane Ca2+-ATPase that pumps Ca2+ to the extracellular medium and the intracellular sarco/endoplasmic reticulum Ca2+-ATPase that transports Ca2+ from the cytosol to the endoplasmic reticulum. Both types of calcium pumps show precise regulatory properties and they are localized in specific subcellular regions. In this review, we describe the functional and regulatory properties of both families of calcium pumps, their distribution in nerve cells, and their involvement in neurological disorders. The functional characterization of neuronal calcium pumps is very important in order to understand the biochemical processes involved in the maintenance of intracellular calcium in synaptic terminals.

  11. Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+

    PubMed Central

    Villalón, M; Hinds, T R; Verdugo, P

    1989-01-01

    Changes of cytosolic [Ca2+] have been proposed to couple stimulation of ciliary movement, however, quantitative measurements of fluctuations of intracellular free [Ca2+] associated with stimulation of ciliated cells have not been investigated. In primary cultures of rabbit oviductal ciliated cells, the stimulation of ciliary activity produced by micromolar concentrations of adenosine triphosphate (ATP) and prostaglandin F2 alpha (PGF2 alpha) was associated with a transient increase of intracellular [Ca2+]. Whereas the increase of cytosolic [Ca2+] and beat frequency produced by ATP were inhibited by the Ca-channel blocker LaCl3, the rise of cytosolic [Ca2+] and frequency of ciliary beat produced by PGF2 alpha was not affected by LaCl3. These results are the first direct demonstration that fluctuations of cytosolic [Ca2+] are associated with increased ciliary beat frequency in mammalian epithelial cells. The present findings suggest two different calcium-dependent mechanisms for stimulus-coupling in ciliary epithelium: ATP acting via purinergic receptor coupled to transmembrane influx of Ca2+, and PGF2 alpha acting via receptor-mediated release of intracellular sequestered Ca. PMID:2611335

  12. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  13. Yeast respond to hypotonic shock with a calcium pulse

    NASA Technical Reports Server (NTRS)

    Batiza, A. F.; Schulz, T.; Masson, P. H.

    1996-01-01

    We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

  14. Spatiotemporal intracellular calcium dynamics during cardiac alternans

    PubMed Central

    Restrepo, Juan G.; Karma, Alain

    2009-01-01

    Cellular calcium transient alternans are beat-to-beat alternations in the peak cytosolic calcium concentration exhibited by cardiac cells during rapid electrical stimulation or under pathological conditions. Calcium transient alternans promote action potential duration alternans, which have been linked to the onset of life-threatening ventricular arrhythmias. Here we use a recently developed physiologically detailed mathematical model of ventricular myocytes to investigate both stochastic and deterministic aspects of intracellular calcium dynamics during alternans. The model combines a spatially distributed description of intracellular calcium cycling, where a large number of calcium release units are spatially distributed throughout the cell, with a full set of ionic membrane currents. The results demonstrate that ion channel stochasticity at the level of single calcium release units can influence the whole-cell alternans dynamics by causing phase reversals over many beats during fixed frequency pacing close to the alternans bifurcation. They also demonstrate the existence of a wide range of dynamical states. Depending on the sign and magnitude of calcium-voltage coupling, calcium alternans can be spatially synchronized or desynchronized, in or out of phase with action potential duration alternans, and the node separating out-of-phase regions of calcium alternans can be expelled from or trapped inside the cell. This range of states is found to be larger than previously anticipated by including a robust global attractor where calcium alternans can be spatially synchronized but out of phase with action potential duration alternans. The results are explained by a combined theoretical analysis of alternans stability and node motion using general iterative maps of the beat-to-beat dynamics and amplitude equations. PMID:19792040

  15. Calcium in diet

    MedlinePlus

    ... of calcium dietary supplements include calcium citrate and calcium carbonate. Calcium citrate is the more expensive form of ... the body on a full or empty stomach. Calcium carbonate is less expensive. It is absorbed better by ...

  16. Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration.

    PubMed Central

    Shannon, T R; Ginsburg, K S; Bers, D M

    2000-01-01

    Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process. PMID:10620297

  17. Calcium - ionized

    MedlinePlus

    ... 245. Read More Acute kidney failure Albumin - blood (serum) test Bone tumor Calcium blood test Hyperparathyroidism Hypoparathyroidism Malabsorption Milk-alkali syndrome Multiple myeloma Osteomalacia Paget disease of the bone Rickets Sarcoidosis Vitamin D Review ...

  18. Calcium Carbonate.

    PubMed

    Al Omari, M M H; Rashid, I S; Qinna, N A; Jaber, A M; Badwan, A A

    2016-01-01

    Calcium carbonate is a chemical compound with the formula CaCO3 formed by three main elements: carbon, oxygen, and calcium. It is a common substance found in rocks in all parts of the world (most notably as limestone), and is the main component of shells of marine organisms, snails, coal balls, pearls, and eggshells. CaCO3 exists in different polymorphs, each with specific stability that depends on a diversity of variables.

  19. Cytosolic Innate Immune Sensing and Signaling upon Infection

    PubMed Central

    Radoshevich, Lilliana; Dussurget, Olivier

    2016-01-01

    Cytosolic sensing of pathogens is essential to a productive immune response. Recent reports have emphasized the importance of signaling platforms emanating from organelles and cytosolic sensors, particularly during the response to intracellular pathogens. Here, we highlight recent discoveries identifying the key mediators of nucleic acid and cyclic nucleotide sensing and discuss their importance in host defense. This review will also cover strategies evolved by pathogens to manipulate these pathways. PMID:27014235

  20. Prevention of bone mineral changes induced by bed rest: Modification by static compression simulating weight bearing, combined supplementation of oral calcium and phosphate, calcitonin injections, oscillating compression, the oral diophosphonatedisodium etidronate, and lower body negative pressure

    NASA Technical Reports Server (NTRS)

    Schneider, V. S.; Hulley, S. B.; Donaldson, C. L.; Vogel, J. M.; Rosen, S. N.; Hantman, D. A.; Lockwood, D. R.; Seid, D.; Hyatt, K. H.; Jacobson, L. B.

    1974-01-01

    The phenomenon of calcium loss during bed rest was found to be analogous to the loss of bone material which occurs in the hypogravic environment of space flight. Ways of preventing this occurrence are investigated. A group of healthy adult males underwent 24-30 weeks of continuous bed rest. Some of them were given an exercise program designed to resemble normal ambulatory activity; another subgroup was fed supplemental potassium phosphate. The results from a 12-week period of treatment were compared with those untreated bed rest periods. The potassium phosphate supplements prevented the hypercalciuria of bed rest, but fecal calcium tended to increase. The exercise program did not diminish the negative calcium balance. Neither treatment affected the heavy loss of mineral from the calcaneus. Several additional studies are developed to examine the problem further.

  1. Calcium Hydroxylapatite

    PubMed Central

    Yutskovskaya, Yana Alexandrovna; Philip Werschler, WM.

    2015-01-01

    Background: Calcium hydroxylapatite is one of the most well-studied dermal fillers worldwide and has been extensively used for the correction of moderate-to-severe facial lines and folds and to replenish lost volume. Objectives: To mark the milestone of 10 years of use in the aesthetic field, this review will consider the evolution of calcium hydroxylapatite in aesthetic medicine, provide a detailed injection protocol for a global facial approach, and examine how the unique properties of calcium hydroxylapatite provide it with an important place in today’s market. Methods: This article is an up-to-date review of calcium hydroxylapatite in aesthetic medicine along with procedures for its use, including a detailed injection protocol for a global facial approach by three expert injectors. Conclusion: Calcium hydroxylapatite is a very effective agent for many areas of facial soft tissue augmentation and is associated with a high and well-established safety profile. Calcium hydroxylapatite combines high elasticity and viscosity with an ability to induce long-term collagen formation making it an ideal agent for a global facial approach. PMID:25610523

  2. Calcium orthophosphates

    PubMed Central

    Dorozhkin, Sergey V.

    2011-01-01

    The present overview is intended to point the readers’ attention to the important subject of calcium orthophosphates. This type of materials is of special significance for human beings, because they represent the inorganic part of major normal (bones, teeth and antlers) and pathological (i.e., those appearing due to various diseases) calcified tissues of mammals. For example, atherosclerosis results in blood vessel blockage caused by a solid composite of cholesterol with calcium orthophosphates, while dental caries and osteoporosis mean a partial decalcification of teeth and bones, respectively, that results in replacement of a less soluble and harder biological apatite by more soluble and softer calcium hydrogenphosphates. Therefore, the processes of both normal and pathological calcifications are just an in vivo crystallization of calcium orthophosphates. Similarly, dental caries and osteoporosis might be considered an in vivo dissolution of calcium orthophosphates. Thus, calcium orthophosphates hold a great significance for humankind, and in this paper, an overview on the current knowledge on this subject is provided. PMID:23507744

  3. Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks.

    PubMed

    Montero, Mayte; Lobatón, Carmen D; Gutierrez-Fernández, Silvia; Moreno, Alfredo; Alvarez, Javier

    2003-12-12

    In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.

  4. Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+

    PubMed Central

    Waldeck-Weiermair, Markus; Malli, Roland; Parichatikanond, Warisara; Gottschalk, Benjamin; Madreiter-Sokolowski, Corina T.; Klec, Christiane; Rost, Rene; Graier, Wolfgang F.

    2015-01-01

    Mitochondrial Ca2+ uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca2+ uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca2+ channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca2+ signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca2+ rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca2+ uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca2+ rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca2+ concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells. PMID:26489515

  5. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

    PubMed Central

    Brandes, R; Figueredo, V M; Camacho, S A; Baker, A J; Weiner, M W

    1993-01-01

    Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+. PMID:8298027

  6. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

    PubMed

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  7. FEL Oscillators

    SciTech Connect

    George Neil

    2003-05-12

    FEL Oscillators have been around since 1977 providing not only a test bed for the physics of Free Electron Lasers and electron/photon interactions but as a workhorse of scientific research. More than 30 FEL oscillators are presently operating around the world spanning a wavelength range from the mm region to the ultraviolet using DC and rf linear accelerators and storage rings as electron sources. The characteristics that have driven the development of these sources are the desire for high peak and average power, high micropulse energies, wavelength tunability, timing flexibility, and wavelengths that are unavailable from more conventional laser sources. Substantial user programs have been performed using such sources encompassing medicine, biology, solid state research, atomic and molecular physics, effects of non-linear fields, surface science, polymer science, pulsed laser vapor deposition, to name just a few.

  8. STABILIZED OSCILLATOR

    DOEpatents

    Jessen, P.L.; Price, H.J.

    1958-03-18

    This patent relates to sine-wave generators and in particular describes a generator with a novel feedback circuit resulting in improved frequency stability. The generator comprises two triodes having a common cathode circuit connected to oscillate at a frequency and amplitude at which the loop galn of the circutt ls unity, and another pair of triodes having a common cathode circuit arranged as a conventional amplifier. A signal is conducted from the osciliator through a frequency selective network to the amplifier and fed back to the osciliator. The unique feature of the feedback circuit is the amplifier operates in the nonlinear portion of its tube characteristics thereby providing a relatively constant feedback voltage to the oscillator irrespective of the amplitude of its input signal.

  9. Doxorubicin Regulates Autophagy Signals via Accumulation of Cytosolic Ca2+ in Human Cardiac Progenitor Cells

    PubMed Central

    Park, Ji Hye; Choi, Sung Hyun; Kim, Hyungtae; Ji, Seung Taek; Jang, Woong Bi; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang Mo

    2016-01-01

    Doxorubicin (DOXO) is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage. Resident cardiac progenitor cells (hCPCs) act as key regulators of homeostasis in myocardial cells. However, little is known about the function of hCPCs in DOXO-induced cardiotoxicity. In this study, we found that DOXO-mediated hCPC toxicity is closely related to calcium-related autophagy signaling and was significantly attenuated by blocking mTOR signaling in human hCPCs. DOXO induced hCPC apoptosis with reduction of SMP30 (regucalcin) and autophagosome marker LC3, as well as remarkable induction of the autophagy-related markers, Beclin-1, APG7, and P62/SQSTM1 and induction of calcium-related molecules, CaM (Calmodulin) and CaMKII (Calmodulin kinase II). The results of an LC3 puncta assay further indicated that DOXO reduced autophagosome formation via accumulation of cytosolic Ca2+. Additionally, DOXO significantly induced mTOR expression in hCPCs, and inhibition of mTOR signaling by rapamycin, a specific inhibitor, rescued DOXO-mediated autophagosome depletion in hCPCs with significant reduction of DOXO-mediated cytosolic Ca2+ accumulation in hCPCs, and restored SMP30 and mTOR expression. Thus, DOXO-mediated hCPC toxicity is linked to Ca2+-related autophagy signaling, and inhibition of mTOR signaling may provide a cardio-protective effect against DOXO-mediated hCPC toxicity. PMID:27735842

  10. Interplay Between Intracellular Ca2+ Oscillations and Ca2+-stimulated Mitochondrial Metabolism

    PubMed Central

    Wacquier, Benjamin; Combettes, Laurent; Van Nhieu, Guy Tran; Dupont, Geneviève

    2016-01-01

    Oscillations of cytosolic Ca2+ concentration are a widespread mode of signalling. Oscillatory spikes rely on repetitive exchanges of Ca2+ between the endoplasmic reticulum (ER) and the cytosol, due to the regulation of inositol 1,4,5-trisphosphate receptors. Mitochondria also sequester and release Ca2+, thus affecting Ca2+ signalling. Mitochondrial Ca2+ activates key enzymes involved in ATP synthesis. We propose a new integrative model for Ca2+ signalling and mitochondrial metabolism in electrically non-excitable cells. The model accounts for (1) the phase relationship of the Ca2+ changes in the cytosol, the ER and mitochondria, (2) the dynamics of mitochondrial metabolites in response to cytosolic Ca2+ changes, and (3) the impacts of cytosol/mitochondria Ca2+ exchanges and of mitochondrial metabolism on Ca2+ oscillations. Simulations predict that as expected, oscillations are slowed down by decreasing the rate of Ca2+ efflux from mitochondria, but also by decreasing the rate of Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU). These predictions were experimentally validated by inhibiting MCU expression. Despite the highly non-linear character of Ca2+ dynamics and mitochondrial metabolism, bioenergetics were found to be robust with respect to changes in frequency and amplitude of Ca2+ oscillations. PMID:26776859

  11. Solar Oscillations

    NASA Technical Reports Server (NTRS)

    Duvall, Thomas

    2004-01-01

    Oscillations were first detected in the solar photosphere in 1962 by Leighton and students. In 1970 it was calculated that these oscillations, with a period near five minutes, were the manifestations of acoustic waves trapped in the interior. The subsequent measurements of the frequencies of global oscillation modes from the spatio-temporal power spectrum of the waves made possible the refinement of solar interior models. Over the years, increased understanding of the nuclear reaction rates, the opacity, the equation of state, convection, and gravitational settling have resulted. Mass flows shift the frequencies of modes leading to very accurate measurements of the interior rotation as a function of radius and latitude. In recent years, analogues of terrestrial seismology have led to a tomography of the interior, including measurements of global north-south flows and flow and wave speed measurements below features such as sunspots. The future of helioseismology seems bright with the approval of NASA's Solar Dynamics Observatory mission, to be launched in 2008.

  12. Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

    PubMed Central

    Asmat, Tauseef M.; Tenenbaum, Tobias; Jonsson, Ann-Beth

    2014-01-01

    The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells. PMID:25464500

  13. Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

    NASA Technical Reports Server (NTRS)

    Johnson, R. F.; Beltz, T. G.; Sharma, R. V.; Xu, Z.; Bhatty, R. A.; Johnson, A. K.

    2001-01-01

    The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

  14. Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions.

    PubMed

    Dombrowski, Yvonne; Peric, Mark; Koglin, Sarah; Kammerbauer, Claudia; Göss, Christine; Anz, David; Simanski, Maren; Gläser, Regine; Harder, Jürgen; Hornung, Veit; Gallo, Richard L; Ruzicka, Thomas; Besch, Robert; Schauber, Jürgen

    2011-05-11

    The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

  15. Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

    PubMed

    Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

    2017-03-16

    Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

  16. Calcium homeostasis in barley aleurone

    SciTech Connect

    Jones, R.L.

    1990-02-21

    Under the auspices of the Department of Energy we investigated calcium homeostasis in aleurone cells of barley. This investigation was initiated to explore the role played by extracellular Ca{sup 2+} in gibberellic acid (GA)-induced synthesis and secretion of hydrolases in the aleurone layer. We have focused our attention on four topics that relate to the role of Ca{sup 2+} in regulating the synthesis of {alpha}-amylase. First, we determined the stoichiometry of Ca{sup 2+} binding to the two principal classes of barley {alpha}-amylase and examined some of the biochemical and physical properties of the native and Ca{sup 2+}-depleted forms of the enzyme. Second, since {alpha}-amylase is a Ca{sup 2+} containing metalloenzyme that binds one atom of Ca{sup 2+} per molecule, we developed methods to determine the concentration of Ca{sup 2+} in the cytosol of the aleurone cell. We developed a technique for introducing Ca{sup 2+}-sensitive dyes into aleurone protoplasts that allows the measurement of Ca{sup 2+} in both cytosol and endoplasmic reticulum (ER). Third, because the results of our Ca{sup 2+} measurements showed higher levels of Ca{sup 2+} in the ER than in the cytosol, we examined Ca{sup 2+} transport into the ER of control and GA-treated aleurone tissue. And fourth, we applied the technique of patch-clamping to the barley aleurone protoplast to examine ion transport at the plasma membrane. Our results with the patch-clamp technique established the presence of K{sup +} channels in the plasma membrane of the aleurone protoplast, and they showed that this cell is ideally suited for the application of this methodology for studying ion transport. 34 refs.

  17. Coupled oscillator model of the dopaminergic neuron of the substantia nigra.

    PubMed

    Wilson, C J; Callaway, J C

    2000-05-01

    Calcium imaging using fura-2 and whole cell recording revealed the effective location of the oscillator mechanism on dopaminergic neurons of the substantia nigra, pars compacta, in slices from rats aged 15-20 days. As previously reported, dopaminergic neurons fired in a slow rhythmic single spiking pattern. The underlying membrane potential oscillation survived blockade of sodium currents with TTX and was enhanced by blockade of voltage-sensitive potassium currents with TEA. Calcium levels increased during the subthreshold depolarizing phase of the membrane potential oscillation and peaked at the onset of the hyperpolarizing phase as expected if the pacemaker potential were due to a low-threshold calcium current and the hyperpolarizing phase to calcium-dependent potassium current. Calcium oscillations were synchronous in the dendrites and soma and were greater in the dendrites than in the soma. Average calcium levels in the dendrites overshot steady-state levels and decayed over the course of seconds after the oscillation was resumed after having been halted by hyperpolarizing currents. Average calcium levels in the soma increased slowly, taking many cycles to achieve steady state. Voltage clamp with calcium imaging revealed the voltage dependence of the somatic calcium current without the artifacts of incomplete spatial voltage control. This showed that the calcium current had little or no inactivation and was half-maximal at -40 to -30 mV. The time constant of calcium removal was measured by the return of calcium to resting levels and depended on diameter. The calcium sensitivity of the calcium-dependent potassium current was estimated by plotting the slow tail current against calcium concentration during the decay of calcium to resting levels at -60 mV. A single compartment model of the dopaminergic neuron consisting of a noninactivating low-threshold calcium current, a calcium-dependent potassium current, and a small leak current reproduced most features of the

  18. Calcium Test

    MedlinePlus

    ... if a person has symptoms of a parathyroid disorder , malabsorption , or an overactive thyroid. A total calcium level is often measured as part of a routine health screening. It is included in the comprehensive metabolic panel (CMP) and the basic metabolic panel (BMP) , ...

  19. Calcium cyanide

    Integrated Risk Information System (IRIS)

    Jump to main content . Integrated Risk Information System Recent Additions | Contact Us Search : All EPA IRIS • You are here : EPA Home • Research • Environmental Assessment • IRIS • IRIS Summaries Redirect Page As of September 28 , 2010 , the assessment summary for calcium cyanide is included in th

  20. Lipid Biosynthesis Coordinates a Mitochondrial-to-Cytosolic Stress Response.

    PubMed

    Kim, Hyun-Eui; Grant, Ana Rodrigues; Simic, Milos S; Kohnz, Rebecca A; Nomura, Daniel K; Durieux, Jenni; Riera, Celine E; Sanchez, Melissa; Kapernick, Erik; Wolff, Suzanne; Dillin, Andrew

    2016-09-08

    Defects in mitochondrial metabolism have been increasingly linked with age-onset protein-misfolding diseases such as Alzheimer's, Parkinson's, and Huntington's. In response to protein-folding stress, compartment-specific unfolded protein responses (UPRs) within the ER, mitochondria, and cytosol work in parallel to ensure cellular protein homeostasis. While perturbation of individual compartments can make other compartments more susceptible to protein stress, the cellular conditions that trigger cross-communication between the individual UPRs remain poorly understood. We have uncovered a conserved, robust mechanism linking mitochondrial protein homeostasis and the cytosolic folding environment through changes in lipid homeostasis. Metabolic restructuring caused by mitochondrial stress or small-molecule activators trigger changes in gene expression coordinated uniquely by both the mitochondrial and cytosolic UPRs, protecting the cell from disease-associated proteins. Our data suggest an intricate and unique system of communication between UPRs in response to metabolic changes that could unveil new targets for diseases of protein misfolding.

  1. Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

    PubMed

    Hou, Bi-Huei; Takanaga, Hitomi; Griesbeck, Oliver; Frommer, Wolf B

    2009-08-01

    Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.

  2. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition.

    PubMed

    Wang, Hui; Klein, Michael G; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-03

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  3. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    SciTech Connect

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  4. Potential mechanisms of cytosolic calcium modulation in interferon-gamma treated U937 cells

    NASA Technical Reports Server (NTRS)

    Klein, Jon B.; Mcleish, Kenneth R.; Sonnenfeld, Gerald; Dean, William L.

    1987-01-01

    The ability of interferon-gamma (IFN-gamma) to alter cytoplasmic Ca(2+) content in the monocytelike cell line U937 was investigated, using a slow Ca-channel blocker, diltiazem. In addition, the Ca-ATPase and the Ca-uptake activities were measured in isolated U937 membranes, together with the effect of inositol trisphosphate (IP3) upon the Ca(2+) release from Ca-loaded membranes. The addition of 50 U/ml INF-gamma to U937 cultures was found to increase internal Ca(2+) by about 100 percent within 3 min. The increase was significantly reduced by incubation in Ca-free buffer or by the addition of diltiazem. A crude membrane preparation from U937 cells was found to contain significant amounts of Ca-ATPase activity and to sequester Ca(2+) to a level of 8 nmol/mg in 30 sec; the addition of IP3 induced release of a portion of the sequestered Ca(2+) which was then resequestered. The results suggest that IFN-gamma causes an increase of cytoplasmic Ca(2+), in part, by the IP3-induced release from the internal storage sites and, in part, from the entry of extracellular Ca through slow channels.

  5. Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance

    PubMed Central

    1991-01-01

    The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm. PMID:2037838

  6. Grid oscillators

    NASA Technical Reports Server (NTRS)

    Popovic, Zorana B.; Kim, Moonil; Rutledge, David B.

    1988-01-01

    Loading a two-dimensional grid with active devices offers a means of combining the power of solid-state oscillators in the microwave and millimeter-wave range. The grid structure allows a large number of negative resistance devices to be combined. This approach is attractive because the active devices do not require an external locking signal, and the combining is done in free space. In addition, the loaded grid is a planar structure amenable to monolithic integration. Measurements on a 25-MESFET grid at 9.7 GHz show power-combining and frequency-locking without an external locking signal, with an ERP of 37 W. Experimental far-field patterns agree with theoretical results obtained using reciprocity.

  7. Oscillator detector

    SciTech Connect

    Potter, B.M.

    1980-05-13

    An alien liquid detector employs a monitoring element and an oscillatory electronic circuit for maintaining the temperature of the monitoring element substantially above ambient temperature. The output wave form, eg., frequency of oscillation or wave shape, of the oscillatory circuit depends upon the temperaturedependent electrical characteristic of the monitoring element. A predetermined change in the output waveform allows water to be discriminated from another liquid, eg., oil. Features of the invention employing two thermistors in two oscillatory circuits include positioning one thermistor for contact with water and the other thermistor above the oil-water interface to detect a layer of oil if present. Unique oscillatory circuit arrangements are shown that achieve effective thermistor action with an economy of parts and energizing power. These include an operational amplifier employed in an astable multivibrator circuit, a discrete transistor-powered tank circuit, and use of an integrated circuit chip.

  8. Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation.

    PubMed

    Rivers, David B; Crawley, Timothy; Bauser, Holly

    2005-02-01

    Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.

  9. Calcium-phospholipid enhanced protein phosphorylation in human placenta

    SciTech Connect

    Moore, J.J.; Moore, R.; Cardaman, R.C.

    1986-07-01

    Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using (..gamma..-/sup 32/P)ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10/sup -6/ M) in combination with phosphatidylserine (50 ..mu..g/ml) significantly enhanced (P < 100) /sup 32/P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal /sup 22/P incorporation was observed with 3.5 x 10/sup -7/ M Ca/sup 2 +/ in the presence of phosphatidylserine (50 ..mu..g/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10/sup -6/ M, /sup 32/P incorporation increased to a maximum at 70 /sup +/g/ml of phosphatidylserine. The increase was suppressed at 150 ..mu..g/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphoproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10/sup -6/ M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specifically inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.

  10. An all-or-nothing rise in cytosolic

    PubMed

    Katoh; Kikuyama

    1997-01-01

    The peritrich ciliate Vorticella sp. exhibits cellular contraction of an all-or-nothing type in response to a mechanical stimulus. Many authors have suggested that the contraction may be controlled by the cytosolic level of Ca2+, since glycerol-extracted Vorticella contracts when Ca2+ is added to the external solution. However, no direct evidence for the increase in cytosolic [Ca2+] has yet been obtained in living Vorticella. In the present study, by injecting a fluorescent Ca2+ indicator into living Vorticella and monitoring the cytosolic [Ca2+] with a confocal microscope, we have demonstrated that a mechanical stimulus evoked an all-or-nothing rise in cytosolic [Ca2+] (Ca2+ 'spike'). The onset of the Ca2+ spike was similar in its time course to that of cellular contraction. Since the Ca2+ spike was recorded in a Ca2+-deprived solution containing 1 mmol l-1 EGTA, we concluded that release of Ca2+ from intracellular Ca2+ storage site(s) is responsible for the Ca2+ spike.

  11. Shared and Distinct Mechanisms of Compartmentalized and Cytosolic Ciliogenesis.

    PubMed

    Avidor-Reiss, Tomer; Leroux, Michel R

    2015-12-07

    Most motile and all non-motile (also known as primary) eukaryotic cilia possess microtubule-based axonemes that are assembled at the cell surface to form hair-like or more elaborate compartments endowed with motility and/or signaling functions. Such compartmentalized ciliogenesis depends on the core intraflagellar transport (IFT) machinery and the associated Bardet-Biedl syndrome complex (BBSome) for dynamic delivery of ciliary components. The transition zone (TZ), an ultrastructurally complex barrier or 'gate' at the base of cilia, also contributes to the formation of compartmentalized cilia. Yet, some ciliated protists do not have IFT components and, like some metazoan spermatozoa, use IFT-independent mechanisms to build axonemes exposed to the cytosol. Moreover, various ciliated protists lack TZ components, whereas Drosophila sperm surprisingly requires the activity of dynamically localized TZ proteins for cytosolic ciliogenesis. Here, we discuss the various ways eukaryotes use IFT and/or TZ proteins to generate the wide assortment of compartmentalized and cytosolic cilia observed in nature. Consideration of the different ciliogenesis pathways allows us to propose how three types of cytosol-exposed cilia (primary, secondary and tertiary), including cilia found in the human sperm proximal segment, are likely generated by evolutionary derivations of compartmentalized ciliogenesis.

  12. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  13. Calcium and bone disease

    PubMed Central

    Blair, Harry C.; Robinson, Lisa J.; Huang, Christopher L.-H.; Sun, Li; Friedman, Peter A.; Schlesinger, Paul H.; Zaidi, Mone

    2013-01-01

    Calcium transport and calcium signaling are of basic importance in bone cells. Bone is the major store of calcium and a key regulatory organ for calcium homeostasis. Bone, in major part, responds to calcium-dependent signals from the parathyroids and via vitamin D metabolites, although bone retains direct response to extracellular calcium if parathyroid regulation is lost. Improved understanding of calcium transporters and calcium-regulated cellular processes has resulted from analysis of genetic defects, including several defects with low or high bone mass. Osteoblasts deposit calcium by mechanisms including phosphate and calcium transport with alkalinization to absorb acid created by mineral deposition; cartilage calcium mineralization occurs by passive diffusion and phosphate production. Calcium mobilization by osteoclasts is mediated by acid secretion. Both bone forming and bone resorbing cells use calcium signals as regulators of differentiation and activity. This has been studied in more detail in osteoclasts, where both osteoclast differentiation and motility are regulated by calcium. PMID:21674636

  14. Intracellular Calcium Dysregulation: Implications for Alzheimer's Disease

    PubMed Central

    Magi, Simona; Castaldo, Pasqualina; Macrì, Maria Loredana; Maiolino, Marta; Matteucci, Alessandra; Bastioli, Guendalina; Gratteri, Santo; Lariccia, Vincenzo

    2016-01-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level. PMID:27340665

  15. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  16. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  17. Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

    PubMed Central

    Park, Hwan-Woo; Park, Haeli; Semple, Ian A.; Jang, Insook; Ro, Seung-Hyun; Kim, Myungjin; Cazares, Victor A.; Stuenkel, Edward L.; Kim, Jung-Jae; Kim, Jeong Sig; Lee, Jun Hee

    2014-01-01

    Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that chronic increase of cytosolic calcium concentration in hepatocytes upon obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than thirty years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients. PMID:25189398

  18. Controlling metabolism and cell death: at the heart of mitochondrial calcium signalling

    PubMed Central

    Murgia, Marta; Giorgi, Carlotta; Pinton, Paolo; Rizzuto, Rosario

    2009-01-01

    Transient increases in intracellular calcium concentration activate and coordinate a wide variety of cellular processes in virtually every cell type. This review describes the main homeostatic mechanisms that control Ca2+ transients, focusing on the mitochondrial checkpoint. We subsequently extend this paradigm to the cardiomyocyte and to the interplay between cytosol, endoplasmic reticulum and mitochondria that occurs beat-to-beat in excitation-contraction coupling. The mechanisms whereby mitochondria decode fast cytosolic calcium spikes are discussed in the light of the results obtained with recombinant photoproteins targeted to the mitochondrial matrix of contracting cardiomyocytes. Mitochondrial calcium homeostasis is then highlighted as a crucial point of convergence of the environmental signals that mediate cardiac cell death, both by necrosis and by apoptosis. Altogether we point to a role of the mitochondrion as an integrator of calcium signalling and fundamental decision maker in cardiomyocyte metabolism and survival. PMID:19285982

  19. Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana.

    PubMed

    Malinova, Irina; Steup, Martin; Fettke, Joerg

    2011-08-15

    Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2). In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids. In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were

  20. Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

    PubMed Central

    Kaufman, Randal J.; Malhotra, Jyoti D.

    2014-01-01

    Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associate changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis [4] [5] [6] [7]. Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered [8] [9] [10]. The next questions are how they are regulated for exquisite tight control of ER – mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. PMID:24690484

  1. Oscillating Permanent Magnets.

    ERIC Educational Resources Information Center

    Michaelis, M. M.; Haines, C. M.

    1989-01-01

    Describes several ways to partially levitate permanent magnets. Computes field line geometries and oscillation frequencies. Provides several diagrams illustrating the mechanism of the oscillation. (YP)

  2. Calcium dynamics in catecholamine-containing secretory vesicles.

    PubMed

    Moreno, Alfredo; Lobatón, Carmen D; Santodomingo, Jaime; Vay, Laura; Hernández-SanMiguel, Esther; Rizzuto, Rosario; Montero, Mayte; Alvarez, Javier

    2005-06-01

    We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

  3. Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

    PubMed Central

    Bose, Jayakumar; Pottosin, Igor I.; Shabala, Stanislav S.; Palmgren, Michael G.; Shabala, Sergey

    2011-01-01

    Transient cytosolic calcium ([Ca2+]cyt) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium “signature” that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms to shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signaling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca2+-permeable channels and efflux systems) taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment. PMID:22639615

  4. Calcium dynamics and buffering in motoneurones of the mouse spinal cord

    PubMed Central

    Palecek, Jiri; Lips, Mario B; Keller, Bernhard U

    1999-01-01

    A quantitative analysis of endogenous calcium homeostasis was performed on 65 motoneurones in slices of the lumbar spinal cord from 2- to 8-day-old mice by simultaneous patch-clamp and microfluorometric calcium measurements. Somatic calcium concentrations were monitored with a temporal resolution in the millisecond time domain. Measurements were performed by using a monochromator for excitation and a photomultiplier detection system. Somatic calcium signalling was investigated during defined voltage-clamp protocols. Calcium responses were observed for membrane depolarizations positive to −50 mV. A linear relation between depolarization time and free calcium concentrations ([Ca2+]i) indicated that voltage-dependent calcium influx dominated the response. Endogenous calcium homeostasis was quantified by using the ‘added buffer’ approach. In the presence of fura-2 and mag-fura-5, calcium transients decayed according to a monoexponential function. Decay-time constants showed a linear dependence on dye concentration and the extrapolated constant in the absence of indicator dye was 371 ± 120 ms (n= 13 cells, 21 °C). For moderate elevations (< 1 μm), recovery kinetics of depolarization-induced calcium transients were characterized by a calcium-independent, ‘effective’ extrusion rate γ = 140 ± 47 s−1 (n= 13 cells, 21 °C). The endogenous calcium binding ratio for fixed buffers in spinal motoneurones was κB’ = 50 ± 17 (n= 13 cells), indicating that less than 2% of cytosolic calcium ions contributed to [Ca2+]i. Endogenous binding ratios in spinal motoneurones were small compared to those found in hippocampal or cerebellar Purkinje neurones. From a functional perspective, they provided motoneurones with rapid dynamics of cytosolic [Ca2+]i for a given set of influx, extrusion and uptake mechanisms. With respect to pathophysiological conditions, our measurements are in agreement with a model where the selective vulnerability of spinal motoneurones during

  5. Calcium signaling and cytotoxicity.

    PubMed Central

    Kass, G E; Orrenius, S

    1999-01-01

    The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery. Images Figure 1 Figure 2 Figure 3 PMID:10229704

  6. Calcium in the regulation of gravitropism by light

    NASA Technical Reports Server (NTRS)

    Perdue, D. O.; LaFavre, A. K.; Leopold, A. C.

    1988-01-01

    The red light requirement for positive gravitropism in roots of corn (Zea mays cv "Merit") provides an entry for examining the participation of calcium in gravitropism. Applications of calcium chelators inhibit the light response. Calcium channel blockers (verapamil, lanthanum) can also inhibit the light response, and a calcium ionophore, A23187, can substitute for light. One can substitute for red light by treatments which have elsewhere been shown to trigger Ca2+ influx into the cytosol, e.g. heat or cold shock. Agents which are known to be agonists of the phosphatidylinositol second messenger system (serotonin, 2,4-dichlorophenoxyacetic acid, deoxycholate) can each partially substitute for the red light, and Li+ can inhibit the light effect. These experiments suggest that the induction of positive gravitropism by red light involves a rise in cytoplasmic Ca2+ concentration, and that a contribution to this end may be made by the phosphatidylinositol second messenger system.

  7. Calcium and Vitamin D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium is required for the bone formation phase of bone remodeling. Typically about 5 nmol (200 mg) of calcium is removed from the adult skeleton and replaced each day. To supply this amount, one would need to consume about 600 mg of calcium, since calcium is not very efficiently absorbed. Calcium ...

  8. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes

    PubMed Central

    Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana

    2015-01-01

    The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases. PMID:26009544

  9. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

    PubMed Central

    Yoon, Mi Na; Kim, Dong Kwan; Kim, Se Hoon

    2017-01-01

    Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells. PMID:28280417

  10. PMCA4 (ATP2B4) mutation in familial spastic paraplegia causes delay in intracellular calcium extrusion

    PubMed Central

    Ho, Philip Wing-Lok; Pang, Shirley Yin-Yu; Li, Miaoxin; Tse, Zero Ho-Man; Kung, Michelle Hiu-Wai; Sham, Pak-Chung; Ho, Shu-Leong

    2015-01-01

    Background Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation (c.803G>A, p.R268Q) in the plasma membrane calcium ATPase (PMCA4, or ATP2B4) gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation. Methods As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4. Results Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin. Conclusion Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP. PMID:25798335

  11. Hypotonic stress-induced calcium signaling in Saccharomyces cerevisiae involves TRP-like transporters on the endoplasmic reticulum membrane.

    PubMed

    Rigamonti, M; Groppi, S; Belotti, F; Ambrosini, R; Filippi, G; Martegani, E; Tisi, R

    2015-02-01

    Saccharomyces cerevisiae cells respond to hypotonic stress (HTS) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. Calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and Cch1-, but not Mid1-, driven manner. HTS-induced calcium release is also negatively regulated by the ER protein Cls2 and involves a poorly characterized protein, FLC2/YAL053W gene product, previously proposed to be required for FAD transport in the ER, albeit, due to its molecular features, it was also previously classified as an ion transporter. A computational analysis revealed that this gene and its three homologs in S. cerevisiae, together with previously identified Schizosaccharomyces pombe pkd2 and Neurospora crassa calcium-related spray protein, belong to a fungal branch of TRP-like ion transporters related to human mucolipin and polycystin 2 calcium transporters. Moreover, disruption of FLC2 gene confers severe sensitivity to Calcofluor white and hyper-activation of the cell wall integrity MAPK cascade, suggesting a role in cell wall maintenance as previously suggested for the fission yeast homolog. Perturbation in cytosolic resting calcium concentration and hyper-activation of calcineurin in exponentially growing cells suggest a role for this transporter in calcium homeostasis in yeast.

  12. Mechanism and evolution of calcium transport across the plant plasma membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium is an essential plant nutrient, thus the influx of Ca(2+) into plant cells is a critical process. In addition, the efflux of Ca(2+) out of a cell is important to prevent toxicity resulting from Ca(2+) excess, and to modulate levels of cytosolic Ca(2+) required for signaling functions. Bioc...

  13. Calcium and Vitamin D

    MedlinePlus

    ... A calcium-rich diet (including dairy, nuts, leafy greens and fish) helps to build and protect your ... yogurt and cheese are high in calcium. Certain green vegetables and other foods contain calcium in smaller ...

  14. Coronary Calcium Scan

    MedlinePlus

    ... Scan Coronary Calcium Scan Related Topics Angina Atherosclerosis Coronary Heart Disease Electrocardiogram Heart Attack Send a link to NHLBI ... calcium, or calcifications, are a sign of atherosclerosis, coronary heart disease, or coronary microvascular disease. A coronary calcium scan ...

  15. Flagellin Polymerisation Control by a Cytosolic Export Chaperone

    PubMed Central

    Auvray, Frédéric; Thomas, Joanne; Fraser, Gillian M.; Hughes, Colin

    2008-01-01

    Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments. PMID:11327763

  16. Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

    PubMed

    Wolfe, L; Bradford, A P; Klarlund, J K; Czech, M P

    1992-05-15

    A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to

  17. Light induced cytosolic drug delivery from liposomes with gold nanoparticles.

    PubMed

    Lajunen, Tatu; Viitala, Lauri; Kontturi, Leena-Stiina; Laaksonen, Timo; Liang, Huamin; Vuorimaa-Laukkanen, Elina; Viitala, Tapani; Le Guével, Xavier; Yliperttula, Marjo; Murtomäki, Lasse; Urtti, Arto

    2015-04-10

    Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells.

  18. A New View of the Bacterial Cytosol Environment

    PubMed Central

    Cossins, Benjamin P.; Jacobson, Matthew P.; Guallar, Victor

    2011-01-01

    The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg2+ ions were prominent in NIMS and almost absent free in solution. Κ+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution. PMID:21695225

  19. Hippocampal cytosolic estrogen receptors regulate fear generalization in females.

    PubMed

    Lynch, Joseph F; Winiecki, Patrick; Vanderhoof, Tyler; Riccio, David C; Jasnow, Aaron M

    2016-04-01

    Generalization of fear responses is a symptom of many anxiety disorders and we have previously demonstrated that female rats generalize fear to a neutral context at a faster rate compared to males. This effect is due in part, to activation of ER and modulation of memory retrieval mechanisms resulting in fear generalization. Given that the effects of estradiol on fear generalization required approximately 24h, our data suggested possible genomic actions on fear generalization. To determine whether these actions were due to cytosolic versus membrane bound receptors, female rats were given infusions of ICI 182,780, a cytosolic estrogen receptor antagonist, into the lateral ventricle or dorsal hippocampus simultaneously with estradiol treatment or with an ER agonist (DPN). Infusions of ICI into the lateral ventricle or the dorsal hippocampus blocked fear generalization induced by peripheral or central treatment with estradiol or DPN, suggesting that estradiol acts through cytosolic ERβ receptors. In further support of these findings, intracerebroventricular or intra-hippocampal infusions of bovine serum conjugated estradiol (E2-BSA), activating membrane-bound estrogen receptors only, did not induce fear generalization. Moreover, rats receiving intra-hippocampal infusions of the ERK/MAPK inhibitor, U0126, continued to display estradiol-induced generalization, again suggesting that membrane-bound estrogen receptors do not contribute to fear generalization. Overall, these data suggest that estradiol-induced enhancements in fear generalization are mediated through activation of cytosolic/nuclear ER within the dorsal hippocampus. This region seems to be an important locus for the effects of estradiol on fear generalization although additional neuroanatomical regions have yet to be identified.

  20. Signaling in the plant cytosol: cysteine or sulfide?

    PubMed

    Gotor, Cecilia; Laureano-Marín, Ana M; Moreno, Inmaculada; Aroca, Ángeles; García, Irene; Romero, Luis C

    2015-10-01

    Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel L-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.

  1. Eukaryotic starch degradation: integration of plastidial and cytosolic pathways.

    PubMed

    Fettke, Joerg; Hejazi, Mahdi; Smirnova, Julia; Höchel, Erik; Stage, Marion; Steup, Martin

    2009-01-01

    Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid.

  2. A reaction-diffusion model of cytosolic hydrogen peroxide.

    PubMed

    Lim, Joseph B; Langford, Troy F; Huang, Beijing K; Deen, William M; Sikes, Hadley D

    2016-01-01

    As a signaling molecule in mammalian cells, hydrogen peroxide (H2O2) determines the thiol/disulfide oxidation state of several key proteins in the cytosol. Localization is a key concept in redox signaling; the concentrations of signaling molecules within the cell are expected to vary in time and in space in manner that is essential for function. However, as a simplification, all theoretical studies of intracellular hydrogen peroxide and many experimental studies to date have treated the cytosol as a well-mixed compartment. In this work, we incorporate our previously reported reduced kinetic model of the network of reactions that metabolize hydrogen peroxide in the cytosol into a model that explicitly treats diffusion along with reaction. We modeled a bolus addition experiment, solved the model analytically, and used the resulting equations to quantify the spatiotemporal variations in intracellular H2O2 that result from this kind of perturbation to the extracellular H2O2 concentration. We predict that micromolar bolus additions of H2O2 to suspensions of HeLa cells (0.8 × 10(9)cells/l) result in increases in the intracellular concentration that are localized near the membrane. These findings challenge the assumption that intracellular concentrations of H2O2 are increased uniformly throughout the cell during bolus addition experiments and provide a theoretical basis for differing phenotypic responses of cells to intracellular versus extracellular perturbations to H2O2 levels.

  3. Cytosolic Replication of Group A Streptococcus in Human Macrophages

    PubMed Central

    O’Neill, Alan M.; Thurston, Teresa L. M.

    2016-01-01

    ABSTRACT As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria. PMID:27073088

  4. Growth factor deprivation induces cytosolic translocation of SIRT1

    NASA Astrophysics Data System (ADS)

    Meng, Chengbo; Xing, Da; Wu, Shengnan; Huang, Lei

    2010-02-01

    Sirtuin type 1 (SIRT1), a NAD+-dependent histone deacetylases, plays a critical role in cellular senescence, aging and longevity. In general, SIRT1 is localized in nucleus and is believed as a nuclear protein. Though overexpression of SIRT1 delays senescence, SIRT1-protein levels decline naturally in thymus and heart during aging. In the present studies, we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidney fibroblast cells (COS-7). Using SIRT1-EGFP fluorescence reporter, we found that SIRT1 localized to nucleus in physiological conditions. We devised a model enabling cell senescence via growth factor deprivation, and we found that SIRT1 partially translocated to cytosol under the treatment, suggesting a reduced level of SIRT1's activity. We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation, because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus. Taken together, we demonstrated that growth factor deprivation induces cytosolic translocation of SIRT1, which suggesting a possible connection between cytoplasm-localized SIRT1 and the aging process.

  5. Routes of Ca2+ Shuttling during Ca2+ Oscillations

    PubMed Central

    Pecze, László; Blum, Walter; Schwaller, Beat

    2015-01-01

    In some cell types, Ca2+ oscillations are strictly dependent on Ca2+ influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca2+ influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca2+ influx played a pivotal role. However, when the Ca2+ transport across the plasma membrane by the “lanthanum insulation method” was blocked prior to the induction of the serum-induced Ca2+ oscillations, mitochondrial Ca2+ transport was found to be able to substitute for the plasmalemmal Ca2+ exchange function, thus rendering the oscillations independent of extracellular Ca2+. However, in a physiological situation, the Ca2+-buffering capacity of mitochondria was found not to be essential for Ca2+ oscillations. Moreover, brief spontaneous Ca2+ changes were observed in the mitochondrial Ca2+ concentration without apparent changes in the cytosolic Ca2+ concentration, indicating the presence of a mitochondrial autonomous Ca2+ signaling mechanism. In the presence of calretinin, a Ca2+-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca2+ ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca2+ shuttling pathways in primary mesothelial cells during Ca2+ oscillations: Ca2+ shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca2+ buffers. PMID:26396196

  6. Mitochondrial calcium in relaxed and tetanized myocardium.

    PubMed Central

    Horikawa, Y; Goel, A; Somlyo, A P; Somlyo, A V

    1998-01-01

    The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+]. PMID:9512053

  7. Chemical oscillator as a generalized Rayleigh oscillator

    NASA Astrophysics Data System (ADS)

    Ghosh, Shyamolina; Ray, Deb Shankar

    2013-10-01

    We derive the conditions under which a set of arbitrary two dimensional autonomous kinetic equations can be reduced to the form of a generalized Rayleigh oscillator which admits of limit cycle solution. This is based on a linear transformation of field variables which can be found by inspection of the kinetic equations. We illustrate the scheme with the help of several chemical and bio-chemical oscillator models to show how they can be cast as a generalized Rayleigh oscillator.

  8. Synchronization of genetic oscillators

    NASA Astrophysics Data System (ADS)

    Zhou, Tianshou; Zhang, Jiajun; Yuan, Zhanjiang; Chen, Luonan

    2008-09-01

    Synchronization of genetic or cellular oscillators is a central topic in understanding the rhythmicity of living organisms at both molecular and cellular levels. Here, we show how a collective rhythm across a population of genetic oscillators through synchronization-induced intercellular communication is achieved, and how an ensemble of independent genetic oscillators is synchronized by a common noisy signaling molecule. Our main purpose is to elucidate various synchronization mechanisms from the viewpoint of dynamics, by investigating the effects of various biologically plausible couplings, several kinds of noise, and external stimuli. To have a comprehensive understanding on the synchronization of genetic oscillators, we consider three classes of genetic oscillators: smooth oscillators (exhibiting sine-like oscillations), relaxation oscillators (displaying jump dynamics), and stochastic oscillators (noise-induced oscillation). For every class, we further study two cases: with intercellular communication (including phase-attractive and repulsive coupling) and without communication between cells. We find that an ensemble of smooth oscillators has different synchronization phenomena from those in the case of relaxation oscillators, where noise plays a different but key role in synchronization. To show differences in synchronization between them, we make comparisons in many aspects. We also show that a population of genetic stochastic oscillators have their own synchronization mechanisms. In addition, we present interesting phenomena, e.g., for relaxation-type stochastic oscillators coupled to a quorum-sensing mechanism, different noise intensities can induce different periodic motions (i.e., inhomogeneous limit cycles).

  9. Regulation of melatonin production and intracellular calcium concentrations in the trout pineal organ.

    PubMed

    Meissl, H; Kroeber, S; Yáñez, J; Korf, H W

    1996-12-01

    The present in vitro study correlates measurements of the melatonin production from trout pineal organs with those of the intracellular calcium concentration in pinealocytes. Melatonin production increases with decreasing irradiance and shows maximal values in darkness. Some pinealocytes exhibit spontaneous calcium oscillations, although most of them have a stable basal calcium concentration. Diminishing extracellular calcium and enhancing magnesium reduces melatonin release in the light-and dark-adapted state. The application of Co2+ decreases melatonin secretion in the mesopic and scotopic range, reversibly blocks spontaneous calcium oscillations, reduces the basal intracellular calcium concentration in non-oscillating pinealocytes, and inhibits the KCl-induced rise in intracellular calcium. Application of glutamate, norepinephrine, isoproterenol, or dopamine has no significant effect on melatonin secretion. Norepinephrine does not influence the calcium concentration in any of the trout pinealocytes. Treatment with the GABAA-receptor agonist muscimol causes a slight reduction of melatonin release in the mesopic and scotopic range of illumination, without affecting intracellular calcium concentrations. Thus, Co2+ and low calcium/high magnesium buffer reduce melatonin release through an action on the calcium concentration in trout pinealocytes and not through a blockade of synaptic transmission. All the data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and that neuronal inputs have a minor, if any, influence on melatonin synthesis.

  10. Yeast and Mammals Utilize Similar Cytosolic Components to Drive Protein Transport through the Golgi Complex

    NASA Astrophysics Data System (ADS)

    Dunphy, William G.; Pfeffer, Suzanne R.; Clary, Douglas O.; Wattenberg, Binks W.; Glick, Benjamin S.; Rothman, James E.

    1986-03-01

    Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

  11. Holographic charge oscillations

    NASA Astrophysics Data System (ADS)

    Blake, Mike; Donos, Aristomenis; Tong, David

    2015-04-01

    The Reissner-Nordström black hole provides the prototypical description of a holographic system at finite density. We study the response of this system to the presence of a local, charged impurity. Below a critical temperature, the induced charge density, which screens the impurity, exhibits oscillations. These oscillations can be traced to the singularities in the density-density correlation function moving in the complex momentum plane. At finite temperature, the oscillations are very similar to the Friedel oscillations seen in Fermi liquids. However, at zero temperature the oscillations in the black hole background remain exponentially damped, while Friedel oscillations relax to a power-law.

  12. In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics.

    PubMed

    Ordenes, Viviana R; Moreno, Ignacio; Maturana, Daniel; Norambuena, Lorena; Trewavas, Anthony J; Orellana, Ariel

    2012-11-01

    The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 μM vs. 0.05 μM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 μM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones.

  13. Imaging calcium in neurons.

    PubMed

    Grienberger, Christine; Konnerth, Arthur

    2012-03-08

    Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.

  14. Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

    PubMed Central

    Nasi, Enrico

    2009-01-01

    In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca2+ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca2+ increase. In contrast, in other species, such as Lima, Ca2+ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (Islow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. Islow obtains after replacing extracellular Na+ with Li+, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. Islow is impervious to anion replacements and can be measured with extracellular Ca2+ as the sole permeant species; Ba can substitute for Ca2+ but Mg2+ cannot. A persistent Ca2+ elevation parallels Islow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca2+ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, Islow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors. PMID

  15. Saturation in coupled oscillators

    NASA Astrophysics Data System (ADS)

    Roman, Ahmed; Hanna, James

    2015-03-01

    We consider a weakly nonlinear system consisting of a resonantly forced oscillator coupled to an unforced oscillator. It has long been known that, for quadratic nonlinearities and a 2:1 resonance between the oscillators, a perturbative solution of the dynamics exhibits a phenomenon known as saturation. At low forcing, the forced oscillator responds, while the unforced oscillator is quiescent. Above a critical value of the forcing, the forced oscillator's steady-state amplitude reaches a plateau, while that of the unforced oscillator increases without bound. We show that, contrary to established folklore, saturation is not unique to quadratically nonlinear systems. We present conditions on the form of the nonlinear couplings and resonance that lead to saturation. Our results elucidate a mechanism for localization or diversion of energy in systems of coupled oscillators, and suggest new approaches for the control or suppression of vibrations in engineered systems.

  16. Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

    PubMed

    Roberts, Stephen K; McAinsh, Martin; Cantopher, Hanna; Sandison, Sean

    2014-01-01

    Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.

  17. Imaging of calcium dynamics in pollen tube cytoplasm.

    PubMed

    Barberini, María Laura; Muschietti, Jorge

    2015-01-01

    Cytoplasmic calcium [(Ca(2+))cyt] is a central component of cellular signal transduction pathways. In plants, many external and internal stimuli transiently elevate (Ca(2+))cyt, initiating downstream responses that control different features of plant development. In pollen tubes the establishment of an oscillatory gradient of calcium at the tip is essential for polarized growth. Disruption of the cytosolic Ca(2+) gradient by chelators or channel blockers inhibits pollen tube growth. To quantify the physiological role of (Ca(2+))cyt in cellular systems, genetically encoded Ca(2+) indicators such as Yellow Cameleons (YCs) have been developed. The Cameleons are based on a fluorescence resonance energy transfer (FRET) process. Here, we describe a method for imaging cytoplasmic Ca(2+) dynamics in growing pollen tubes that express the fluorescent calcium indicator Yellow Cameleon 3.6 (YC 3.6), using laser-scanning confocal microscopy.

  18. Stochastic Modeling of Calcium in 3D Geometry

    PubMed Central

    Mazel, Tomáš; Raymond, Rebecca; Raymond-Stintz, Mary; Jett, Stephen; Wilson, Bridget S.

    2009-01-01

    Release of inflammatory mediators by mast cells in type 1 immediate-hypersensitivity allergic reactions relies on antigen-dependent increases in cytosolic calcium. Here, we used a series of electron microscopy images to build a 3D reconstruction representing a slice through a rat tumor mast cell, which then served as a basis for stochastic modeling of inositol-trisphosphate-mediated calcium responses. The stochastic approach was verified by reaction-diffusion modeling within the same geometry. Local proximity of the endoplasmic reticulum to either the plasma membrane or mitochondria is predicted to differentially impact local inositol trisphosphate receptor transport. The explicit consideration of organelle spatial relationships represents an important step toward building a comprehensive, realistic model of cellular calcium dynamics. PMID:19254531

  19. Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin

    NASA Technical Reports Server (NTRS)

    Yang, T.; Poovaiah, B. W.

    2002-01-01

    Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

  20. Calcium signaling in plant cell organelles delimited by a double membrane.

    PubMed

    Xiong, Tou-Cheu; Bourque, Stéphane; Lecourieux, David; Amelot, Nicolas; Grat, Sabine; Brière, Christian; Mazars, Christian; Pugin, Alain; Ranjeva, Raoul

    2006-11-01

    Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.

  1. CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor.

    PubMed

    Eraso-Pichot, A; Larramona-Arcas, R; Vicario-Orri, E; Villalonga, R; Pardo, L; Galea, E; Masgrau, R

    2017-03-01

    Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

  2. Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases.

    PubMed

    Ko, Kyounga; Kurogi, Katsuhisa; Davidson, Garrett; Liu, Ming-Yih; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2012-09-01

    Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.

  3. Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer

    PubMed Central

    Perez, Itxaro; Blanco, Lorena; Sanz, Begoña; Errarte, Peio; Ariz, Usue; Beitia, Maider; Fernández, Ainhoa; Loizate, Alberto; Candenas, M Luz; Pinto, Francisco M; Gil, Javier; López, José I.; Larrinaga, Gorka

    2015-01-01

    Background and Objective: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. Methods: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). Results: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall suvival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. Conclusions: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis. PMID:26078706

  4. Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

    PubMed Central

    Baird, Nicholas L.; York, Joanne

    2012-01-01

    Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis. PMID:22875974

  5. Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins

    PubMed Central

    Dou, Zhicheng; McGovern, Olivia L.; Di Cristina, Manlio

    2014-01-01

    ABSTRACT The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. PMID:25028423

  6. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent.

    PubMed

    Davis, F M; Azimi, I; Faville, R A; Peters, A A; Jalink, K; Putney, J W; Goodhill, G J; Thompson, E W; Roberts-Thomson, S J; Monteith, G R

    2014-05-01

    Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of

  7. Covariant harmonic oscillators and coupled harmonic oscillators

    NASA Technical Reports Server (NTRS)

    Han, Daesoo; Kim, Young S.; Noz, Marilyn E.

    1995-01-01

    It is shown that the system of two coupled harmonic oscillators shares the basic symmetry properties with the covariant harmonic oscillator formalism which provides a concise description of the basic features of relativistic hadronic features observed in high-energy laboratories. It is shown also that the coupled oscillator system has the SL(4,r) symmetry in classical mechanics, while the present formulation of quantum mechanics can accommodate only the Sp(4,r) portion of the SL(4,r) symmetry. The possible role of the SL(4,r) symmetry in quantum mechanics is discussed.

  8. SHOCK-EXCITED OSCILLATOR

    DOEpatents

    Creveling, R.

    1957-12-17

    S> A shock-excited quartz crystal oscillator is described. The circuit was specifically designed for application in micro-time measuring work to provide an oscillator which immediately goes into oscillation upon receipt of a trigger pulse and abruptly ceases oscillation when a second pulse is received. To achieve the instant action, the crystal has a prestressing voltage applied across it. A monostable multivibrator receives the on and off trigger pulses and discharges a pulse through the crystal to initiate or terminate oscillation instantly.

  9. Live imaging of calcium spikes during double fertilization in Arabidopsis

    PubMed Central

    Hamamura, Yuki; Nishimaki, Moe; Takeuchi, Hidenori; Geitmann, Anja; Kurihara, Daisuke; Higashiyama, Tetsuya

    2014-01-01

    Ca2+ waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca2+ in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca2+ spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca2+ oscillations on pollen tube arrival. The two synergid cells showed distinct Ca2+ dynamics depending on their respective roles in tube reception. These Ca2+ dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants. PMID:25146889

  10. Nature's Autonomous Oscillators

    NASA Technical Reports Server (NTRS)

    Mayr, H. G.; Yee, J.-H.; Mayr, M.; Schnetzler, R.

    2012-01-01

    Nonlinearity is required to produce autonomous oscillations without external time dependent source, and an example is the pendulum clock. The escapement mechanism of the clock imparts an impulse for each swing direction, which keeps the pendulum oscillating at the resonance frequency. Among nature's observed autonomous oscillators, examples are the quasi-biennial oscillation and bimonthly oscillation of the Earth atmosphere, and the 22-year solar oscillation. The oscillations have been simulated in numerical models without external time dependent source, and in Section 2 we summarize the results. Specifically, we shall discuss the nonlinearities that are involved in generating the oscillations, and the processes that produce the periodicities. In biology, insects have flight muscles, which function autonomously with wing frequencies that far exceed the animals' neural capacity; Stretch-activation of muscle contraction is the mechanism that produces the high frequency oscillation of insect flight, discussed in Section 3. The same mechanism is also invoked to explain the functioning of the cardiac muscle. In Section 4, we present a tutorial review of the cardio-vascular system, heart anatomy, and muscle cell physiology, leading up to Starling's Law of the Heart, which supports our notion that the human heart is also a nonlinear oscillator. In Section 5, we offer a broad perspective of the tenuous links between the fluid dynamical oscillators and the human heart physiology.

  11. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling.

    PubMed

    Bernard, Stéphanie M; Habash, Dimah Z

    2009-01-01

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  12. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    SciTech Connect

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  13. No warmup crystal oscillator

    NASA Technical Reports Server (NTRS)

    Phillips, D. H.

    1982-01-01

    During warmup, crystal oscillators often show a frequency offset as large as 1 part in 10 to the 5th power. If timing information is transferred to the oscillator and then the oscillator is allowed to warmup, a timing error greater than 1 millisecond will occur. For many applications, it is unsuitable to wait for the oscillator to warmup. For medium accuracy timing requirements where overall accuracies in the order of 1 millisecond are required, a no warmup crystal concept was developed. The concept utilizes two crystal oscillator, used sequentially to avoid using a crystal oscillator for timing much higher frequency accuracy once warmed up. The accuracy achieved with practical TCXOs at initial start over a range of temperatures is discussed. A second design utilizing two oven controlled oscillators is also discussed.

  14. Non-linear oscillations

    NASA Astrophysics Data System (ADS)

    Hagedorn, P.

    The mathematical pendulum is used to provide a survey of free and forced oscillations in damped and undamped systems. This simple model is employed to present illustrations for and comparisons between the various approximation schemes. A summary of the Liapunov stability theory is provided. The first and the second method of Liapunov are explained for autonomous as well as for nonautonomous systems. Here, a basic familiarity with the theory of linear oscillations is assumed. La Salle's theorem about the stability of invariant domains is explained in terms of illustrative examples. Self-excited oscillations are examined, taking into account such oscillations in mechanical and electrical systems, analytical approximation methods for the computation of self-excited oscillations, analytical criteria for the existence of limit cycles, forced oscillations in self-excited systems, and self-excited oscillations in systems with several degrees of freedom. Attention is given to Hamiltonian systems and an introduction to the theory of optimal control is provided.

  15. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT WITH SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  16. Calcium ions affect the hepatitis B virus core assembly

    SciTech Connect

    Choi, Yongwook; Gyoo Park, Sung; Yoo, Jun-hi; Jung, Guhung . E-mail: drjung@snu.ac.kr

    2005-02-05

    Previous report showed that cytosolic Ca{sup 2+} induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca{sup 2+} through the sucrose density gradient and the analytical ultracentrifuge analysis. Also (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.

  17. [Do cows drink calcium?].

    PubMed

    Geishauser, T; Lechner, S; Plate, I; Heidemann, B

    2008-03-01

    The objective of this study was to investigate how well cows drink the Propeller calcium drink, and it's effect on blood calcium concentration. Drinking was tested in 120 cows right after calving, before cows drank anything else. 60 cows each were offered 20 liters of Propeller calcium drink or 20 liters of water. Cows drank the Propeller as good as water. 72% of all cows drank all 20 liters, 18% drank on average 8.2 liters and 10% drank less than 1 liter. Blood calcium concentration was studied in 16 cows right after calving. Eight cows each were offered 20 liters of Propeller calcium drink or no calcium drink. Blood calcium significantly increased ten minutes after Propeller intake and stayed significantly elevated for 24 hours. Without calcium drink blood calcium levels decreased significantly. Advantages of the new Propeller calcium drink over calcium gels or boli could be that cows now drink calcium themselves and that the Propeller increases blood calcium concentration rapidly and long lasting.

  18. Calcium sensitive ring-like oligomers formed by synaptotagmin

    PubMed Central

    Wang, Jing; Bello, Oscar; Auclair, Sarah M.; Wang, Jing; Coleman, Jeff; Pincet, Frederic; Krishnakumar, Shyam S.; Sindelar, Charles V.; Rothman, James E.

    2014-01-01

    The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. PMID:25201968

  19. Computational model for amoeboid motion: Coupling membrane and cytosol dynamics.

    PubMed

    Moure, Adrian; Gomez, Hector

    2016-10-01

    A distinguishing feature of amoeboid motion is that the migrating cell undergoes large deformations, caused by the emergence and retraction of actin-rich protrusions, called pseudopods. Here, we propose a cell motility model that represents pseudopod dynamics, as well as its interaction with membrane signaling molecules. The model accounts for internal and external forces, such as protrusion, contraction, adhesion, surface tension, or those arising from cell-obstacle contacts. By coupling the membrane and cytosol interactions we are able to reproduce a realistic picture of amoeboid motion. The model results are in quantitative agreement with experiments and show how cells may take advantage of the geometry of their microenvironment to migrate more efficiently.

  20. A high molecular weight protease in liver cytosol.

    PubMed

    Rose, I A; Warms, J V; Hershko, A

    1979-09-10

    A high molecular weight (greater than 400,000) protease active with [3H]leucine-labeled globin has been found in the postmicrosomal fraction of mouse kidney, brain, heart, spleen, and tumor cells and is most active in liver. The presence in liver was unexpected because liver cytosol is very ineffective in the breakdown of endogenous, labeled proteins. The enzyme has a number of properties that distinguish it from known cathepsins in addition to its high molecular weight. It is most active at pH approximately 7.5. When purified, it is unstable above 20 degrees C and is stabilized by metal chelating agents such as citrate, creatine-P, and glycerate-3-P. It is an -SH protease, but its thermal instability is not affected by 1 mM dithiothreitol. The enzyme is not lysosomal.

  1. Nod-Like Receptors: Cytosolic Watchdogs for Immunity against Pathogens

    PubMed Central

    Sirard, Jean-Claude; Vignal, Cécile; Dessein, Rodrigue; Chamaillard, Mathias

    2007-01-01

    In mammals, tissue-specific sets of pattern-recognition molecules, including Nod-like receptors (NLR), enable concomitant and sequential detection of microbial-associated molecular patterns from both the extracellular and intracellular microenvironment. Repressing and de-repressing the cytosolic surveillance machinery contributes to vital immune homeostasis and protective responses within specific tissues. Conversely, defective biology of NLR drives the development of recurrent infectious, autoimmune and/or inflammatory diseases by failing to mount barrier functions against pathogens, to tolerate commensals, and/or to instruct the adaptive immune response against microbes. Better decoding microbial strategies that are evolved to circumvent NLR sensing will provide clues for the development of rational therapies aimed at curing and/or preventing common and emerging immunopathologies. PMID:18166077

  2. Computational model for amoeboid motion: Coupling membrane and cytosol dynamics

    NASA Astrophysics Data System (ADS)

    Moure, Adrian; Gomez, Hector

    2016-10-01

    A distinguishing feature of amoeboid motion is that the migrating cell undergoes large deformations, caused by the emergence and retraction of actin-rich protrusions, called pseudopods. Here, we propose a cell motility model that represents pseudopod dynamics, as well as its interaction with membrane signaling molecules. The model accounts for internal and external forces, such as protrusion, contraction, adhesion, surface tension, or those arising from cell-obstacle contacts. By coupling the membrane and cytosol interactions we are able to reproduce a realistic picture of amoeboid motion. The model results are in quantitative agreement with experiments and show how cells may take advantage of the geometry of their microenvironment to migrate more efficiently.

  3. Calcium and Mitosis

    NASA Technical Reports Server (NTRS)

    Hepler, P.

    1983-01-01

    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  4. Trisulfate Disaccharide Decreases Calcium Overload and Protects Liver Injury Secondary to Liver Ischemia/Reperfusion

    PubMed Central

    Vasques, Enio Rodrigues; Cunha, Jose Eduardo Monteiro; Coelho, Ana Maria Mendonca; Sampietre, Sandra N.; Patzina, Rosely Antunes; Abdo, Emilio Elias; Nader, Helena B.; Tersariol, Ivarne L. S.; Lima, Marcelo Andrade; Godoy, Carlos M. G.; Rodrigues, Tiago; Chaib, Eleazar; D’Albuquerque, Luiz A. C.

    2016-01-01

    Background Ischemia and reperfusion (I/R) causes tissue damage and intracellular calcium levels are a factor of cell death. Sodium calcium exchanger (NCX) regulates calcium extrusion and Trisulfated Disaccharide (TD) acts on NCX decreasing intracellular calcium through the inhibition of the exchange inhibitory peptide (XIP). Objectives The aims of this research are to evaluate TD effects in liver injury secondary to I/R in animals and in vitro action on cytosolic calcium of hepatocytes cultures under calcium overload. Methods Wistar rats submitted to partial liver ischemia were divided in groups: Control: (n = 10): surgical manipulation with no liver ischemia; Saline: (n = 15): rats receiving IV saline before reperfusion; and TD: (n = 15): rats receiving IV TD before reperfusion. Four hours after reperfusion, serum levels of AST, ALT, TNF-α, IL-6, and IL-10 were measured. Liver tissue samples were collected for mitochondrial function and malondialdehyde (MDA) content. Pulmonary vascular permeability and histologic parameters of liver were determined. TD effect on cytosolic calcium was evaluated in BRL3A hepatic rat cell cultures stimulated by thapsigargin pre and after treatment with TD. Results AST, ALT, cytokines, liver MDA, mitochondrial dysfunction and hepatic histologic injury scores were less in TD group when compared to Saline Group (p<0.05) with no differences in pulmonary vascular permeability. In culture cells, TD diminished the intracellular calcium raise and prevented the calcium increase pre and after treatment with thapsigargin, respectively. Conclusion TD decreases liver cell damage, preserves mitochondrial function and increases hepatic tolerance to I/R injury by calcium extrusion in Ca2+ overload situations. PMID:26901764

  5. Inhibiting cytosolic translation and autophagy improves health in mitochondrial disease

    PubMed Central

    Peng, Min; Ostrovsky, Julian; Kwon, Young Joon; Polyak, Erzsebet; Licata, Joseph; Tsukikawa, Mai; Marty, Eric; Thomas, Jeffrey; Felix, Carolyn A.; Xiao, Rui; Zhang, Zhe; Gasser, David L.; Argon, Yair; Falk, Marni J.

    2015-01-01

    Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2kd/kd mice with complexes I–III/II–III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse

  6. Calcium and bones

    MedlinePlus

    ... very bad at absorbing calcium. Most people absorb only 15% to 20% of the calcium they eat in their diet. Vitamin D is the hormone that helps the gut absorb more calcium. Many older adults have common risks that make bone health worse. ...

  7. Get Enough Calcium

    MedlinePlus

    ... a food with 45% DV of calcium. Check food labels. The Daily Value (DV) on a food label tells you the amount of a nutrient (like ... serving, or 60% DV. Learn how to check food labels for calcium information. Use this calcium shopping list ...

  8. Paradoxes of neutrino oscillations

    SciTech Connect

    Akhmedov, E. Kh.; Smirnov, A. Yu.

    2009-08-15

    Despite the theory of neutrino oscillations being rather old, some of its basic issues are still being debated in the literature. We discuss a number of such issues, including the relevance of the 'same energy' and 'same momentum' assumptions, the role of quantum-mechanical uncertainty relations in neutrino oscillations, the dependence of the coherence and localization conditions that ensure the observability of neutrino oscillations on neutrino energy and momentum uncertainties, the question of (in)dependence of the oscillation probabilities on the neutrino production and detection processes, and the applicability limits of the stationary-source approximation. We also develop a novel approach to calculation of the oscillation probability in the wave-packet approach, based on the summation/integration conventions different from the standard one, which allows a new insight into the 'same energy' vs. 'same momentum' problem. We also discuss a number of apparently paradoxical features of the theory of neutrino oscillations.

  9. Does calcium channel blockade have a role in prevention of expression of sepsis in renal transplant recipients?

    PubMed Central

    D’Elia, John A; Gleason, Ray E; Monaco, Anthony P; Weinrauch, Larry A

    2016-01-01

    Many antihypertensive agents have been demonstrated to assist in preservation of kidney function, among them those that modulate calcium channels. Calcium channel blockers may also be of value in protecting hemodialysis patients from complications of sepsis. In diabetic recipients of kidney transplant allografts treated with cyclosporine, calcium channel blockade has been retrospectively linked to improved graft preservation and to fewer episodes of sepsis. This brief review outlines clinical and experimental publications on potential protection from sepsis by addition of calcium channel blockers to standard antibiotic therapy in individuals who may or may not have normal kidney function, or in the presence or absence of immunosuppression. Such mechanisms include blockade of antibiotic cytosolic extrusion in the cases of Pneumococci, Mycobacterium tuberculosis, Plasmodium falciparum malaria, or Schistosoma mansoni; blockade of the calcineurin/calmodulin pathway (in immunosuppressed patients allowing for lower dosage of cyclosporine); stabilization of calcium movement at the level of sarcoplasmic reticulum by which shock (vasopressor instability) is prevented; or of cytosolic calcium influx and cell death (in the case of allograft acute tubular necrosis). Given the high cost of development of new antibiotics, a role for generic calcium channel blockade in sepsis prevention should be pursued by additional studies to investigate potential links between blockade of calcium channels and expression of sepsis in at-risk populations. PMID:27920569

  10. Bax inhibitor 1, a modulator of calcium homeostasis, confers affective resilience.

    PubMed

    Hunsberger, Joshua G; Machado-Vieira, Rodrigo; Austin, Daniel R; Zarate, Carlos; Chuang, De-Maw; Chen, Guang; Reed, John C; Manji, Husseini K

    2011-07-27

    The endoplasmic reticulum (ER) is a critical site for intracellular calcium storage as well as protein synthesis, folding, and trafficking. Disruption of these processes is gaining support for contributing to heritable vulnerability of certain diseases. Here, we investigated Bax inhibitor 1 (BI-1), an anti-apoptotic protein that primarily resides in the ER and associates with B-cell lymphoma 2 (Bcl-2) and Bcl-XL, as an affective resiliency factor through its modulation of calcium homeostasis. We found that transgenic (TG) mice with BI-1 reinforced expression, via the neuronal specific enolase promoter, showed protection against the learned helplessness (LH) paradigm, an animal model to test stress coping. TG mice were also protected against anhedonia following both serotonin and catecholamine depletion as measured in two different models, the female urine sniffing test and the saccharine preference test. In addition, we used primary mouse cortical cultures to explore the ability of BI-1 to influence calcium homeostasis under basal conditions and also following challenge with thapsigargin (THPS), an inhibitor of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) that disrupts calcium homeostasis. TG neurons showed decreased basal cytosolic calcium levels and decreased Ca(2+) cytosolic accumulation following challenge with THPS as compared to WT neuronal cultures. Together, these data suggest that BI-1, through its actions on calcium homeostasis, may confer affective resiliency in multiple animal models of depression and anhedonia.

  11. Bax inhibitor 1, a modulator of calcium homeostasis, confers affective resilience

    PubMed Central

    Hunsberger, Joshua G.; Machado-Vieira, Rodrigo; Austin, Daniel R.; Zarate, Carlos; Chuang, De-Maw; Chen, Guang; Reed, John C.; Manji, Husseini K.

    2011-01-01

    The endoplamic reticulum (ER) is a critical site for intracellular calcium storage as well as protein synthesis, folding, and trafficking. Disruption of these processes is gaining support for contributing to heritable vulnerability of certain diseases. Here, we investigated Bax inhibitor 1 (BI-1), an anti-apoptotic protein that primarily resides in the ER and associates with B-cell lymphoma 2 (Bcl-2) and Bcl-XL, as an affective resiliency factor through its modulation of calcium homeostasis. We found that transgenic (TG) mice with BI-1 reinforced expression, via the neuronal specific enolase promoter, showed protection against the learned helplessness (LH) paradigm, an animal model to test stress coping. TG mice were also protected against anhedonia following both serotonin and catecholamine depletion as measured in two different models, the female urine sniffing test and the saccharine preference test. In addition, we used primary mouse cortical cultures to explore the ability of BI-1 to influence calcium homeostasis under basal conditions and also following challenge with thapsigargin (THPS), an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) that disrupts calcium homeostasis. TG neurons showed decreased basal cytosolic calcium levels and decreased Ca2+ cytosolic accumulation following challenge with THPS as compared to WT neuronal cultures. Together, these data suggest that BI-1, through its actions on calcium homeostasis, may confer affective resiliency in multiple animal models of depression and anhedonia. PMID:21718971

  12. Oscillations in stellar atmospheres

    NASA Technical Reports Server (NTRS)

    Costa, A.; Ringuelet, A. E.; Fontenla, J. M.

    1989-01-01

    Atmospheric excitation and propagation of oscillations are analyzed for typical pulsating stars. The linear, plane-parallel approach for the pulsating atmosphere gives a local description of the phenomenon. From the local analysis of oscillations, the minimum frequencies are obtained for radially propagating waves. The comparison of the minimum frequencies obtained for a variety of stellar types is in good agreement with the observed periods of the oscillations. The role of the atmosphere in the globar stellar pulsations is thus emphasized.

  13. Workshop on Harmonic Oscillators

    NASA Technical Reports Server (NTRS)

    Han, D. (Editor); Kim, Y. S. (Editor); Zachary, W. W. (Editor)

    1993-01-01

    Proceedings of a workshop on Harmonic Oscillators held at the College Park Campus of the University of Maryland on March 25 - 28, 1992 are presented. The harmonic oscillator formalism is playing an important role in many branches of physics. This is the simplest mathematical device which can connect the basic principle of physics with what is observed in the real world. The harmonic oscillator is the bridge between pure and applied physics.

  14. Self-oscillation

    NASA Astrophysics Data System (ADS)

    Jenkins, Alejandro

    2013-04-01

    Physicists are very familiar with forced and parametric resonance, but usually not with self-oscillation, a property of certain dynamical systems that gives rise to a great variety of vibrations, both useful and destructive. In a self-oscillator, the driving force is controlled by the oscillation itself so that it acts in phase with the velocity, causing a negative damping that feeds energy into the vibration: no external rate needs to be adjusted to the resonant frequency. The famous collapse of the Tacoma Narrows bridge in 1940, often attributed by introductory physics texts to forced resonance, was actually a self-oscillation, as was the swaying of the London Millennium Footbridge in 2000. Clocks are self-oscillators, as are bowed and wind musical instruments. The heart is a “relaxation oscillator”, i.e., a non-sinusoidal self-oscillator whose period is determined by sudden, nonlinear switching at thresholds. We review the general criterion that determines whether a linear system can self-oscillate. We then describe the limiting cycles of the simplest nonlinear self-oscillators, as well as the ability of two or more coupled self-oscillators to become spontaneously synchronized (“entrained”). We characterize the operation of motors as self-oscillation and prove a theorem about their limit efficiency, of which Carnot’s theorem for heat engines appears as a special case. We briefly discuss how self-oscillation applies to servomechanisms, Cepheid variable stars, lasers, and the macroeconomic business cycle, among other applications. Our emphasis throughout is on the energetics of self-oscillation, often neglected by the literature on nonlinear dynamical systems.

  15. Mycobacterium tuberculosis Meets the Cytosol: The Role of cGAS in Anti-mycobacterial Immunity.

    PubMed

    Majlessi, Laleh; Brosch, Roland

    2015-06-10

    The intracellular fate of Mycobacterium tuberculosis is a subject of long debate. In this issue of Cell Host & Microbe, three independent studies reveal the detection of cytosolic mycobacterial DNA by the nucleotidyltransferase cGAS, emphasizing the concept of cytosolic access by M. tuberculosis and its role in balancing immune-protection and immune-pathogenesis.

  16. Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah E.; Tsou, Pei-Lan; Robertson, Dominique; Brown, C. S. (Principal Investigator)

    2002-01-01

    Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

  17. Growth control in colon epithelial cells: gadolinium enhances calcium-mediated growth regulation.

    PubMed

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K; Varani, James

    2012-12-01

    Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1-5 μM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet.

  18. A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

    PubMed

    Assinger, Alice; Volf, Ivo; Schmid, Diethart

    2015-01-01

    Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar(+)-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

  19. A Novel, Rapid Method to Quantify Intraplatelet Calcium Dynamics by Ratiometric Flow Cytometry

    PubMed Central

    Assinger, Alice; Volf, Ivo; Schmid, Diethart

    2015-01-01

    Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics. PMID:25849642

  20. Adenine Nucleotide Levels in the Cytosol, Chloroplasts, and Mitochondria of Wheat Leaf Protoplasts 1

    PubMed Central

    Stitt, Mark; Lilley, Ross McC.; Heldt, Hans W.

    1982-01-01

    Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. `Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria. PMID:16662653

  1. Electronically Tuned Microwave Oscillator

    NASA Technical Reports Server (NTRS)

    Lakshminarayana, Mysore

    1987-01-01

    Features include low phase noise and frequency stability. Bias-tuned, low-phase-noise microwave oscillator circuit based on npn bipolar transistor and dielectric resonator. Operating at frequency of about 8.4 GHz, oscillator adjusted to give low phase noise, relatively flat power output versus frequency, and nearly linear frequency versus bias voltage.

  2. Investigating Magnetic Oscillations.

    ERIC Educational Resources Information Center

    Brueningsen, Christopher A.

    1993-01-01

    Studies magnetic oscillation using an air track. Ceramic magnets are attached to the cart and also are used as dampeners in place of the springs. The resulting oscillations are fairly sinusoidal and is a good example of simple harmonic motion. (MVL)

  3. Active-bridge oscillator

    DOEpatents

    Wessendorf, Kurt O.

    2001-01-01

    An active bridge oscillator is formed from a differential amplifier where positive feedback is a function of the impedance of one of the gain elements and a relatively low value common emitter resistance. This use of the nonlinear transistor parameter h stabilizes the output and eliminates the need for ALC circuits common to other bridge oscillators.

  4. HIGH POWER PULSED OSCILLATOR

    DOEpatents

    Singer, S.; Neher, L.K.

    1957-09-24

    A high powered, radio frequency pulse oscillator is described for generating trains of oscillations at the instant an input direct voltage is impressed, or immediately upon application of a light pulse. In one embodiment, the pulse oscillator comprises a photo-multiplier tube with the cathode connected to the first dynode by means of a resistor, and adjacent dynodes are connected to each other through adjustable resistors. The ohmage of the resistors progressively increases from a very low value for resistors adjacent the cathode to a high value adjacent the plate, the last dynode. Oscillation occurs with this circuit when a high negative voltage pulse is applied to the cathode and the photo cathode is bombarded. Another embodiment adds capacitors at the resistor connection points of the above circuit to increase the duration of the oscillator train.

  5. Active and passive calcium transport systems in plant cells. Progress report, May 1986--January 1991

    SciTech Connect

    Sze, H.

    1991-12-31

    The ability to change cytoplasmic Ca{sup 2+} levels ([Ca{sup 2+}]) by cells has made this cation a key regulator of many biological processes. Cytoplasmic [Ca{sup 2+}] is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic [Ca{sup 2+}] and active Ca{sup 2+} transport systems that lower cytosolic [Ca{sup 2+}]. The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  6. Spautin-1 Ameliorates Acute Pancreatitis via Inhibiting Impaired Autophagy and Alleviating Calcium Overload

    PubMed Central

    Xiao, Juan; Feng, Xueping; Huang, Xiao-Ying; Huang, Zhongshi; Huang, Yanqiang; Li, Chaogan; Li, Genliang; Nong, Song; Wu, Ruoshi; Huang, Yongzhi; Long, Xi-Dai

    2016-01-01

    Acute pancreatitis is characterized by zymogen preactivation. Severe inflammation caused by zymogen activation can eventually lead to multiple organ dysfunctions which contribute to the high mortality rate of severe acute pancreatitis. However, there is no specific treatment available for acute pancreatitis therapy. Here, we show that spautin-1, which effectively inhibits autophagy flux, ameliorated the pathogenesis of acute pancreatitis induced by cerulein or L-arginine. CaMKII phosphorylation due to cytosolic calcium overload was revealed in this paper. It was also demonstrated that autophagic protein aggregates degradation blockade accompanied by impaired autophagy correlated positively with intra-acinar cell digestive aymogen activation stimulated by cerulein or L-arginine. The role of spautin-1 in ameliorating acute pancreatitis was shown here to be associated with impaired autophagy inhibition and Ca2+ overload alleviation. We provide a promising therapy for acute pancreatitis through targeting both impaired autophagy and increased cytosolic calcium. PMID:27579473

  7. Simplified model of cytosolic Ca2+ dynamics in the presence of one or several clusters of Ca2+ -release channels

    NASA Astrophysics Data System (ADS)

    Solovey, G.; Fraiman, D.; Pando, B.; Ponce Dawson, S.

    2008-10-01

    Calcium release from intracellular stores plays a key role in the regulation of a variety of cellular activities. In various cell types this release occurs through inositol-triphosphate (IP3) receptors which are Ca2+ channels whose open probability is modulated by the cytosolic Ca2+ concentration itself. Thus, the combination of Ca2+ release and Ca2+ diffusion evokes a variety of Ca2+ signals depending on the number and relative location of the channels that participate of them. In fact, a hierarchy of Ca2+ signals has been observed in Xenopus laevis oocytes, ranging from very localized events (puffs and blips) to waves that propagate throughout the cell. In this cell type channels are organized in clusters. The behavior of individual channels within a cluster cannot be resolved with current optical techniques. Therefore, a combination of experiments and mathematical modeling is unavoidable to understand these signals. However, the numerical simulation of a detailed mathematical model of the problem is very hard given the large range of spatial and temporal scales that must be covered. In this paper we present an alternative model in which the cluster region is modeled using a relatively fine grid but where several approximations are made to compute the cytosolic Ca2+ concentration ([Ca2+]) distribution. The inner-cluster [Ca2+] distribution is used to determine the openings and closings of the channels of the cluster. The spatiotemporal [Ca2+] distribution outside the cluster is determined using a coarser grid in which each (active) cluster is represented by a point source whose current is proportional to the number of open channels determined before. A full reaction-diffusion system is solved on this coarser grid.

  8. A Cytosolic STIM2 Preprotein Created by Signal Peptide Inefficiency Activates ORAI1 in a Store-independent Manner*

    PubMed Central

    Graham, Sarah J. L.; Dziadek, Marie A.; Johnstone, Lorna S.

    2011-01-01

    Calcium (Ca2+) influx through the plasma membrane store-operated Ca2+ channel ORAI1 is controlled by Ca2+ sensors of the stromal interaction molecule (STIM) family. STIM1 responds to endoplasmic reticulum (ER) Ca2+ store depletion by redistributing and activating ORAI1 from regions of the ER juxtaposed to the plasma membrane. Unlike STIM1, STIM2 can regulate ORAI1 in a store-dependent and store-independent manner, but the mechanism by which this is achieved is unknown. Here we find that STIM2 is translated from a highly conserved methionine residue and is directed to the ER by an incredibly long 101-amino acid signal peptide. We find that although the majority of the total STIM2 population resides on the ER membrane, a second population escapes ER targeting to accumulate as a full-length preprotein in the cytosol, signal peptide intact. Unlike STIM2, preSTIM2 localizes to the inner leaflet of the plasma membrane where it interacts with ORAI1 to regulate basal Ca2+ concentration and Ca2+-dependent gene transcription in a store-independent manner. Furthermore, a third protein comprising a fragment of the STIM2 signal peptide is released from the ER membrane into the cytosol where it regulates gene transcription in a Ca2+-independent manner. This study establishes a new model for STIM2-mediated regulation of ORAI1 in which two distinct proteins, STIM2 and preSTIM2, control store-dependent and store-independent modes of ORAI1 activation. PMID:21383014

  9. Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx.

    PubMed

    Jaffe, Lionel F

    2007-03-01

    For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.

  10. Oscillators and Oscillations in the Basal Ganglia

    PubMed Central

    Wilson, Charles J.

    2015-01-01

    What is the meaning of an action potential? There must be different answers for neurons that oscillate spontaneously, firing action potentials even in the absence of any synaptic input, and those driven to fire from a resting membrane potential. In spontaneously firing neurons, the occurrence of the next action potential is guaranteed. Only variations in its timing can carry the message. Among cells of this type are all those making up the deeper nuclei of the basal ganglia, including both segments of the globus pallidus, the substantia nigra, and the subthalamic nucleus. These cells receive thousands of excitatory and inhibitory synaptic inputs, but no input is required to maintain the firing of the cells; they fire at approximately the same rate when the synapses are silenced. Instead, synaptic inputs produce brief changes in spike timing and firing rate. The interactions among oscillating cells within and among the basal ganglia nuclei produce a complex resting pattern of activity. Normally, this pattern is highly irregular and decorrelates the network, so that the firing of each cell is statistically independent of the others. This maximizes the potential information that may be transmitted by the basal ganglia to its target structures. In Parkinson’s disease, the resting pattern of activity is dominated by a slow oscillation shared by all the neurons. Treatment with deep brain stimulation may gain its therapeutic value by disrupting this shared pathological oscillation, and restoring independent action by each neuron in the network. PMID:25449134

  11. Changes in the distribution of lens calcium during development of x-ray cataract

    SciTech Connect

    Hightower, K.R.; Giblin, F.J.; Reddy, V.N.

    1983-09-01

    The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks post x-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase.

  12. Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407).

    PubMed

    Yada, T; Oiki, S; Ueda, S; Okada, Y

    1986-06-16

    Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.

  13. Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy

    PubMed Central

    Fraysse, Bodvaël; Guicheney, Pascale

    2016-01-01

    ABSTRACT Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca2+ and releasable Ca2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content. PMID:27870637

  14. Cleavage by MALT1 induces cytosolic release of A20.

    PubMed

    Malinverni, Claire; Unterreiner, Adeline; Staal, Jens; Demeyer, Annelies; Galaup, Marion; Luyten, Marcel; Beyaert, Rudi; Bornancin, Frédéric

    2010-10-01

    The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never been detected directly, thus limiting insight into the functional consequences of MALT1-mediated cleavage of A20. Here, various antibodies were tested, including newly generated hA20p50 and hA20p37 specific antibodies, leading to detection of the hA20p50 fragment produced after MALT1-mediated cleavage of ectopically expressed as well as endogenous A20 proteins. The properties of both A20 fragments, generated upon co-expression with a constitutively active MALT1 protein, were further studied by sub-cellular fractionation and fluorescence microscopy. In contrast to full-length A20 which is particulate and insoluble, we found hA20p50 to be soluble and readily released into the cytosol whereas hA20p37 was partially soluble, thus suggesting loss of compartmentalization as a possible mechanism for MALT1-mediated dampening of A20 function.

  15. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

    PubMed

    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  16. Role of Cytosolic Carboxypeptidase 5 in Neuronal Survival and Spermatogenesis

    PubMed Central

    Wu, Hui-Yuan; Wei, Peng; Morgan, James I.

    2017-01-01

    Proteins may undergo a type of posttranslational modification – polyglutamylation, where a glutamate residue is enzymatically linked to the γ-carboxyl group of a glutamate in the primary sequence of proteins and additional glutamates are then sequentially added via α-carboxyl–linkages to the growing glutamate side chain. Nna1 (a.k.a. CCP1) defines the 6-member cytosolic carboxypeptidase (CCP) family that metabolizes polyglutamate side chain and its loss results in neurodegeneration and male infertility. Whereas most CCPs catalyze hydrolysis of α-carboxyl-linked glutamates, CCP5 uniquely metabolizes the γ-carboxyl linked, branch point glutamate. Using purified recombinant mouse CCP5, we confirmed that it metabolized γ-carboxyl-linked glutamate of synthetic substrates and tubulin. Despite this unique feature and its indispensible functions in lower species, we found that unlike Nna1, CCP5 is not essential for neuronal survival in mouse. CCP5 deficiency does cause male infertility. However, the mechanism by which this occurs is distinct from that of Nna1 loss. Instead, it is phenotypically reminiscent of the infertility of olt mice. Our findings suggest that Nna1 and CCP5 do not work coordinately in the same pathway in either the nervous system or spermatogenesis. This is the first study addressing the function of CCP5 in mammals. PMID:28128286

  17. Regulation of the cytosolic sulfotransferases by nuclear receptors

    PubMed Central

    Runge-Morris, Melissa; Kocarek, Thomas A.; Falany, Charles N.

    2013-01-01

    The cytosolic sulfotransferases (SULTs) are a multigene family of enzymes that catalyze the transfer of a sulfonate group from the physiologic sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, to a nucleophilic substrate to generate a polar product that is more amenable to elimination from the body. As catalysts of both xenobiotic and endogenous metabolism, the SULTs are major points of contact between the external and physiological environments, and modulation of SULT-catalyzed metabolism can not only affect xenobiotic disposition, but it can also alter endogenous metabolic processes. Therefore, it is not surprising that SULT expression is regulated by numerous members of the nuclear receptor (NR) superfamily that function as sensors of xenobiotics as well as endogenous molecules, such as fatty acids, bile acids, and oxysterols. These NRs include the peroxisome proliferator-activated receptors, pregnane X receptor, constitutive androstane receptor, vitamin D receptor, liver X receptors, farnesoid X receptor, retinoid-related orphan receptors, and estrogen-related receptors. This review summarizes current information about NR regulation of SULT expression. Because species differences in SULT subfamily composition and tissue-, sex-, development-, and inducer-dependent regulation are prominent, these differences will be emphasized throughout the review. In addition, because of the central role of the SULTs in cellular physiology, the effect of NR-mediated SULT regulation on physiological and pathophysiological processes will be discussed. Gaps in current knowledge that require further investigation are also highlighted. PMID:23330539

  18. Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles

    PubMed Central

    Liu, Shu; Hossinger, André; Hofmann, Julia P.; Denner, Philip

    2016-01-01

    ABSTRACT Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast (Saccharomyces cerevisiae) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. PMID:27406566

  19. Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer.

    PubMed

    Nikolic, Ivana; Kastratovic, Tatjana; Zelen, Ivanka; Zivanovic, Aleksandar; Arsenijevic, Slobodan; Mitrovic, Marina

    2010-04-30

    Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic "BH3-only" BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase's downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.

  20. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1

    PubMed Central

    Drmota Prebil, Sara; Slapšak, Urška; Pavšič, Miha; Ilc, Gregor; Puž, Vid; de Almeida Ribeiro, Euripedes; Anrather, Dorothea; Hartl, Markus; Backman, Lars; Plavec, Janez; Lenarčič, Brigita; Djinović-Carugo, Kristina

    2016-01-01

    The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin. PMID:27272015

  1. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

    PubMed

    Drmota Prebil, Sara; Slapšak, Urška; Pavšič, Miha; Ilc, Gregor; Puž, Vid; de Almeida Ribeiro, Euripedes; Anrather, Dorothea; Hartl, Markus; Backman, Lars; Plavec, Janez; Lenarčič, Brigita; Djinović-Carugo, Kristina

    2016-06-07

    The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

  2. A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

    PubMed

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

    The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

  3. Ultrastable Cryogenic Microwave Oscillators

    NASA Astrophysics Data System (ADS)

    Mann, Anthony G.

    Ultrastable cryogenic microwave oscillators are secondary frequency standards in the microwave domain. The best of these oscillators have demonstrated a short term frequency stability in the range 10-14 to a few times 10-16. The main application for these oscillators is as flywheel oscillators for the next generation of passive atomic frequency standards, and as local oscillators in space telemetry ground stations to clean up the transmitter close in phase noise. Fractional frequency stabilities of passive atomic frequency standards are now approaching 3 x10^-14 /τ where τ is the measurement time, limited only by the number of atoms that are being interrogated. This requires an interrogation oscillator whose short-term stability is of the order of 10-14 or better, which cannot be provided by present-day quartz technology. Ultrastable cryogenic microwave oscillators are based on resonators which have very high electrical Q-factors. The resolution of the resonator's linewidth is typically limited by electronics noise to about 1ppm and hence Q-factors in excess of 108 are required. As these are only attained in superconducting cavities or sapphire resonators at low temperatures, use of liquid helium cooling is mandatory, which has so far restricted these oscillators to the research or metrology laboratory. Recently, there has been an effort to dispense with the need for liquid helium and make compact flywheel oscillators for the new generation of primary frequency standards. Work is under way to achieve this goal in space-borne and mobile liquid-nitrogen-cooled systems. The best cryogenic oscillators developed to date are the ``whispering gallery'' (WG) mode sapphire resonator-oscillators of NASA's Jet Propulsion Laboratory (JPL) and the University of Western Australia (UWA), as well as Stanford University's superconducting cavity stabilized oscillator (SCSO). All of these oscillators have demonstrated frequency

  4. Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.

    PubMed

    Shen, Yu J; Le Bert, Nina; Chitre, Anuja A; Koo, Christine Xing'Er; Nga, Xing H; Ho, Samantha S W; Khatoo, Muznah; Tan, Nikki Y; Ishii, Ken J; Gasser, Stephan

    2015-04-21

    The DNA damage response (DDR) induces the expression of type I interferons (IFNs), but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  5. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25.

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  6. A mathematical model of calcium dynamics in HSY cells

    PubMed Central

    Han, Jung Min; Tanimura, Akihiko; Kirk, Vivien; Sneyd, James

    2017-01-01

    Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+) plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. PMID:28199326

  7. Quasi-Fibonacci oscillators

    NASA Astrophysics Data System (ADS)

    Gavrilik, A. M.; Kachurik, I. I.; Rebesh, A. P.

    2010-06-01

    We study the properties of the sequences of the energy eigenvalues for some generalizations of q-deformed oscillators including the p, q-oscillator, and the three-, four- and five-parameter deformed oscillators given in the literature. It is shown that most of the considered models belong to the class of so-called Fibonacci oscillators for which any three consecutive energy levels satisfy the relation En + 1 = λEn + ρEn - 1 with real constants λ, ρ. On the other hand, for a certain μ-oscillator known since 1993, we prove its non-Fibonacci nature. Possible generalizations of the three-term Fibonacci relation are discussed, among which for the μ-oscillator we choose, as the most adequate, the so-called quasi-Fibonacci (or local Fibonacci) property of the energy levels. The property is encoded in the three-term quasi-Fibonacci (QF) relation with the non-constant, n-dependent coefficients λ and ρ. Various aspects of the QF relation are elaborated for the μ-oscillator and some of its extensions.

  8. Francisella Inflammasomes: Integrated Responses to a Cytosolic Stealth Bacterium.

    PubMed

    Wallet, Pierre; Lagrange, Brice; Henry, Thomas

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium causing tularemia, a zoonotic disease. Francisella replicates in the macrophage cytosol and eventually triggers cytosolic immune responses. In murine macrophages, Francisella novicida and Francisella tularensis live vaccine strain lyse in the host cytosol and activate the cytosolic DNA receptor Aim2. Here, we review the mechanisms leading or contributing to Aim2 inflammasome activation, including the role of TLRs and of IFN signaling and the implication of the guanylate-binding proteins 2 and 5 in triggering cytosolic bacteriolysis. Furthermore, we present how this cytosolic Gram-negative bacterium escapes recognition by caspase-11 but can trigger a non-canonical caspase-8 inflammasome. In addition, we highlight the differences in inflammasome activation in murine and human cells with pyrin, NLRP3, and AIM2 involved in sensing Francisella in human phagocytes. From a bacterial prospective, we describe the hiding strategy of Francisella to escape recognition by innate sensors and to resist to bacteriolysis in the host cytosol. Finally, we discuss the inability of the inflammasome sensors to detect F. tularensis subspecies tularensis strains, making them highly pathogenic stealth microbes.

  9. Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages.

    PubMed

    Manzanillo, Paolo S; Shiloh, Michael U; Portnoy, Daniel A; Cox, Jeffery S

    2012-05-17

    Cytosolic bacterial pathogens activate the cytosolic surveillance pathway (CSP) and induce innate immune responses, but how the host detects vacuolar pathogens like Mycobacterium tuberculosis is poorly understood. We show that M. tuberculosis also initiates the CSP upon macrophage infection via limited perforation of the phagosome membrane mediated by the ESX-1 secretion system. Although the bacterium remains within the phagosome, this permeabilization results in phagosomal and cytoplasmic mixing and allows extracellular mycobacterial DNA to access host cytosolic receptors, thus blurring the distinction between "vacuolar" and "cytosolic" pathogens. Activation of cytosolic receptors induces signaling through the Sting/Tbk1/Irf3 axis, resulting in IFN-β production. Surprisingly, Irf3(-/-) mice, which cannot respond to cytosolic DNA, are resistant to long-term M. tuberculosis infection, suggesting that the CSP promotes M. tuberculosis infection. Thus, cytosolic sensing of mycobacterial DNA plays a key role in M. tuberculosis pathogenesis and likely contributes to the high type I IFN signature in tuberculosis.

  10. Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

    NASA Astrophysics Data System (ADS)

    Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

    1996-07-01

    In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

  11. Cytosolic Na+ controls and epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.

    PubMed Central

    Komwatana, P; Dinudom, A; Young, J A; Cook, D I

    1996-01-01

    In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-. Images Fig. 4 PMID:8755611

  12. The cytosolic tail of the tumor marker protein Trop2 - a structural switch triggered by phosphorylation

    NASA Astrophysics Data System (ADS)

    Pavšič, Miha; Ilc, Gregor; Vidmar, Tilen; Plavec, Janez; Lenarčič, Brigita

    2015-05-01

    Trop2 is a transmembrane signaling glycoprotein upregulated in stem and carcinoma cells. Proliferation-enhancing signaling involves regulated intramembrane proteolytic release of a short cytoplasmic fragment, which is later engaged in a cytosolic signaling complex. We propose that Trop2 function is modulated by phosphorylation of a specific serine residue within this cytosolic region (Ser303), and by proximity effects exerted on the cytosolic tail by Trop2 dimerization. Structural characterization of both the transmembrane (Trop2TM) and cytosolic regions (Trop2IC) support this hypothesis, and shows that the central region of Trop2IC forms an α-helix. Comparison of NMR structures of non-phosphorylated and phosphorylated forms suggest that phosphorylation of Trop2IC triggers salt bridge reshuffling, resulting in significant conformational changes including ordering of the C-terminal tail. In addition, we demonstrate that the cytosolic regions of two Trop2 subunits can be brought into close proximity via transmembrane part dimerization. Finally, we show that Ser303-phosphorylation significantly affects the structure and accessibility of functionally important regions of the cytosolic tail. These observed structural features of Trop2 at the membrane-cytosol interface could be important for regulation of Trop2 signaling activity.

  13. Cerebellar network plasticity: from genes to fast oscillation.

    PubMed

    Cheron, G; Servais, L; Dan, B

    2008-04-22

    The role of the cerebellum has been increasingly recognized not only in motor control but in sensory, cognitive and emotional learning and regulation. Purkinje cells, being the sole output from the cerebellar cortex, occupy an integrative position in this network. Plasticity at this level is known to critically involve calcium signaling. In the last few years, electrophysiological study of genetically engineered mice has demonstrated the topical role of several genes encoding calcium-binding proteins (calretinin, calbindin, parvalbumin). Specific inactivation of these genes results in the emergence of a fast network oscillation (ca. 160 Hz) throughout the cerebellar cortex in alert animals, associated with ataxia. This oscillation is produced by synchronization of Purkinje cells along the parallel fiber beam. It behaves as an electrophysiological arrest rhythm, being blocked by sensorimotor stimulation. Pharmacological manipulations showed that the oscillation is blocked by GABA(A) and NMDA antagonists as well as gap junction blockers. This cerebellar network oscillation has also been documented in mouse models of human conditions with complex developmental cerebellar dysfunction, such as Angelman syndrome and fetal alcohol syndrome. Recent evidence suggests a relationship between fast oscillation and cerebellar long term depression (LTD). This may have major implications for future therapeutic targeting.

  14. The plasma membrane calcium ATPase and disease.

    PubMed

    Tempel, B L; Shilling, D J

    2007-01-01

    The plasma membrane calcium ATPase (PMCA) uses energy to pump calcium (Ca2+) ions out of the cytosol into the extracellular milieu, usually against a strong chemical gradient. This energy expenditure is necessary to maintain a relatively low intracellular net Ca2+ load. Mammals have four genes (ATP2B1-ATP2B4), encoding the proteins PMCA1 through PMCA4. Transcripts from each of these genes are alternatively spliced to generate several variant proteins that are in turn post-translationally modified in a variety of ways. Expressed ubiquitously and with some level of functional redundancy in most vital tissues, only one of the four genes--Atp2b2--has been causally linked through naturally occuring mutations to disease in mammals: specifically to deafness and ataxia in spontaneous mouse mutants. In humans, a missense amino acid substitution in PMCA2 modifies the severity of hearing loss. Targeted null mutations of the Atp2b1 and Atp2b4 genes in mouse are embryonic lethal and cause a sperm motility defect, respectively. These phenotypes point to complex human diseases like hearing loss, cardiac function and infertility. Changes in PMCA expression are associated with other diseases including cataract formation, carciniogenesis, diabetes, and cardiac hypertension and hypertrophy. Severity of these diseases may be affected by subtle changes in expression of the PMCA isoforms expressed in those tissues.

  15. Calcium hydroxide poisoning

    MedlinePlus

    These products contain calcium hydroxide: Cement Limewater Many industrial solvents and cleaners (hundreds to thousands of construction products, flooring strippers, brick cleaners, cement thickening products, and many ...

  16. Calcium: total or ionized?

    PubMed

    Schenck, Patricia A; Chew, Dennis J

    2008-05-01

    Measurement of serum total calcium (tCa) has been relied on for assessment of calcium status, despite the fact that it is the ionized calcium (iCa) fraction that has biologic activity. Serum tCa does not accurately predict iCa status in many clinical conditions. For accurate assessment of iCa status, iCa should be directly measured. Anaerobic measurement of serum iCa under controlled conditions provides the most reliable assessment of calcium status; aerobic measurement of iCa with species-specific pH correction is highly correlated with anaerobic measurements.

  17. Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

    PubMed Central

    Taylor, Michael; Banerjee, Tuhina; VanBennekom, Neyda; Teter, Ken

    2012-01-01

    AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12. To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin. PMID:22231143

  18. Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels

    PubMed Central

    Mukherjee, Indrani; Barlowe, Charles

    2016-01-01

    SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca2+/Mn2+ transporter and suggests that Sly41 influences cellular Ca2+ and Mn2+ homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca2+ concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca2+ on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport. PMID:27030673

  19. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  20. Towards the Physics of Calcium Signalling in Plants

    PubMed Central

    Vaz Martins, Teresa; Evans, Matthew J.; Woolfenden, Hugh C.; Morris, Richard J.

    2013-01-01

    Calcium is an abundant element with a wide variety of important roles within cells. Calcium ions are inter- and intra-cellular messengers that are involved in numerous signalling pathways. Fluctuating compartment-specific calcium ion concentrations can lead to localised and even plant-wide oscillations that can regulate downstream events. Understanding the mechanisms that give rise to these complex patterns that vary both in space and time can be challenging, even in cases for which individual components have been identified. Taking a systems biology approach, mathematical and computational techniques can be employed to produce models that recapitulate experimental observations and capture our current understanding of the system. Useful models make novel predictions that can be investigated and falsified experimentally. This review brings together recent work on the modelling of calcium signalling in plants, from the scale of ion channels through to plant-wide responses to external stimuli. Some in silico results that have informed later experiments are highlighted. PMID:27137393

  1. Undamped fritting oscillations

    NASA Astrophysics Data System (ADS)

    Titov, V. A.

    2013-01-01

    Fritting oscillations in a glasslike film of methane and chlorine rapidly attenuate. A change in the boundary condition makes them weakly damped, while dosed synchronized injections of vacancies with high-energy particles make it possible to obtain a self-oscillatory system. The mechanism of fritting oscillations is described in detail. An oscillating dissipative structure is formed in the active medium of nonequilibrium glass supersaturated with vacancies and exhibiting a liquid-like behavior. A capillary flow of the medium plays a special role in its evolution.

  2. Solar atmosphere neutrino oscillations

    SciTech Connect

    Fogli, G.L.; Lisi, E.; Mirizzi, A.; Montanino, D.; Serpico, P.D.; /Fermilab

    2007-02-01

    The Sun is a source of high energy neutrinos (E > 10 GeV) produced by cosmic ray interactions in the solar atmosphere. We study the impact of three-flavor oscillations on the solar atmosphere neutrino fluxes observable at Earth. We find that peculiar matter oscillation effects in the Sun do exist, but are significantly suppressed by averaging over the production region and over the neutrino and antineutrino components. In particular, the relation between the neutrino fluxes at the Sun and at the Earth can be approximately expressed in terms of phase-averaged ''vacuum'' oscillations, dominated by a single mixing parameter (the angle {theta}{sub 23}).

  3. LSND neutrino oscillation results

    SciTech Connect

    Louis, W.C.

    1996-06-01

    In the past several years, a number of experiments have searched for neutrino oscillations, where a neutrino of one type (say {bar {nu}}{sub {mu}}) spontaneously transforms into a neutrino of another type (say {bar {nu}}{sub e}). For this phenomenon to occur, neutrinos must be massive and the apparent conservation law of lepton families must be violated. In 1995 the LSND experiment published data showing candidate events that are consistent with {bar {nu}}{sub {mu}} oscillations. Additional data are reported here which provide stronger evidence for neutrino oscillations.

  4. Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy

    PubMed Central

    Acosta, Yassel; Zhang, Qi; Rahaman, Arifur; Ouellet, Hugues; Xiao, Chuan; Sun, Jianjun; Li, Chunqiang

    2014-01-01

    Transition from latency to active tuberculosis requires Mycobacterium tuberculosis (Mtb) to penetrate the phagosomal membrane and translocate to the cytosol of the host macrophage. Quantitative two-photon fluorescence resonance energy transfer (FRET) microscopy is developed to measure cytosolic translocation using Mycobacterium marinum (Mm) as a model organism for Mtb. Macrophages were infected with Mm or non-pathogenic Mycobacterium smegmatis (Ms) as a control, then loaded with a FRET substrate. Once translocation occurs, mycobacterium-bearing β-lactamase cleaves the substrate, resulting in decrease of FRET signal. Quantification of this FRET signal change revealed that Mm, but not Ms, is capable of translocating to the cytosol. PMID:25426325

  5. Collective Calcium Dynamics in Networks of Communicating Cells

    NASA Astrophysics Data System (ADS)

    Byrd, Tommy; Potter, Garrett; Sun, Bo; Mugler, Andrew

    Cells can sense and encode information about their environment with remarkable precision. These properties have been studied extensively for single cells, but intercellular communication is known to be important for both single- and multicellular organisms. Here, we examine calcium dynamics of fibroblast cells exposed to external ATP stimuli, and the effects of communication and stimulus strength on cells' response. Experimental results show that increasing communication strength induces a greater fraction of cells to exhibit oscillatory calcium dynamics, but the frequencies of oscillation do not systematically shift with ATP strength. We developed a model of calcium signaling by adding noise, communication, and cell-to-cell variability to the model of Tang and Othmer. This model reproduces cells' increased tendency to oscillate as a function of communication strength, and frequency encoding is nearly removed at the global level. Our model therefore suggests that the propensity of cells to oscillate, rather than frequency encoding, determines the response to external ATP. These results suggest that the system lies near a critical boundary separating non-oscillatory and oscillatory calcium dynamics.

  6. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  7. Entraining synthetic genetic oscillators

    NASA Astrophysics Data System (ADS)

    Wagemakers, Alexandre; Buldú, Javier M.; Sanjuán, Miguel A. F.; de Luis, Oscar; Izquierdo, Adriana; Coloma, Antonio

    2009-09-01

    We propose a new approach for synchronizing a population of synthetic genetic oscillators, which consists in the entrainment of a colony of repressilators by external modulation. We present a model where the repressilator dynamics is affected by periodic changes in temperature. We introduce an additional plasmid in the bacteria in order to correlate the temperature variations with the enhancement of the transcription rate of a certain gene. This can be done by introducing a promoter that is related to the heat shock response. This way, the expression of that gene results in a protein that enhances the overall oscillations. Numerical results show coherent oscillations of the population for a certain range of the external frequency, which is in turn related to the natural oscillation frequency of the modified repressilator. Finally we study the transient times related with the loss of synchronization and we discuss possible applications in biotechnology of large-scale production coupled to synchronization events induced by heat shock.

  8. Oscillating fluid power generator

    DOEpatents

    Morris, David C

    2014-02-25

    A system and method for harvesting the kinetic energy of a fluid flow for power generation with a vertically oriented, aerodynamic wing structure comprising one or more airfoil elements pivotably attached to a mast. When activated by the moving fluid stream, the wing structure oscillates back and forth, generating lift first in one direction then in the opposite direction. This oscillating movement is converted to unidirectional rotational movement in order to provide motive power to an electricity generator. Unlike other oscillating devices, this device is designed to harvest the maximum aerodynamic lift forces available for a given oscillation cycle. Because the system is not subjected to the same intense forces and stresses as turbine systems, it can be constructed less expensively, reducing the cost of electricity generation. The system can be grouped in more compact clusters, be less evident in the landscape, and present reduced risk to avian species.

  9. High frequency nanotube oscillator

    DOEpatents

    Peng, Haibing [Houston, TX; Zettl, Alexander K [Kensington, TX

    2012-02-21

    A tunable nanostructure such as a nanotube is used to make an electromechanical oscillator. The mechanically oscillating nanotube can be provided with inertial clamps in the form of metal beads. The metal beads serve to clamp the nanotube so that the fundamental resonance frequency is in the microwave range, i.e., greater than at least 1 GHz, and up to 4 GHz and beyond. An electric current can be run through the nanotube to cause the metal beads to move along the nanotube and changing the length of the intervening nanotube segments. The oscillator can operate at ambient temperature and in air without significant loss of resonance quality. The nanotube is can be fabricated in a semiconductor style process and the device can be provided with source, drain, and gate electrodes, which may be connected to appropriate circuitry for driving and measuring the oscillation. Novel driving and measuring circuits are also disclosed.

  10. A novel photonic oscillator

    NASA Technical Reports Server (NTRS)

    Yao, X. S.; Maleki, L.

    1995-01-01

    We report a novel oscillator for photonic RF systems. This oscillator is capable of generating high-frequency signals up to 70 GHz in both electrical and optical domains and is a special voltage-controlled oscillator with an optical output port. It can be used to make a phase-locked loop (PLL) and perform all functions that a PLL is capable of for photonic systems. It can be synchronized to a reference source by means of optical injection locking, electrical injection locking, and PLL. It can also be self-phase locked and self-injection locked to generate a high-stability photonic RF reference. Its applications include high-frequency reference regeneration and distribution, high-gain frequency multiplication, comb-frequecy and square-wave generation, carrier recovery, and clock recovery. We anticipate that such photonic voltage-controlled oscillators (VCOs) will be as important to photonic RF systems as electrical VCOs are to electrical RF systems.

  11. Oscillating Filaments. I. Oscillation and Geometrical Fragmentation

    NASA Astrophysics Data System (ADS)

    Gritschneder, Matthias; Heigl, Stefan; Burkert, Andreas

    2017-01-01

    We study the stability of filaments in equilibrium between gravity and internal as well as external pressure using the grid-based AMR code RAMSES. A homogeneous, straight cylinder below a critical line mass is marginally stable. However, if the cylinder is bent, such as with a slight sinusoidal perturbation, an otherwise stable configuration starts to oscillate, is triggered into fragmentation, and collapses. This previously unstudied behavior allows a filament to fragment at any given scale, as long as it has slight bends. We call this process “geometrical fragmentation.” In our realization, the spacing between the cores matches the wavelength of the sinusoidal perturbation, whereas up to now, filaments were thought to be only fragmenting on the characteristic scale set by the mass-to-line ratio. Using first principles, we derive the oscillation period as well as the collapse timescale analytically. To enable a direct comparison with observations, we study the line-of-sight velocity for different inclinations. We show that the overall oscillation pattern can hide the infall signature of cores.

  12. Intracellular calcium and the relationship to contractility in an avian model of heart failure

    PubMed Central

    Kim, C. S.; Doye, A. A.; Davidoff, A. J.; Maki, T. M.

    2005-01-01

    Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates. PMID:10935520

  13. Disruption of the vacuolar calcium-ATPases in arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium (Ca2+) signals regulate many aspects of plant development, including the Hypersensitive Response (HR) that triggers a programmed cell death response to protect a plant from a pathogen. A transient increase in cytosolic Ca2+ ([Ca2+]cyt ) results from Ca2+ entry from the apoplast or release fr...

  14. Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy

    PubMed Central

    Burr, A R; Molkentin, J D

    2015-01-01

    Muscular dystrophy (MD) refers to a clinically and genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. Although the primary defect underlying most forms of MD typically results from a loss of sarcolemmal integrity, the secondary molecular mechanisms leading to muscle degeneration and myofiber necrosis is debated. One hypothesis suggests that elevated or dysregulated cytosolic calcium is the common transducing event, resulting in myofiber necrosis in MD. Previous measurements of resting calcium levels in myofibers from dystrophic animal models or humans produced equivocal results. However, recent studies in genetically altered mouse models have largely solidified the calcium hypothesis of MD, such that models with artificially elevated calcium in skeletal muscle manifest fulminant dystrophic-like disease, whereas models with enhanced calcium clearance or inhibited calcium influx are resistant to myofiber death and MD. Here, we will review the field and the recent cadre of data from genetically altered mouse models, which we propose have collectively mostly proven the hypothesis that calcium is the primary effector of myofiber necrosis in MD. This new consensus on calcium should guide future selection of drugs to be evaluated in clinical trials as well as gene therapy-based approaches. PMID:26088163

  15. Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland.

    PubMed

    Barbosa, Roseli; Scialfa, Julieta Helena; Terra, Ilza Mingarini; Cipolla-Neto, José; Simonneaux, Valérie; Afeche, Solange Castro

    2008-02-27

    Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

  16. Current oscillations in nanopores

    NASA Astrophysics Data System (ADS)

    Hyland, Brittany

    We develop a simple phenomenological model to describe current oscillations in single, conically shaped nanopores. The model utilizes aspects of reaction rate theory, electrochemical oscillators, and nonlinear dynamical systems. Time series of experimental data were analyzed and compared to time series simulated using the model equations. There is good qualitative agreement between experiment and simulation, though the model needs to be improved in order to obtain better quantitative agreement.

  17. Ultrastable Multigigahertz Photonic Oscillator

    NASA Technical Reports Server (NTRS)

    Logan, Ronald T., Jr.

    1996-01-01

    Novel photonic oscillator developed to serve as ultrastable source of microwave and millimeter-wave signals. In system, oscillations generated photonically, then converted to electronic form. Includes self-mode-locked semiconductor laser producing stream of pulses, detected and fed back to laser as input. System also includes fiber-optic-delay-line discriminator, which detects fluctuations of self-mode-locking frequency and generates error signal used in negative-feedback loop to stabilize pulse-repetition frequency.

  18. Rocket Engine Oscillation Diagnostics

    NASA Technical Reports Server (NTRS)

    Nesman, Tom; Turner, James E. (Technical Monitor)

    2002-01-01

    Rocket engine oscillating data can reveal many physical phenomena ranging from unsteady flow and acoustics to rotordynamics and structural dynamics. Because of this, engine diagnostics based on oscillation data should employ both signal analysis and physical modeling. This paper describes an approach to rocket engine oscillation diagnostics, types of problems encountered, and example problems solved. Determination of design guidelines and environments (or loads) from oscillating phenomena is required during initial stages of rocket engine design, while the additional tasks of health monitoring, incipient failure detection, and anomaly diagnostics occur during engine development and operation. Oscillations in rocket engines are typically related to flow driven acoustics, flow excited structures, or rotational forces. Additional sources of oscillatory energy are combustion and cavitation. Included in the example problems is a sampling of signal analysis tools employed in diagnostics. The rocket engine hardware includes combustion devices, valves, turbopumps, and ducts. Simple models of an oscillating fluid system or structure can be constructed to estimate pertinent dynamic parameters governing the unsteady behavior of engine systems or components. In the example problems it is shown that simple physical modeling when combined with signal analysis can be successfully employed to diagnose complex rocket engine oscillatory phenomena.

  19. Oscillating asymmetric dark matter

    SciTech Connect

    Tulin, Sean; Yu, Hai-Bo; Zurek, Kathryn M. E-mail: haiboyu@umich.edu

    2012-05-01

    We study the dynamics of dark matter (DM) particle-antiparticle oscillations within the context of asymmetric DM. Oscillations arise due to small DM number-violating Majorana-type mass terms, and can lead to recoupling of annihilation after freeze-out and washout of the DM density. Asymmetric DM oscillations 'interpolate' between symmetric and asymmetric DM freeze-out scenarios, and allow for a larger DM model-building parameter space. We derive the density matrix equations for DM oscillations and freeze-out from first principles using nonequilibrium field theory, and our results are qualitatively different than in previous studies. DM dynamics exhibits particle-vs-antiparticle 'flavor' effects, depending on the interaction type, analogous to neutrino oscillations in a medium. 'Flavor-sensitive' DM interactions include scattering or annihilation through a new vector boson, while 'flavor-blind' interactions include scattering or s-channel annihilation through a new scalar boson. In particular, we find that flavor-sensitive annihilation does not recouple when coherent oscillations begin, and that flavor-blind scattering does not lead to decoherence.

  20. Oscillating asymmetric dark matter

    NASA Astrophysics Data System (ADS)

    Tulin, Sean; Yu, Hai-Bo; Zurek, Kathryn M.

    2012-05-01

    We study the dynamics of dark matter (DM) particle-antiparticle oscillations within the context of asymmetric DM. Oscillations arise due to small DM number-violating Majorana-type mass terms, and can lead to recoupling of annihilation after freeze-out and washout of the DM density. Asymmetric DM oscillations "interpolate" between symmetric and asymmetric DM freeze-out scenarios, and allow for a larger DM model-building parameter space. We derive the density matrix equations for DM oscillations and freeze-out from first principles using nonequilibrium field theory, and our results are qualitatively different than in previous studies. DM dynamics exhibits particle-vs-antiparticle "flavor" effects, depending on the interaction type, analogous to neutrino oscillations in a medium. "Flavor-sensitive" DM interactions include scattering or annihilation through a new vector boson, while "flavor-blind" interactions include scattering or s-channel annihilation through a new scalar boson. In particular, we find that flavor-sensitive annihilation does not recouple when coherent oscillations begin, and that flavor-blind scattering does not lead to decoherence.

  1. Calcium fluoride window mounting

    NASA Astrophysics Data System (ADS)

    Berger, D. Douglas

    1982-10-01

    A technique has been developed for joining a large calcium fluoride crystal to a stainless-steel flange by means of a silver transition ring. The process involves both vacuum brazing using a copper-silver alloy and air brazing using silver chloride. This paper describes the procedure used in fabricating a high-vacuum leak-tight calcium fluoride window assembly.

  2. Calcium and bones (image)

    MedlinePlus

    ... for the growth, maintenance, and reproduction of the human body. Bones, like other tissues in the body, are continually being re-formed and incorporate calcium into their structure. Calcium is essential for the formation of and maintenance of healthy teeth.

  3. Kidney and calcium homeostasis.

    PubMed

    Jeon, Un Sil

    2008-12-01

    Plasma calcium concentration is maintained within a narrow range (8.5-10.5 mg/dL) by the coordinated action of parathyroid hormone (PTH), 1,25(OH)2D3, calcitonin, and ionized calcium (iCa(2+)) itself. The kidney plays a key role in this process by the fine regulation of calcium excretion. More than 95% of filtered calcium is reabsorbed along the renal tubules. In the proximal tubules, 60% of filtered calcium is reabsorbed by passive mechanisms. In the thick ascending limb, 15% of calcium is reabsorbed by paracellular diffusion through paracellin-1 (claudin-16). The calcium sensing receptor (CaSR) in the basolateral membrane of the thick ascending limb senses the change in iCa(2+) and inhibits calcium reabsorption independent to PTH and 1,25(OH)2D3. The fine regulation of calcium excretion occurs in the distal convoluted tubules and connecting tubules despite the fact that only 10-15% of filtered calcium is reabsorbed there. Transient receptor potential vanilloid 5 (TRPV5) and 6 (TRPV6) in the apical membrane act as the main portal of entry, calbindin-D28K delivers Ca(2+) in the cytoplasm, and then Na(2+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase in the basolateral membrane serve as an exit. In the cortical collecting duct, TRPV6 is expressed, but the role might be negligible. In addition to PTH and 1,25(OH)2D3, acid-base disturbance, diuretics, and estrogen affect on these calcium channels. Recently, klotho and fibroblast growth factor 23 (FGF23) are suggested as new players in the calcium metabolism. Klotho is exclusively expressed in the kidney and co-localized with TRPV5, NCX1, and calbindin-D28K. Klotho increases calcium reabsorption through trafficking of TRPV5 to the plasma membrane, and also converts FGF receptor to the specific FGF23 receptor. FGF23:klotho complex bound to FGF receptor inhibits 1α-hydroxylase of vitamin D, and contributes to calcium reabsorption and phosphate excretion in the kidney.

  4. The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells

    NASA Astrophysics Data System (ADS)

    Wu, Zu-Hui; Zhu, Dan; Chen, Ji-Yao; Zhou, Lu-Wei

    2012-05-01

    The effects of thermal stimuli on rat basophilic leukemia mast cells were studied. The cells in calcium-contained or calcium-free buffers were thermally stimulated in the temperature range of 25-60 °C. The corresponding calcium ion concentration in cells [Ca2+]i as well as the released histamine from cells was measured with fluorescence staining methods. The ruthenium red (RR), a block of membrane calcium channels (transient receptor potential family V (TRPV)), was used in experiments. Under the stimulus of 25-50 °C, no significant difference on [Ca2+]i was found between these three groups of the cells in calcium-contained buffer without or with RR and cells in calcium-free saline, indicating that the increased calcium in cytosol did not result from the extracellular buffer but came from the intracellular calcium stores. The [Ca2+]i continuously increased under the temperature of 50-60 °C, but the RR and calcium-free saline can obviously diminish the [Ca2+]i increase at these high temperatures, reflecting that the opening of the TRPV2 channels leads to a calcium influx resulting in the [Ca2+]i increment. The histamine release also became significant in these cases. Since the released histamine is a well-known mediator for the microcirculation promotion, the histamine release from mast cells could be one of the mechanisms of thermal therapy.

  5. Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol.

    PubMed

    Sauer, John-Demian; Witte, Chelsea E; Zemansky, Jason; Hanson, Bill; Lauer, Peter; Portnoy, Daniel A

    2010-05-20

    A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogen's intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.

  6. Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming.

    PubMed

    Schellenberg, Barbara; Wang, Pengbo; Keeble, James A; Rodriguez-Enriquez, Ricardo; Walker, Scott; Owens, Thomas W; Foster, Fiona; Tanianis-Hughes, Jolanta; Brennan, Keith; Streuli, Charles H; Gilmore, Andrew P

    2013-03-07

    The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax's dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity.

  7. Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

    PubMed Central

    Hendrix, Jelle; Baumgärtel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kräusslich, Hans-Georg; Müller, Barbara

    2015-01-01

    Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly. PMID:26283800

  8. Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin

    NASA Astrophysics Data System (ADS)

    Wlodarski, Michal; Raciti, Bianca; Kotar, Jurij; Cosentino Lagomarsino, Marco; Fraser, Gillian M.; Cicuta, Pietro

    2017-02-01

    While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as μNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

  9. Cytosolic pressure provides a propulsive force comparable to actin polymerization during lamellipod protrusion

    NASA Astrophysics Data System (ADS)

    Manoussaki, Daphne; Shin, William D.; Waterman, Clare M.; Chadwick, Richard S.

    2015-07-01

    Does cytosolic pressure facilitate f-actin polymerization to push the leading edge of a cell forward during self-propelled motion? AFM force-distance (f-d) curves obtained from lamellipodia of live cells often exhibit a signal from which the tension, bending modulus, elastic modulus and thickness in the membrane-cortex complex can be estimated close to the contact point. These measurements permit an estimate of the cytosolic pressure via the canonical Laplace force balance. The deeper portion of the f-d curve allows estimation of the bulk modulus of the cytoskeleton after removal of the bottom effect artifact. These estimates of tension, pressure, cortex thickness and elastic moduli imply that cytosolic pressure both pushes the membrane forward and compresses the actin cortex rearward to facilitate f-actin polymerization. We also estimate that cytosolic pressure fluctuations, most likely induced by myosin, provide a propulsive force comparable to that provided by f-actin polymerization in a lamellipod.

  10. Characterization of the kinetics of cardiac cytosolic malate dehydrogenase and comparative analysis of cytosolic and mitochondrial isoforms.

    PubMed

    Dasika, Santosh K; Vinnakota, Kalyan C; Beard, Daniel A

    2015-01-20

    Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.

  11. A Computational Model of Cytosolic and Mitochondrial [Ca2+] in Paced Rat Ventricular Myocytes

    PubMed Central

    Choi, Seong Woo; Jang, Chang Han; Kim, Hyoung Kyu; Leem, Chae Hun; Kim, Nari; Han, Jin

    2011-01-01

    We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial Ca2+ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1~3 Hz) made both the diastolic and systolic [Ca2+] bigger in mitochondria as well as in cytosol. As L-type Ca2+ channel has key influence on the amplitude of Ca2+-induced Ca2+ release, the relation between stimulus frequency and the amplitude of Ca2+ transients was examined under the low density (1/10 of control) of L-type Ca2+ channel in model simulation, where the relation was reversed. In experiment, block of Ca2+ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial Ca2+ transients, while it failed to affect the cytosolic Ca2+ transients. In computer simulation, the amplitude of cytosolic Ca2+ transients was not affected by removal of Ca2+ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [Ca2+] in cytosol and eventually abolished the Ca2+ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type Ca2+ channel to total transsarcolemmal Ca2+ flux could determine whether the cytosolic Ca2+ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic Ca2+ affects mitochondrial Ca2+ in a beat-to-beat manner, however, removal of Ca2+ influx mechanism into mitochondria does not affect the amplitude of cytosolic Ca2+ transients. PMID:21994480

  12. Biochemical Issues in Estimation of Cytosolic Free NAD/NADH Ratio

    PubMed Central

    Xie, Jiansheng; Hu, Xun

    2012-01-01

    Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P. PMID:22570687

  13. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle.

    PubMed

    Park, S; Scheffler, T L; Gunawan, A M; Shi, H; Zeng, C; Hannon, K M; Grant, A L; Gerrard, D E

    2009-01-01

    Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

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

    SciTech Connect

    Chen, Min; Wang, Yanru; Qu, Aijuan

    2010-06-11

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

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

    PubMed

    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.

  16. The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development.

    PubMed

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-05-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family.

  17. The Secretory Pathway Calcium ATPase PMR-1/SPCA1 Has Essential Roles in Cell Migration during Caenorhabditis elegans Embryonic Development

    PubMed Central

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B.; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-01-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family. PMID:23696750

  18. Cotransin induces accumulation of a cytotoxic clusterin variant that cotranslationally rerouted to the cytosol

    SciTech Connect

    Choi, Ilho; Kim, Jiyeon; Park, Joong-Yeol; Kang, Sang-Wook

    2013-05-01

    Although clusterin (CLU) was originally identified as a secreted glycoprotein that plays cytoprotective role, several intracellular CLU variants have been recently identified in the diverse pathological conditions. The mechanistic basis of these variants is now believed to be alternative splicing and retrotranslocation. Here, we uncovered, an unglycosylated and signal sequence-unprocessed, CLU variant in the cytosol. This variant proved to be a product that cotranslationally rerouted to the cytosol during translocation. Cytosolic CLU was prone to aggregation at peri-nuclear region of cells and induced cell death. Signal sequence is shown to be an important determinant for cytosolic CLU generation and aggregation. These results provide not only a new mechanistic insight into the cytosolic CLU generation but also an idea for therapeutic mislocalization of CLU as a strategy for cancer treatment. - Highlights: ► Intracellular CLU variants have been recently identified in the diverse pathological conditions. ► Translocation of clusterin is less efficient than that of Prl. ► We identified a new cytotoxic clusterin variant whose signal sequence was unprocessed. ► This variant proved to be a product that cotranslationally rerouted to cytosol.

  19. Neutrino Oscillation Physics

    SciTech Connect

    Kayser, Boris

    2012-06-01

    To complement the neutrino-physics lectures given at the 2011 International School on Astro Particle Physics devoted to Neutrino Physics and Astrophysics (ISAPP 2011; Varenna, Italy), at the 2011 European School of High Energy Physics (ESHEP 2011; Cheila Gradistei, Romania), and, in modified form, at other summer schools, we present here a written description of the physics of neutrino oscillation. This description is centered on a new way of deriving the oscillation probability. We also provide a brief guide to references relevant to topics other than neutrino oscillation that were covered in the lectures. Neutrinos and photons are by far the most abundant elementary particles in the universe. Thus, if we would like to comprehend the universe, we must understand the neutrinos. Of course, studying the neutrinos is challenging, since the only known forces through which these electrically-neutral leptons interact are the weak force and gravity. Consequently, interactions of neutrinos in a detector are very rare events, so that very large detectors and intense neutrino sources are needed to make experiments feasible. Nevertheless, we have confirmed that the weak interactions of neutrinos are correctly described by the Standard Model (SM) of elementary particle physics. Moreover, in the last 14 years, we have discovered that neutrinos have nonzero masses, and that leptons mix. These discoveries have been based on the observation that neutrinos can change from one 'flavor' to another - the phenomenon known as neutrino oscillation. We shall explain the physics of neutrino oscillation, deriving the probability of oscillation in a new way. We shall also provide a very brief guide to references that can be used to study some major neutrino-physics topics other than neutrino oscillation.

  20. A newly identified tomato peptide induces cytosolic calcium and may correspond to pathogen defense-related endogenous peptides in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants recognize a variety of stimuli that invoke defenses against attacking pathogens and herbivores. This recognition primes the plant to mount defenses against herbivory and disease. These stimuli include molecules called damage-associated molecular patterns or DAMPs, among them signaling peptide...

  1. Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds*

    PubMed Central

    Fedosejevs, Eric T.; Ying, Sheng; Park, Joonho; Anderson, Erin M.; Mullen, Robert T.; She, Yi-Min; Plaxton, William C.

    2014-01-01

    Sucrose synthase (SUS) catalyzes the UDP-dependent cleavage of sucrose into UDP-glucose and fructose and has become an important target for improving seed crops via metabolic engineering. A UDP-specific SUS homotetramer composed of 93-kDa subunits was purified to homogeneity from the triacylglyceride-rich endosperm of developing castor oil seeds (COS) and identified as RcSUS1 by mass spectrometry. RcSUS1 transcripts peaked during early development, whereas levels of SUS activity and immunoreactive 93-kDa SUS polypeptides maximized during mid-development, becoming undetectable in fully mature COS. The cytosolic location of the enzyme was established following transient expression of RcSUS1-enhanced YFP in tobacco suspension cells and fluorescence microscopy. Immunological studies using anti-phosphosite-specific antibodies revealed dynamic and high stoichiometric in vivo phosphorylation of RcSUS1 at its conserved Ser-11 residue during COS development. Incorporation of 32Pi from [γ-32P]ATP into a RcSUS1 peptide substrate, alongside a phosphosite-specific ELISA assay, established the presence of calcium-dependent RcSUS1 (Ser-11) kinase activity. Approximately 10% of RcSUS1 was associated with COS microsomal membranes and was hypophosphorylated relative to the remainder of RcSUS1 that partitioned into the soluble, cytosolic fraction. Elimination of sucrose supply caused by excision of intact pods of developing COS abolished RcSUS1 transcription while triggering the progressive dephosphorylation of RcSUS1 in planta. This did not influence the proportion of RcSUS1 associated with microsomal membranes but instead correlated with a subsequent marked decline in SUS activity and immunoreactive RcSUS1 polypeptides. Phosphorylation at Ser-11 appears to protect RcSUS1 from proteolysis, rather than influence its kinetic properties or partitioning between the soluble cytosol and microsomal membranes. PMID:25313400

  2. Biochemical and molecular characterization of RcSUS1, a cytosolic sucrose synthase phosphorylated in vivo at serine 11 in developing castor oil seeds.

    PubMed

    Fedosejevs, Eric T; Ying, Sheng; Park, Joonho; Anderson, Erin M; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2014-11-28

    Sucrose synthase (SUS) catalyzes the UDP-dependent cleavage of sucrose into UDP-glucose and fructose and has become an important target for improving seed crops via metabolic engineering. A UDP-specific SUS homotetramer composed of 93-kDa subunits was purified to homogeneity from the triacylglyceride-rich endosperm of developing castor oil seeds (COS) and identified as RcSUS1 by mass spectrometry. RcSUS1 transcripts peaked during early development, whereas levels of SUS activity and immunoreactive 93-kDa SUS polypeptides maximized during mid-development, becoming undetectable in fully mature COS. The cytosolic location of the enzyme was established following transient expression of RcSUS1-enhanced YFP in tobacco suspension cells and fluorescence microscopy. Immunological studies using anti-phosphosite-specific antibodies revealed dynamic and high stoichiometric in vivo phosphorylation of RcSUS1 at its conserved Ser-11 residue during COS development. Incorporation of (32)P(i) from [γ-(32)P]ATP into a RcSUS1 peptide substrate, alongside a phosphosite-specific ELISA assay, established the presence of calcium-dependent RcSUS1 (Ser-11) kinase activity. Approximately 10% of RcSUS1 was associated with COS microsomal membranes and was hypophosphorylated relative to the remainder of RcSUS1 that partitioned into the soluble, cytosolic fraction. Elimination of sucrose supply caused by excision of intact pods of developing COS abolished RcSUS1 transcription while triggering the progressive dephosphorylation of RcSUS1 in planta. This did not influence the proportion of RcSUS1 associated with microsomal membranes but instead correlated with a subsequent marked decline in SUS activity and immunoreactive RcSUS1 polypeptides. Phosphorylation at Ser-11 appears to protect RcSUS1 from proteolysis, rather than influence its kinetic properties or partitioning between the soluble cytosol and microsomal membranes.

  3. Role of calcium signaling in epithelial bicarbonate secretion.

    PubMed

    Jung, Jinsei; Lee, Min Goo

    2014-06-01

    Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.

  4. The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+

    PubMed Central

    1996-01-01

    The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes. PMID:8854341

  5. Peroxisomes are required for in vivo nitric oxide accumulation in the cytosol following salinity stress of Arabidopsis plants.

    PubMed

    Corpas, Francisco J; Hayashi, Makoto; Mano, Shoji; Nishimura, Mikio; Barroso, Juan B

    2009-12-01

    Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mM NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO(-)) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress.

  6. Dynamic modulation of spike timing-dependent calcium influx during corticostriatal upstates

    PubMed Central

    Evans, R. C.; Maniar, Y. M.

    2013-01-01

    The striatum of the basal ganglia demonstrates distinctive upstate and downstate membrane potential oscillations during slow-wave sleep and under anesthetic. The upstates generate calcium transients in the dendrites, and the amplitude of these calcium transients depends strongly on the timing of the action potential (AP) within the upstate. Calcium is essential for synaptic plasticity in the striatum, and these large calcium transients during the upstates may control which synapses undergo plastic changes. To investigate the mechanisms that underlie the relationship between calcium and AP timing, we have developed a realistic biophysical model of a medium spiny neuron (MSN). We have implemented sophisticated calcium dynamics including calcium diffusion, buffering, and pump extrusion, which accurately replicate published data. Using this model, we found that either the slow inactivation of dendritic sodium channels (NaSI) or the calcium inactivation of voltage-gated calcium channels (CDI) can cause high calcium corresponding to early APs and lower calcium corresponding to later APs. We found that only CDI can account for the experimental observation that sensitivity to AP timing is dependent on NMDA receptors. Additional simulations demonstrated a mechanism by which MSNs can dynamically modulate their sensitivity to AP timing and show that sensitivity to specifically timed pre- and postsynaptic pairings (as in spike timing-dependent plasticity protocols) is altered by the timing of the pairing within the upstate. These findings have implications for synaptic plasticity in vivo during sleep when the upstate-downstate pattern is prominent in the striatum. PMID:23843436

  7. A New Neutrino Oscillation

    SciTech Connect

    Parke, Stephen J.; /Fermilab

    2011-07-01

    Starting in the late 1960s, neutrino detectors began to see signs that neutrinos, now known to come in the flavors electron ({nu}{sub e}), muon ({nu}{sub {mu}}), and tau ({nu}{sub {tau}}), could transform from one flavor to another. The findings implied that neutrinos must have mass, since massless particles travel at the speed of light and their clocks, so to speak, don't tick, thus they cannot change. What has since been discovered is that neutrinos oscillate at two distinct scales, 500 km/GeV and 15,000 km/GeV, which are defined by the baseline (L) of the experiment (the distance the neutrino travels) divided by the neutrino energy (E). Neutrinos of one flavor can oscillate into neutrinos of another flavor at both L/E scales, but the amplitude of these oscillations is different for the two scales and depends on the initial and final flavor of the neutrinos. The neutrino states that propogate unchanged in time, the mass eigenstates {nu}1, {nu}2, {nu}3, are quantum mechanical mixtures of the electron, muon, and tau neutrino flavors, and the fraction of each flavor in a given mass eigenstate is controlled by three mixing angles and a complex phase. Two of these mixing angles are known with reasonable precision. An upper bound exists for the third angle, called {theta}{sub 13}, which controls the size of the muon neutrino to electron neutrino oscillation at an L/E of 500 km/GeV. The phase is completely unknown. The existence of this phase has important implications for the asymmetry between matter and antimatter we observe in the universe today. Experiments around the world have steadily assembled this picture of neutrino oscillation, but evidence of muon neutrino to electron neutrino oscillation at 500 km/GeV has remained elusive. Now, a paper from the T2K (Tokai to Kamioka) experiment in Japan, reports the first possible observation of muon neutrinos oscillating into electron neutrinos at 500 km/GeV. They see 6 candidate signal events, above an expected background

  8. [Calcium and health].

    PubMed

    Ortega Anta, Rosa M; Jiménez Ortega, Ana I; López-Sobaler, Ana M

    2015-04-07

    An adequate intake of calcium is only not limited to avoid the risk of osteoporosis and its benefits in longterm bone health, but also it has been linked to protection against various major diseases, such as hypertension, cancer, kidney stones, insulin resistance, diabetes... and several investigations suggest its importance in preventing and controlling obesity. Studies conducted in Spanish representative samples show that a high percentage of adults and children (> 75%) don't achieve the recommended intake of calcium. Moreover, are growing trends among the population suggesting that calcium intake and dairy consumption (main food source of the mineral) are high, and even excessive, in many individuals. This misconception results in that the calcium intake is increasingly far from the recommended one. The maximum tolerable intake of the mineral is fixed at 2.500 mg/day, but this intake is unusual, and it's more disturbing and frequent, to find intakes below the recommended calcium intakes (1.000 and 1.200 mg/day in adults, men and women, respectively). Data from different studies highlight the risk of an inadequate calcium intake and the damages that may affect the health in a long term. It is not about transmitting indiscriminate guidelines in order to increase the intake of calcium / dairy, but the recommended intakes must be met to achieve both the nutritional and health benefits. Also activities for demystification of misconceptions are need, increasingly frequent, that may impair health population.

  9. Calcium Signaling and Neurodegeneration

    PubMed Central

    2010-01-01

    Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so–called “calcium hypothesis of neurodegenerative diseases.” The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early–stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy. PMID:22649630

  10. Oscillate boiling from microheaters

    NASA Astrophysics Data System (ADS)

    Li, Fenfang; Gonzalez-Avila, S. Roberto; Nguyen, Dang Minh; Ohl, Claus-Dieter

    2017-01-01

    We report about an intriguing boiling regime occurring for small heaters embedded on the boundary in subcooled water. The microheater is realized by focusing a continuous wave laser beam to about 10 μ m in diameter onto a 165-nm-thick layer of gold, which is submerged in water. After an initial vaporous explosion a single bubble oscillates continuously and repeatedly at several 100 kHz albeit with constant laser power input. The microbubble's oscillations are accompanied with bubble pinch-off, leading to a stream of gaseous bubbles in the subcooled water. The self-driven bubble oscillation is explained with a thermally kicked oscillator caused by surface attachment and by the nonspherical collapses. Additionally, Marangoni stresses induce a recirculating streaming flow which transports cold liquid towards the microheater, reducing diffusion of heat along the substrate and therefore stabilizing the phenomenon to many million cycles. We speculate that this oscillate boiling regime may overcome the heat transfer thresholds observed during the nucleate boiling crisis and offers a new pathway for heat transfer under microgravity conditions.

  11. Oscillations following periodic reinforcement.

    PubMed

    Monteiro, Tiago; Machado, Armando

    2009-06-01

    Three experiments examined behavior in extinction following periodic reinforcement. During the first phase of Experiment 1, four groups of pigeons were exposed to fixed interval (FI 16s or FI 48s) or variable interval (VI 16s or VI 48s) reinforcement schedules. Next, during the second phase, each session started with reinforcement trials and ended with an extinction segment. Experiment 2 was similar except that the extinction segment was considerably longer. Experiment 3 replaced the FI schedules with a peak procedure, with FI trials interspersed with non-food peak interval (PI) trials that were four times longer. One group of pigeons was exposed to FI 20s PI 80s trials, and another to FI 40s PI 160s trials. Results showed that, during the extinction segment, most pigeons trained with FI schedules, but not with VI schedules, displayed pause-peck oscillations with a period close to, but slightly greater than the FI parameter. These oscillations did not start immediately after the onset of extinction. Comparing the oscillations from Experiments 1 and 2 suggested that the alternation of reconditioning and re-extinction increases the reliability and earlier onset of the oscillations. In Experiment 3 the pigeons exhibited well-defined pause-peck cycles since the onset of extinction. These cycles had periods close to twice the value of the FI and lasted for long intervals of time. We discuss some hypotheses concerning the processes underlying behavioral oscillations following periodic reinforcement.

  12. Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation–contraction coupling

    PubMed Central

    Pitake, Saumitra

    2015-01-01

    The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation–contraction coupling. However, the mechanism for subsequent Ca2+ release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca2+ under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation–contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca2+ from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca2+ was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca2+ concentration in the media. Ca2+ entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca2+ entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca2+ release from the ryanodine receptor to the cytosol. PMID:26643865

  13. Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

    PubMed

    Pitake, Saumitra; Ochs, Raymond S

    2016-04-01

    The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation-contraction coupling. However, the mechanism for subsequent Ca(2+) release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca(2+) under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation-contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca(2+) from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca(2+) was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca(2+) concentration in the media. Ca(2+) entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca(2+) entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca(2+) release from the ryanodine receptor to the cytosol.

  14. Differential CaMKII regulation by voltage-gated calcium channels in the striatum.

    PubMed

    Pasek, Johanna G; Wang, Xiaohan; Colbran, Roger J

    2015-09-01

    Calcium signaling regulates synaptic plasticity and many other functions in striatal medium spiny neurons to modulate basal ganglia function. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major calcium-dependent signaling protein that couples calcium entry to diverse cellular changes. CaMKII activation results in autophosphorylation at Thr286 and sustained calcium-independent CaMKII activity after calcium signals dissipate. However, little is known about the mechanisms regulating striatal CaMKII. To address this, mouse brain slices were treated with pharmacological modulators of calcium channels and punches of dorsal striatum were immunoblotted for CaMKII Thr286 autophosphorylation as an index of CaMKII activation. KCl depolarization increased levels of CaMKII autophosphorylation ~2-fold; this increase was blocked by an LTCC antagonist and was mimicked by treatment with pharmacological LTCC activators. The chelation of extracellular calcium robustly decreased basal CaMKII autophosphorylation within 5min and increased levels of total CaMKII in cytosolic fractions, in addition to decreasing the phosphorylation of CaMKII sites in the GluN2B subunit of NMDA receptors and the GluA1 subunit of AMPA receptors. We also found that the maintenance of basal levels of CaMKII autophosphorylation requires low-voltage gated T-type calcium channels, but not LTCCs or R-type calcium channels. Our findings indicate that CaMKII activity is dynamically regulated by multiple calcium channels in the striatum thus coupling calcium entry to key downstream substrates.

  15. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  16. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  17. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  18. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  19. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT OF CENTER WITH TOP OF SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  20. Knocking Out Cytosolic Cysteine Synthesis Compromises the Antioxidant Capacity of the Cytosol to Maintain Discrete Concentrations of Hydrogen Peroxide in Arabidopsis1[W

    PubMed Central

    López-Martín, M. Carmen; Becana, Manuel; Romero, Luis C.; Gotor, Cecilia

    2008-01-01

    Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (Cys) biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidopsis (Arabidopsis thaliana). Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favor of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to cadmium. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under nonstress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. Evidences that the mutation caused a perturbation in H2O2 homeostasis are that, in the knockout, H2O2 production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H2O2 homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis. PMID:18441224

  1. Digital numerically controlled oscillator

    NASA Technical Reports Server (NTRS)

    Cellier, A.; Huey, D. C.; Ma, L. N. (Inventor)

    1980-01-01

    The frequency and phase of an output signal from an oscillator circuit are controlled with accuracy by a digital input word. Positive and negative alterations in output frequency are both provided for by translating all values of input words so that they are positive. The oscillator reference frequency is corrected only in one direction, by adding phase to the output frequency of the oscillator. The input control word is translated to a single algebraic sign and the digital 1 is added thereto. The translated input control word is then accumulated. A reference clock signal having a frequency at an integer multiple of the desired frequency of the output signal is generated. The accumulated control word is then compared with a threshold level. The output signal is adjusted in a single direction by dividing the frequency of the reference clock signal by a first integer or by an integer different from the first integer.

  2. Magnetic vortex oscillators

    NASA Astrophysics Data System (ADS)

    Hrkac, Gino; Keatley, Paul S.; Bryan, Matthew T.; Butler, Keith

    2015-11-01

    The magnetic vortex has sparked the interest of the academic and industrial communities over the last few decades. From their discovery in the 1970s for bubble memory devices to their modern application as radio frequency oscillators, magnetic vortices have been adopted to modern telecommunication and sensor applications. Basic properties of vortex structures in the static and dynamic regime, from a theoretical and experimental point of view, are presented as well as their application in spin torque driven nano-pillar and magnetic tunnel junction devices. Single vortex excitations and phase locking phenomena of coupled oscillators are discussed with an outlook of vortex oscillators in magnetic hybrid structures with imprinted domain confinement and dynamic encryption devices.

  3. Chalcogenide optical parametric oscillator.

    PubMed

    Ahmad, Raja; Rochette, Martin

    2012-04-23

    We demonstrate the first optical parametric oscillator (OPO) based on chalcogenide glass. The parametric gain medium is an As(2)Se(3) chalcogenide microwire coated with a layer of polymer. The doubly-resonant OPO oscillates simultaneously at a Stokes and an anti Stokes wavelength shift of >50 nm from the pump wavelength that lies at λ(P) = 1,552 nm. The oscillator has a peak power threshold of 21.6 dBm and a conversion efficiency of >19%. This OPO experiment provides an additional application of the chalcogenide microwire technology; and considering the transparency of As(2)Se(3) glass extending far in the mid-infrared (mid-IR) wavelengths, the device holds promise for realizing mid-IR OPOs utilizing existing optical sources in the telecommunications wavelength region.

  4. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    PubMed Central

    Casciano, Jessica C.; Duchemin, Nicholas J.; Lamontagne, R. Jason; Steel, Laura F.; Bouchard, Michael J.

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases

  5. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

    PubMed

    Casciano, Jessica C; Duchemin, Nicholas J; Lamontagne, R Jason; Steel, Laura F; Bouchard, Michael J

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

  6. Intrinsic Oscillations of Neocortex Generated by Layer 5 Pyramidal Neurons

    NASA Astrophysics Data System (ADS)

    Silva, Laurie R.; Amitai, Yael; Connors, Barry W.

    1991-01-01

    Rhythmic activity in the neocortex varies with different behavioral and pathological states and in some cases may encode sensory information. However, the neural mechanisms of these oscillations are largely unknown. Many pyramidal neurons in layer 5 of the neocortex showed prolonged, 5- to 12-hertz rhythmic firing patterns at threshold. Rhythmic firing was due to intrinsic membrane properties, sodium conductances were essential for rhythmicity, and calcium-dependent conductances strongly modified rhythmicity. Isolated slices of neocortex generated epochs of 4- to 10-hertz synchronized activity when N-methyl-D-aspartate receptor-mediated channels were facilitated. Layer 5 was both necessary and sufficient to produce these synchronized oscillations. Thus, synaptic networks of intrinsically rhythmic neurons in layer 5 may generate or promote certain synchronized oscillations of the neocortex.

  7. Cytosolic Phospholipase A2 and Lysophospholipids in Tumor Angiogenesis

    PubMed Central

    Linkous, Amanda G.

    2010-01-01

    Background Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA2) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA2 as a novel molecular target for antiangiogenesis therapy. Methods Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA2 expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA2α+/+) or deficient in (cPLA2α−/−) cPLA2α, the predominant isoform in endothelium (n = 6–7 mice per group). The effect of inhibiting cPLA2 activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA2 inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA2α+/+ and cPLA2α−/− mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA2α−/− MPMEC. All statistical tests were two-sided. Results GL261 tumor progression proceeded normally in cPLA2α+/+ mice, whereas no GL261 tumors formed in cPLA2α−/− mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA2α−/− mice. Immunohistochemical examination of the remaining tumors from cPLA2α−/− mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA2α+/+ mice. Inhibition of cPLA2 activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average

  8. Micromechanical Oscillating Mass Balance

    NASA Technical Reports Server (NTRS)

    Altemir, David A. (Inventor)

    1997-01-01

    A micromechanical oscillating mass balance and method adapted for measuring minute quantities of material deposited at a selected location, such as during a vapor deposition process. The invention comprises a vibratory composite beam which includes a dielectric layer sandwiched between two conductive layers. The beam is positioned in a magnetic field. An alternating current passes through one conductive layers, the beam oscillates, inducing an output current in the second conductive layer, which is analyzed to determine the resonant frequency of the beam. As material is deposited on the beam, the mass of the beam increases and the resonant frequency of the beam shifts, and the mass added is determined.

  9. New sensitive marginal oscillator

    NASA Astrophysics Data System (ADS)

    Rahf, L.

    1981-09-01

    A new type of a sensitive marginal oscillator has been developed for the determination of high magnetic inductions by means of nuclear magnetic resonance. Obtaining a high sensitivity with this measuring principle demands a soft behavior of the oscillator which is a particular feature of the circuit presented. It is shown that this behavior is due to the fact that a very weak positive feedback is established by the inner capacitances of the single field effect transistor used in the circuit. Optimal values for the operation parameters are calculated.

  10. Two different effects of calcium on aquaporins in salinity-stressed pepper plants.

    PubMed

    Martínez-Ballesta, M Carmen; Cabañero, Francisco; Olmos, Enrique; Periago, Paula María; Maurel, Christophe; Carvajal, Micaela

    2008-06-01

    Two different effects of calcium were studied, respectively, in plasma membrane vesicles and in protoplasts isolated from roots of control pepper plants (Capsicum annuum L cv. California) or of plants treated with 50 mM NaCl, 10 mM CaCl(2) or 10 mM CaCl(2) + 50 mM NaCl. Under saline conditions, osmotic water permeability (P ( f )) values decreased in protoplasts and plasma membrane vesicles, and the same reduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein abundance. The cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was reduced by salinity, as observed by confocal microscope analysis. Two different actions of Ca(2+) were observed. On the one hand, increase in free cytosolic calcium concentrations associated with stress perception may lead to aquaporin closure. On the other hand, when critical requirements of Ca(2+) were reduced (by salinity), and extra-calcium would lead to an upregulation of aquaporins, indicating that a positive role of calcium at whole plant level combined with an inhibitory mechanism at aquaporin level may work in the regulation of pepper root water transport under salt stress. However, a link between these observations and other cell signalling in relation to water channel gating remains to be established.

  11. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

    PubMed Central

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  12. Calcium Channel Blockers

    MedlinePlus

    ... such as high blood pressure, chest pain and Raynaud's disease. Find out more about this class of medication. ... Irregular heartbeats (arrhythmia) Some circulatory conditions, such as Raynaud's disease For black people and older people, calcium channel ...

  13. Stoichiometry of Calcium Medicines

    ERIC Educational Resources Information Center

    Pinto, Gabriel

    2005-01-01

    The topic of calcium supplement and its effects on human lives is presented in the way of questions to the students. It enables the students to realize the relevance of chemistry outside the classroom surrounding.

  14. Calcium-D-glucarate.

    PubMed

    2002-08-01

    Calcium-D-glucarate is the calcium salt of D-glucaric acid, a substance produced naturally in small amounts by mammals, including humans. Glucaric acid is also found in many fruits and vegetables with the highest concentrations to be found in oranges, apples, grapefruit, and cruciferous vegetables. Oral supplementation of calcium-D-glucarate has been shown to inhibit beta-glucuronidase, an enzyme produced by colonic microflora and involved in Phase II liver detoxification. Elevated beta-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as breast, prostate, and colon cancers. Other potential clinical applications of oral calcium-D-glucarate include regulation of estrogen metabolism and as a lipid-lowering agent.

  15. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  16. Coupled opto-electronic oscillator

    NASA Technical Reports Server (NTRS)

    Yao, X. Steve (Inventor); Maleki, Lute (Inventor)

    1999-01-01

    A coupled opto-electronic oscillator that directly couples a laser oscillation with an electronic oscillation to simultaneously achieve a stable RF oscillation at a high frequency and ultra-short optical pulsation by mode locking with a high repetition rate and stability. Single-mode selection can be achieved even with a very long opto-electronic loop. A multimode laser can be used to pump the electronic oscillation, resulting in a high operation efficiency. The optical and the RF oscillations are correlated to each other.

  17. A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

    PubMed

    Huang, Cathy Chia-Yu; Ko, Michael Lee; Ko, Gladys Yi-Ping

    2013-01-01

    In the retina, the L-type voltage-gated calcium channels (L-VGCCs) are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC) signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities.

  18. Hormone-induced increase in free cytosolic calcium and glycogen phosphorylase activation in rat hepatocytes incubated in normal and low-calcium media.

    PubMed Central

    Binet, A; Berthon, B; Claret, M

    1985-01-01

    The action of alpha 1-adrenergic agonists (noradrenaline in the presence of propranolol), vasopressin and angiotensin on the intracellular free Ca2+ concentration, [Ca2+]i, was determined by using the fluorescent dye quin2 in isolated rat liver cells. In the presence of external Ca2+ (1.8 mM), 1 microM-noradrenaline induced an increase in [Ca2+]i up to about 800 nM without apparent delay, whereas 10 nM-vasopressin and 1 nM-angiotensin increased [Ca2+]i to values higher than 1500 nM with a lag period of about 6s. The successive addition of the hormones and of their specific antagonists indicated that the actions of the three Ca2+-mobilizing hormones occurred without apparent desensitization (over 6 min) and via independent receptors. The relative contributions of internal and external Ca2+ pools to the cell response were determined by studying the hormone-mediated [Ca2+]i increase and glycogen phosphorylase activation in low-Ca2+ media (22 microM). In this medium: (1) [Ca2+]i was lowered and the hormones initiated a transient instead of a sustained increase in [Ca2+]i; subsequent addition (2 min) of a second hormone promoted a lesser increase in [Ca2+]i; in contrast, the subsequent addition (2 min) of Ca2+ (1.8 mM) caused [Ca2+]i to increase to a value close to that initiated by the hormone in control conditions, the amplitude of the latter response being dependent on the concentration of Ca2+ added to the medium; (2) returning to normal Ca2+ (1.8 mM) restored the resting [Ca2+]i and allowed the hormone added 2 min later to promote a large increase in [Ca2+]i whose final amplitude was also dependent on the concentration of Ca2+ added beforehand. Similar results were found when the same protocol was applied to the glycogen phosphorylase activation. It is concluded that Ca2+ influx is required for a maximal and sustained response and to reload the hormone-sensitive stores. PMID:4026798

  19. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  20. Calcium-sensing receptors.

    PubMed

    Goodman, William G

    2004-01-01

    It is now known that variations in extracellular calcium concentration exert diverse physiologic effects in a variety of tissues that are mediated by a calcium-sensing receptor (CaSRs). In parathyroid tissue, the CaSR represents the molecular mechanism by which parathyroid cells detect changes in blood ionized calcium concentration, modulate parathyroid hormone (PTH) secretion accordingly, and thus maintain serum calcium levels within a narrow physiologic range. In the kidney, the CaSR regulates renal calcium excretion and influences the transepithelial movement of water and other electrolytes. More generally, activation of the CaSR represents an important signal transduction pathway in intestine, placenta, brain, and perhaps bone. Some of these actions involve cell cycle regulation, changes that may be relevant to understanding the pathogenesis of parathyroid gland hyperplasia in secondary hyperparathyroidism caused by chronic kidney disease. The CaSR represents an appealing target for therapeutic agents designed to modify parathyroid gland function in vivo, offering the prospect of novel therapies for selected disorders of bone and mineral metabolism. Other receptors capable of responding to extracellular calcium ions also have been identified, but the functional importance of these interactions remains to be determined.

  1. Collective Calcium Signaling of Defective Multicellular Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    2015-03-01

    A communicating multicellular network processes environmental cues into collective cellular dynamics. We have previously demonstrated that, when excited by extracellular ATP, fibroblast monolayers generate correlated calcium dynamics modulated by both the stimuli and gap junction communication between the cells. However, just as a well-connected neural network may be compromised by abnormal neurons, a tissue monolayer can also be defective with cancer cells, which typically have down regulated gap junctions. To understand the collective cellular dynamics in a defective multicellular network we have studied the calcium signaling of co-cultured breast cancer cells and fibroblast cells in various concentrations of ATP delivered through microfluidic devices. Our results demonstrate that cancer cells respond faster, generate singular spikes, and are more synchronous across all stimuli concentrations. Additionally, fibroblast cells exhibit persistent calcium oscillations that increase in regularity with greater stimuli. To interpret these results we quantitatively analyzed the immunostaining of purigenic receptors and gap junction channels. The results confirm our hypothesis that collective dynamics are mainly determined by the availability of gap junction communications.

  2. Oxidative calcium release from catechol.

    PubMed

    Riley, Patrick A; Stratford, Michael R L

    2015-04-01

    Oxidation of 4-methylcatechol previously exposed to aqueous calcium chloride was shown by ion chromatography to be associated with release of calcium ions. The catechol was oxidised to the corresponding orthoquinone by the use of tyrosinase from Agaricus bisporus. The oxidative release of calcium from the catechol is ascribed to the diminution of the available hydroxyl functions able to act as chelating groups. Our results suggest that the redox status of melanin may regulate calcium binding and influence calcium levels in pigmented cells.

  3. Oscillating Reactions: Two Analogies

    ERIC Educational Resources Information Center

    Petruševski, Vladimir M.; Stojanovska, Marina I.; Šoptrajanov, Bojan T.

    2007-01-01

    Oscillating chemical reactions are truly spectacular phenomena, and demonstrations are always appreciated by the class. However, explaining such reactions to high school or first-year university students is problematic, because it may seem that no acceptable explanation is possible unless the students have profound knowledge of both physical…

  4. Wein bridge oscillator circuit

    NASA Technical Reports Server (NTRS)

    Lipoma, P. C.

    1971-01-01

    Circuit with minimum number of components provides stable outputs of 2 to 8 volts at frequencies of .001 to 100 kHz. Oscillator exhibits low power consumption, portability, simplicity, and drive capability, it has application as loudspeaker tester and audible alarm, as well as in laboratory and test generators.

  5. A simple violin oscillator

    NASA Technical Reports Server (NTRS)

    Jones, R. T.

    1976-01-01

    For acoustic tests the violin is driven laterally at the bridge by a small speaker of the type commonly found in pocket transistor radios. An audio oscillator excites the tone which is picked up by a sound level meter. Gross patterns of vibration modes are obtained by the Chladni method.

  6. Voltage-Controlled Oscillator

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Integrated Component Systems, Inc. incorporated information from a NASA Tech Briefs article into a voltage-controlled oscillator it designed for a customer. The company then applied the technology to its series of phase-locked loop synthesizers, which offer superior phase noise performance.

  7. LSND neutrino oscillation results

    SciTech Connect

    Louis, W.C.; LSND Collaboration

    1997-06-01

    The LSND experiment at Los Alamos has conducted searches for {anti {nu}}{sub {mu}} {r_arrow} {anti {nu}}{sub e} oscillations using {anti {nu}}{sub {mu}} from U{sup +} decay at rest and for {nu}{sub {mu}} {r_arrow} {nu}{sub e} oscillations using {nu}{sub {mu}} from {pi}{sup +} decay in flight. For the {anti {nu}}{sub {mu}} {r_arrow} {anti {nu}}{sub e} search, a total excess of 51.8{sub {minus}16.9}{sup +18.7} {+-} 8.0 events is observed with e{sup +} energy between 20 and 60 MeV, while for the {nu}{sub {mu}} {r_arrow} {nu}{sub e} search, a total excess of 18.1 {+-} 6.6 {+-} 4.0 events is observed with e{sup {minus}} energy between 60 and 200 MeV. If attributed to neutrino oscillations, these excesses correspond to oscillation probabilities (averaged over the experimental energies and spatial acceptances) of (0.31 {+-} 0.12 {+-} 0.05)% and (0.26 {+-} 0.10 {+-} 0.05)%, respectively.

  8. Relativistic harmonic oscillator revisited

    SciTech Connect

    Bars, Itzhak

    2009-02-15

    The familiar Fock space commonly used to describe the relativistic harmonic oscillator, for example, as part of string theory, is insufficient to describe all the states of the relativistic oscillator. We find that there are three different vacua leading to three disconnected Fock sectors, all constructed with the same creation-annihilation operators. These have different spacetime geometric properties as well as different algebraic symmetry properties or different quantum numbers. Two of these Fock spaces include negative norm ghosts (as in string theory), while the third one is completely free of ghosts. We discuss a gauge symmetry in a worldline theory approach that supplies appropriate constraints to remove all the ghosts from all Fock sectors of the single oscillator. The resulting ghost-free quantum spectrum in d+1 dimensions is then classified in unitary representations of the Lorentz group SO(d,1). Moreover, all states of the single oscillator put together make up a single infinite dimensional unitary representation of a hidden global symmetry SU(d,1), whose Casimir eigenvalues are computed. Possible applications of these new results in string theory and other areas of physics and mathematics are briefly mentioned.

  9. A fast sensor for in vivo quantification of cytosolic phosphate in Saccharomyces cerevisiae.

    PubMed

    Zhang, Jinrui; Sassen, Tom; ten Pierick, Angela; Ras, Cor; Heijnen, Joseph J; Wahl, Sebastian Aljoscha

    2015-05-01

    Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processes which are distributed over different subcellular compartments. Currently, available metabolite measurement protocols allow to measure metabolite whole cell amounts which hinder progress to describe the in vivo dynamics in different compartments, which are driven by compartment specific concentrations. Phosphate (Pi) is an essential component for: (1) the metabolic balance of upper and lower glycolytic flux; (2) Together with ATP and ADP determines the phosphorylation energy. Especially, the cytosolic Pi has a critical role in disregulation of glycolysis in tps1 knockout. Here we developed a method that enables us to monitor the cytosolic Pi concentration in S. cerevisiae using an equilibrium sensor reaction: maltose + Pi < = > glucose + glucose-1-phosphate. The required enzyme, maltose phosphorylase from L. sanfranciscensis was overexpressed in S. cerevisiae. With this reaction in place, the cytosolic Pi concentration was obtained from intracellular glucose, G1P and maltose concentrations. The cytosolic Pi concentration was determined in batch and chemostat (D = 0.1 h(-1) ) conditions, which was 17.88 µmol/gDW and 25.02 µmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D = 0.1 h(-1) ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 µmol/gDW vs. 25.02 µmol/gDW). In response to a Pi pulse, the cytosolic Pi increased very rapidly, together with the concentration of sugar phosphates. Main sources of the rapid Pi increase are vacuolar Pi (and not the polyPi), as well as Pi uptake from the extracellular space. The temporal increase of cytosolic Pi increases the driving force of GAPDH reaction of the lower glycolytic reactions. The novel cytosol specific Pi concentration measurements provide new insight into the thermodynamic driving force for ATP hydrolysis, GAPDH

  10. Calcium Signaling in Interstitial Cells: Focus on Telocytes

    PubMed Central

    Radu, Beatrice Mihaela; Banciu, Adela; Banciu, Daniel Dumitru; Radu, Mihai; Cretoiu, Dragos; Cretoiu, Sanda Maria

    2017-01-01

    In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca2+ oscillations, the inositol triphosphate (IP3)/Ca2+ signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies. PMID:28208829

  11. Calcium Signaling in Interstitial Cells: Focus on Telocytes.

    PubMed

    Radu, Beatrice Mihaela; Banciu, Adela; Banciu, Daniel Dumitru; Radu, Mihai; Cretoiu, Dragos; Cretoiu, Sanda Maria

    2017-02-13

    In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca(2+) oscillations, the inositol triphosphate (IP₃)/Ca(2+) signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies.

  12. Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release.

    PubMed

    Mukherjee, Sreya; Karolak, Aleksandra; Debant, Marjolaine; Buscaglia, Paul; Renaudineau, Yves; Mignen, Olivier; Guida, Wayne C; Brooks, Wesley H

    2017-02-27

    Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

  13. The fragile X mental retardation protein developmentally regulates the strength and fidelity of calcium signaling in Drosophila mushroom body neurons.

    PubMed

    Tessier, Charles R; Broadie, Kendal

    2011-01-01

    Fragile X syndrome (FXS) is a broad-spectrum neurological disorder characterized by hypersensitivity to sensory stimuli, hyperactivity and severe cognitive impairment. FXS is caused by loss of the fragile X mental retardation 1 (FMR1) gene, whose FMRP product regulates mRNA translation downstream of synaptic activity to modulate changes in synaptic architecture, function and plasticity. Null Drosophila FMR1 (dfmr1) mutants exhibit reduced learning and loss of protein synthesis-dependent memory consolidation, which is dependent on the brain mushroom body (MB) learning and memory center. We targeted a transgenic GFP-based calcium reporter to the MB in order to analyze calcium dynamics downstream of neuronal activation. In the dfmr1 null MB, there was significant augmentation of the calcium transients induced by membrane depolarization, as well as elevated release of calcium from intracellular organelle stores. The severity of these calcium signaling defects increased with developmental age, although early stages were characterized by highly variable, low fidelity calcium regulation. At the single neuron level, both calcium transient and calcium store release defects were exhibited by dfmr1 null MB neurons in primary culture. Null dfmr1 mutants exhibit reduced brain mRNA expression of calcium-binding proteins, including calcium buffers calmodulin and calbindin, predicting that the inability to appropriately sequester cytosolic calcium may be the common mechanistic defect causing calcium accumulation following both influx and store release. Changes in the magnitude and fidelity of calcium signals in the absence of dFMRP likely contribute to defects in neuronal structure/function, leading to the hallmark learning and memory dysfunction of FXS.

  14. Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death

    PubMed Central

    Thurston, Teresa L. M.; Matthews, Sophie A.; Jennings, Elliott; Alix, Eric; Shao, Feng; Shenoy, Avinash R.; Birrell, Mark A.; Holden, David W.

    2016-01-01

    Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria. PMID:27808091

  15. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum

    PubMed Central

    Takahashi, Kei; Toyota, Taro

    2017-01-01

    Background: The transformation of the supported lipid bilayer (SLB) membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level), contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane. PMID:28272354

  16. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  17. Cytosolic Ca(2+) Signals Enhance the Vacuolar Ion Conductivity of Bulging Arabidopsis Root Hair Cells.

    PubMed

    Wang, Yi; Dindas, Julian; Rienmüller, Florian; Krebs, Melanie; Waadt, Rainer; Schumacher, Karin; Wu, Wei-Hua; Hedrich, Rainer; Roelfsema, M Rob G

    2015-11-02

    Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 mV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca(2+) sensor R-GECO1, rapid elevation of the cytosolic Ca(2+) concentration was observed, after impalement with microelectrodes, or injection of the Ca(2+) chelator BAPTA. Elevation of the cytosolic Ca(2+) level stimulated the activity of voltage-independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca(2+) level in cells injected with fluorescent Ca(2+) indicator FURA-2. These data thus show that cytosolic Ca(2+) signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.

  18. Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).

    PubMed

    Mayerhofer, Hubert; Panneerselvam, Saravanan; Kaljunen, Heidi; Tuukkanen, Anne; Mertens, Haydyn D T; Mueller-Dieckmann, Jochen

    2015-01-30

    Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.

  19. Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

    PubMed

    Troll, H; Winckler, T; Lascu, I; Müller, N; Saurin, W; Véron, M; Mutzel, R

    1993-12-05

    We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

  20. AlF4- induces Ca2+ oscillations in guinea-pig ileal smooth muscle.

    PubMed

    Himpens, B; Missiaen, L; Droogmans, G; Casteels, R

    1991-02-01

    The effects of different compounds that inhibit the isolated plasma-membrane Ca2+/Mg2(+)-ATPase on the cytosolic free Ca2+ concentration ([Ca2+]i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F-, in the presence of Al3+, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca2+]i while the phasic contractions were accompanied by concomitant oscillations in [Ca2+]i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca2+]i and of force elicited by AlF4- were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca2+ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF4-, vanadate and calmidazolium induce cytoplasmic Ca2+ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca2+/Mg2(+)-ATPase. The generation of membrane-linked Ca2+ oscillations could therefore be related to an inhibition of the plasma-membrane Ca2+ pump resulting in an increase of [Ca2+]i. This change in [Ca2+]i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.

  1. Monolithic Millimeter Wave Oscillator

    NASA Astrophysics Data System (ADS)

    Wang, Nan-Lei

    There is an increasing interest in the millimeter -wave spectrum for use in communications and for military and scientific applications. The concept of monolithic integration aims to produce very-high-frequency circuits in a more reliable, reproducible way than conventional electronics, and also at lower cost, with smaller size and lighter weight. In this thesis, a negative resistance device is integrated monolithically with a resonator to produce an effective oscillator. This work fills the void resulting from the exclusion of the local oscillator from the monolithic millimeter-wave integrated circuit (MMMIC) receiver design. For convenience a microwave frequency model was used to design the resonator circuit. A 5 GHz hybrid oscillator was first fabricated to test the design; the necessary GaAs process technology was developed for the fabrication. Negative resistance devices and oscillator theory were studied, and a simple but practical model of the Gunn diode was devised to solve the impedance matching problem. Monolithic oscillators at the Ka band (35 GHz) were built and refined. All devices operated in CW mode. By means of an electric-field probe, the output power was coupled into a metallic waveguide for measurement purposes. The best result was 3.63 mW of power output, the highest efficiency was 0.43% and the frequency stability was better than 10-4. In the future, an IMPATT diode could replace the Gunn device to give much higher power and efficiency. A varactor-tuned circuit also suitable for large-scale integration is under study.

  2. Orthogonal polynomials and deformed oscillators

    NASA Astrophysics Data System (ADS)

    Borzov, V. V.; Damaskinsky, E. V.

    2015-10-01

    In the example of the Fibonacci oscillator, we discuss the construction of oscillator-like systems associated with orthogonal polynomials. We also consider the question of the dimensions of the corresponding Lie algebras.

  3. Master oscillator stability requirements considerations

    SciTech Connect

    Schwarz, H.; Vancraeynest, J.

    1986-06-24

    This note attempts to point out some ideas about the required stability of the 476 MHz master oscillator, assuming that the phase noise of the oscillator is the only source of noise in the accelerator system.

  4. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

    PubMed Central

    Sbano, Luigi; Bonora, Massimo; Marchi, Saverio; Baldassari, Federica; Medina, Diego L.; Ballabio, Andrea; Giorgi, Carlotta; Pinton, Paolo

    2017-01-01

    Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. PMID:28084445

  5. Ionization oscillations in Hall accelerators

    NASA Astrophysics Data System (ADS)

    Barral, S.; Peradzyński, Z.

    2010-01-01

    The underlying mechanism of low-frequency oscillations in Hall accelerators is investigated theoretically. It is shown that relaxation oscillations arise from a competition between avalanche ionization and the advective transport of the working gas. The model derived recovers the slow progression and fast recession of the ionization front. Analytical approximations of the shape of current pulses and of the oscillation frequency are provided for the case of large amplitude oscillations.

  6. Many-body Bloch oscillations

    NASA Astrophysics Data System (ADS)

    Haque, Masud

    2014-03-01

    We consider Bloch oscillations of interacting quantum particles in a one-dimensional lattice subject to a linear potential gradient (a tilt). For hard-core bosons and for free fermions, we show perfectly periodic behavior of density and momentum distributions. The oscillations can be predominantly position oscillations, or predominantly width oscillations, depending on the initial state. We show how the periodic behavior is modified for weak and strong interactions.

  7. [Microbial geochemical calcium cycle].

    PubMed

    Zavarzin, G A

    2002-01-01

    The participation of microorganisms in the geochemical calcium cycle is the most important factor maintaining neutral conditions on the Earth. This cycle has profound influence on the fate of inorganic carbon, and, thereby, on the removal of CO2 from the atmosphere. The major part of calcium deposits was formed in the Precambrian, when prokaryotic biosphere predominated. After that, calcium recycling based on biogenic deposition by skeletal organisms became the main process. Among prokaryotes, only a few representatives, e.g., cyanobacteria, exhibit a special calcium function. The geochemical calcium cycle is made possible by the universal features of bacteria involved in biologically mediated reactions and is determined by the activities of microbial communities. In the prokaryotic system, the calcium cycle begins with the leaching of igneous rock predominantly through the action of the community of organotrophic organisms. The release of carbon dioxide to the soil air by organotrophic aerobes leads to leaching with carbonic acid and soda salinization. Under anoxic conditions, of major importance is the organic acid production by primary anaerobes (fermentative microorganisms). Calcium carbonate is precipitated by secondary anaerobes (sulfate reducers) and to a smaller degree by methanogens. The role of the cyanobacterial community in carbonate deposition is exposed by stromatolites, which are the most common organo-sedimentary Precambrian structures. Deposition of carbonates in cyanobacterial mats as a consequence of photoassimilation of CO2 does not appear to be a significant process. It is argued that carbonates were deposited at the boundary between the "soda continent", which emerged as a result of subaerial leaching with carbonic acid, and the ocean containing Ca2+. Such ecotones provided favorable conditions for the development of the benthic cyanobacterial community, which was a precursor of stromatolites.

  8. Peroxisomal plant nitric oxide synthase (NOS) protein is imported by peroxisomal targeting signal type 2 (PTS2) in a process that depends on the cytosolic receptor PEX7 and calmodulin.

    PubMed

    Corpas, Francisco J; Barroso, Juan B

    2014-06-05

    Nitric oxide (NO) production in plant peroxisomes by l-arginine-dependent NO synthase activity has been proven. The PEX5 and PEX7 PTS receptors, which recognize PTS1- and PTS2-containing proteins, are localized in the cytosol. Using AtPex5p and AtPex7p knockdown in Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i, we found that the l-arginine-dependent protein responsible for NO generation in peroxisomes appears to be imported through an N-terminal PTS2. Pharmacological analyzes using a calcium channel blocker and calmodulin (CaM) antagonist show that the import of the peroxisomal NOS protein also depends on calcium and calmodulin.

  9. ER-Dependent Ca++-mediated Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic and ATM-JNK Signal Pathways in Gallic Acid-treated Human Oral Cancer Cells.

    PubMed

    Lu, Yao-Cheng; Lin, Meng-Liang; Su, Hong-Lin; Chen, Shih-Shun

    2016-02-01

    Release of calcium (Ca(++)) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using inhibitor of ER Ca(++) release dantrolene and inhibitor of mitochondrial Ca(++) uptake Ru-360, we demonstrated that Ca(++) release from the ER was associated with generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Small interfering RNA-mediated suppression of protein kinase RNA-like endoplasmic reticulum kinase inhibited tunicamycin-induced induction of 78 kDa glucose-regulated protein, C/EBP homologous protein, pro-caspase-12 cleavage, cytosolic Ca(++) increase and apoptosis, but did not attenuate the increase in cytosolic Ca(++) level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)-mediated c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis by GA was blocked by dantrolene. The specificity of ROS-mediated ATM-JNK activation was confirmed by treatment with N-acetylcysteine, a ROS scavenger. Blockade of ATM activation by specific inhibitor KU55933, short hairpin RNA, or kinase-dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro-caspase-3 cleavage, and apoptosis induced by GA. Taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic and ATM-JNK signal pathways, likely through ER Ca(++)-mediated ROS production.

  10. The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance.

    PubMed

    Fernández-Niño, Miguel; Marquina, Maribel; Swinnen, Steve; Rodríguez-Porrata, Boris; Nevoigt, Elke; Ariño, Joaquín

    2015-11-01

    It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH.

  11. Metal exposures to native populations of the caddisfly Hydropsyche (Trichoptera: Hydropsychidae) determined from cytosolic and whole body metal concentrations

    USGS Publications Warehouse

    Cain, D.J.; Luoma, S.N.

    1998-01-01

    Metal concentrations of the soluble fraction of the cytoplasm (cytosol) and the whole body were determined in the caddisfly Hydropsyche spp. (Trichoptera). Metal accumulation in the cytosol and the whole body were compared in samples collected along 380 kms of a contamination gradient in the Clark Fork river in four consecutive years (1992-1995), and from a contaminated tributary (Flint Creek). Samples from the contaminated sites were compared to an uncontaminated tributary (Blackfoot River). Relations between cytosolic metal concentration and cytosolic protein (used as a general biomarker of protein metabolism) also were examined in 1994 and 1995. Relative to whole body concentrations, cytosolic metal concentrations varied among metals and years. Spatial patterns in whole body and cytosolic Cd, Cu and Pb concentrations were qualitatively similar each year, and these concentrations generally corresponded to contamination levels measured in bed sediments. The proportions of metals recovered in the cytosol of ranged from 12 to 64% for Cd and Cu and from 2 to 38% for Pb. Zinc in the whole body also was consistent with contamination levels, but cytosolic Zn concentrations increased only at the highest whole body Zn concentrations. As a result, the proportion of Zn recovered in the cytosol ranged from 16 to 63% and tended to be inversely related to whole body Zn concentrations. The proportions of cytosolic metals varied significantly among years and, as a result, interannual differences in metal concentrations were greater in the cytosol than in the whole body. The results demonstrated that Hydropsyche in the river were chronically exposed to biologically available metals. Some features of this exposure were not evident from whole body concentrations. In general, protein levels did not correspond to cytosolic metal concentrations. A variety of environmental factors could interact with metal exposures to produce complex responses in protein metabolism. Systematic study

  12. Neutrino Oscillations with Reactor Neutrinos

    NASA Astrophysics Data System (ADS)

    Cabrera, Anatael

    2007-06-01

    Prospect measurements of neutrino oscillations with reactor neutrinos are reviewed in this document. The following items are described: neutrinos oscillations status, reactor neutrino experimental strategy, impact of uncertainties on the neutrino oscillation sensitivity and, finally, the experiments in the field. This is the synthesis of the talk delivered during the NOW2006 conference at Otranto (Italy) during September 2006.

  13. Specific Mechanical and Structural Responses of Cortical and Cytosolic Cytoskeleton in Living Adherent Cells

    NASA Astrophysics Data System (ADS)

    Laurent, Valérie M.; Fodil, Redouane; Cañadas, Patrick; Planus, Emmanuelle; Isabey, Daniel

    We studied the relation between actin structural changes and cytoskeleton mechanical properties in living adherent epithelial alveolar cells, before and during actin depolymerization. The mechanical response of adherent alveolar epithelial cells was measured using magnetic twisting cytometry and a two-component model representing the cortical and the cytosolic elastic components at equilibrium. Chemiluminescent staining of the actin cytoskeleton was performed in the same living cells to estimate the intracellular actin density distribution for each cytoskeleton component. We found that (i) cytoskeleton alterations induced by actin depolymerization differed between the cortical and cytosolic cytoskeleton components (e.g., -30% and -49%, respectively, at a stress of 31 Pa) and that (ii) the concomitant change in actin distribution was also different (e.g., actin volume decrease was -7% and -19% for the cortical and cytosolic components, respectively).

  14. Listeria monocytogenes that lyse in the macrophage cytosol trigger AIM2-mediated pyroptosis

    PubMed Central

    Sauer, John-Demian; Witte, Chelsea E.; Zemansky, Jason; Hanson, Bill; Lauer, Peter; Portnoy, Daniel A.

    2010-01-01

    Summary To gain insight into the mechanisms by which host cells detect cytosolic invasion by intracellular pathogens, a genetic screen was performed to identify Listeria monocytogenes mutants that induced altered levels of host cell death. A mutation in lmo2473 resulted in hyper-stimulation of host cell death and IL-1β secretion (pyroptosis) following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 receptors, but dependent on ASC and AIM2. Importantly, wild type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. Since AIM2 is activated by DNA, these data suggested that pyroptosis is triggered by bacterial DNA released during lysis. PMID:20417169

  15. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels.

    PubMed

    Yang, Huanghe; Hu, Lei; Shi, Jingyi; Delaloye, Kelli; Horrigan, Frank T; Cui, Jianmin

    2007-11-13

    The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.

  16. Modulation of the cytosolic androgen receptor in striated muscle by sex steroids

    NASA Technical Reports Server (NTRS)

    Rance, N. E.; Max, S. E.

    1982-01-01

    The influence of orchiectomy (GDX) and steroid administration on the level of the cytosolic androgen receptor in the rat levator ani muscle and in rat skeletal muscles (tibialis anterior and extensor digitorum longus) was studied. Androgen receptor binding to muscle cytosol was measured using H-3 methyltrienolone (R1881) as ligand, 100 fold molar excess unlabeled R1881 to assess nonspecific binding, and 500 fold molar excess of triamcinolone acetonide to prevent binding to glucocorticoid and progestin receptors. Results demonstrate that modification of the levels of sex steroids can alter the content of androgen receptors of rat striated muscle. Data suggest that: (1) cytosolic androgen receptor levels increase after orchiectomy in both levator ani muscle and skeletal muscle; (2) the acute increase in receptor levels is blocked by an inhibitor of protein synthesis; and (3) administration of estradiol-17 beta to castrated animals increases receptor binding in levator ani muscle but not in skeletal muscle.

  17. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    PubMed

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  18. Altered Calcium Handling in Reperfusion Injury.

    PubMed

    Bompotis, Georgios C; Deftereos, Spyridon; Angelidis, Christos; Choidis, Efthymios; Panagopoulou, Vasiliki; Kaoukis, Andreas; Vassilikos, Vassilios P; Cleman, Michael W; Giannopoulos, Georgios

    2016-01-01

    Coronary Heart Disease (CHD) is the major mortality cause in the Western Hemisphere. Reinstituting blood flow in the acutely occluded coronary vessel became the standard intervention to prevent Myocardial Infarct (MI) progression. Ever since their conception, thrombolysis, Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG) have been at the forefront of CHD treatment, limiting MI size. However, it quickly became apparent that after a period of ischemia, reperfusion itself sets off a cascade of events leading to cell injury. It seems that cellular changes in the ischemic period, prime the cell for a loss of homeostasis once blood flow returns. Loss of calcium (Ca(2+)) regulation has been found to be a main culprit in both ischemia and reperfusion. Indeed, sarcoplasmic Ca(2+) overload during reperfusion is related to hypercontracture, proteolysis and mitochondrial failure--the so-called Reperfusion Injury (RI). Ca(2+) channels of the sarcolemma (SL) (L-Type Ca((2+)) Channels, Sodium / Calcium Exchanger) initiate Ca(2+) flux and those of the Sarcoplasmic Reticulum (SR) (Ca(2+) ATPase, Ca(2+) release channel) sustain the rise in intracellular Ca(2+) concentration. Ensuing interplay between Ca(2+), SR, mitochondria, myofilaments and proteolytic cascades i.e. calpain activation, results in cell injury. Novel insight about this interplay and details about the extent by which each of these players contributes to the RI, may allow scientists to devise and design proper interventions that ultimately reduce RI in clinical practice. The present article reviews the literature about key subcellular players participating in the sustained rise of cardiac myocyte cytosolic Ca(2+) during ischemia and reperfusion.

  19. Genome-derived cytosolic DNA contributes to type I interferon expression and immunogenicity of B-cell lymphoma cells.

    PubMed

    Shen, Yu J; Ho, Samantha S W; Tan, Nikki Y; Koo, Christine Xing'Er; Khatoo, Muznah; Cheung, Florence S G; Gasser, Stephan

    2015-12-01

    We recently provided evidence that genome-derived DNA is present in the cytosol of many tumor cells. Genomic loci that give rise to cytosolic DNA can potentially form non-B DNA structures including triple-stranded RNA:DNA structures (R-loops). The RNA:DNA-specific endonuclease RNaseh1 reduced the levels of cytosolic DNA and type I interferon-dependent rejection of B-cell lymphoma suggesting that cytosolic DNA may contribute to immune surveillance of B-cell lymphoma.

  20. Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway1[OPEN

    PubMed Central

    Durand, Astrid Nagels; Ritter, Andrés; Klassen, Roland; Tohge, Takayuki; Fernie, Alisdair R.; Leidel, Sebastian A.; Pauwels, Laurens

    2016-01-01

    Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17. Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions. PMID:27503603

  1. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    PubMed

    Böldicke, Thomas

    2017-03-08

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, non aggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. This article is protected by copyright. All rights reserved.

  2. Role of cytosolic phospholipase A2 in cytokine-stimulated prostaglandin release by human gallbladder cells.

    PubMed

    Grossmann, E M; Longo, W E; Mazuski, J E; Panesar, N; Kaminski, D L

    2000-01-01

    Eicosanoids are involved in gallbladder inflammation, epithelial water transport, and mucous secretion. Phospholipase Asubscript2 enzymes liberate arachidonic acid from membrane phospholipids for the synthesis of eicosanoids. The purpose of this study was to determine the effect of selective cytoplasmic and secretory phospholipase A2 inhibitors on basal and stimulated arachidonic acid and prostaglandin E2 release in gallbladder cells. Western immunoblotting was employed to evaluate both cytosolic and secretory phospholipase A2 enzymes in human gallbladder cells. Cells were incubated for 22 hours with (3)H-labeled arachidonic acid. Arachidonic acid and prostaglandin E2 release was then measured in the supernate after 2 hours of exposure to human interleukin-1beta, alone or after pretreatment for 1 hour with the inhibitors. Unstimulated gallbladder cells express both 85 kDa cytosolic and 14 kDa secretory phospholipase A2++. The 85 kDa phospholipase A2 was induced by interleukin-1beta, whereas there was no apparent change in secretory phospholipase A2 enzyme concentrations. Both the secretory phospholipase A2 inhibitor p-bromophenylacyl bromide and the cytosolic phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone decreased basal and interleukin-1beta-stimulated arachidonic acid release. In contrast, only inhibition of cytosolic phospholipase A2 led to a decrease in interleukin-1beta-stimulated prostaglandin E2 release. Basal and interleukin-1beta-stimulated arachidonic acid release appears to be the result of the activity of both cytosolic and secretory phospholipase A2. Interleukin-1beta-stimulated prostaglandin E2 release appears to be dependent on the activity of cytosolic phospholipase A2.

  3. Hyaluronan in cytosol--microinjection-based probing of its existence and suggested functions.

    PubMed

    Siiskonen, Hanna; Rilla, Kirsi; Kärnä, Riikka; Bart, Genevieve; Jing, Wei; Haller, Michael F; DeAngelis, Paul L; Tammi, Raija H; Tammi, Markku I

    2013-02-01

    Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

  4. Calcium metabolism and correcting calcium deficiencies.

    PubMed

    Emkey, Ronald D; Emkey, Gregory R

    2012-09-01

    Calcium is the most abundant cation in the human body, of which approximately 99% occurs in bone, contributing to its rigidity and strength. Bone also functions as a reservoir of Ca for its role in multiple physiologic and biochemical processes. This article aims to provide a thorough understanding of the absorptive mechanisms and factors affecting these processes to enable one to better appreciate an individual's Ca needs, and to provide a rationale for correcting Ca deficiencies. An overview of Ca requirements and suggested dosing regimens is presented, with discussion of various Ca preparations and potential toxicities of Ca treatment.

  5. Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.

    PubMed

    Urbano, Francisco J; Luster, Brennon R; D'Onofrio, Stasia; Mahaffey, Susan; Garcia-Rill, Edgar

    2016-09-14

    Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV. Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

  6. Voltage-controlled photonic oscillator.

    PubMed

    Savchenkov, A A; Ilchenko, V S; Liang, W; Eliyahu, D; Matsko, A B; Seidel, D; Maleki, L

    2010-05-15

    We report the development and demonstration of an X-band voltage-controlled photonic oscillator based on a whispering gallery mode resonator made of an electro-optic crystalline material. The oscillator has good spectral purity and wide, agile, linear tunability. We have modified the existing theoretical model of the opto-electronic oscillator to describe the performance of our tunable oscillator and have found a good agreement between the theoretical predictions and the measurement results. We show that the device is promising for higher-frequency applications where high-performance tunable oscillators with wide tunability do not exist.

  7. Buffer regulation of calcium puff sequences.

    PubMed

    Fraiman, Daniel; Dawson, Silvina Ponce

    2014-02-01

    Puffs are localized Ca(2 +) signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP3). They are the result of the liberation of Ca(2 +) from the endoplasmic reticulum through the coordinated opening of IP3 receptor/channels clustered at a functional release site. The presence of buffers that trap Ca(2 +) provides a mechanism that enriches the spatio-temporal dynamics of cytosolic calcium. The expression of different types of buffers along the cell's life provides a tool with which Ca(2 +) signals and their responses can be modulated. In this paper we extend the stochastic model of a cluster of IP3R-Ca(2 +) channels introduced previously to elucidate the effect of buffers on sequences of puffs at the same release site. We obtain analytically the probability laws of the interpuff time and of the number of channels that participate of the puffs. Furthermore, we show that under typical experimental conditions the effect of buffers can be accounted for in terms of a simple inhibiting function. Hence, by exploring different inhibiting functions we are able to study the effect of a variety of buffers on the puff size and interpuff time distributions. We find the somewhat counter-intuitive result that the addition of a fast Ca(2 +) buffer can increase the average number of channels that participate of a puff.

  8. Stable local oscillator module.

    SciTech Connect

    Brocato, Robert Wesley

    2007-11-01

    This report gives a description of the development of a Stable Local Oscillator (StaLO) multi-chip module (MCM). It is a follow-on report to SAND2006-6414, Stable Local Oscillator Microcircuit. The StaLO accepts a 100MHz input signal and produces output signals at 1.2, 3.3, and 3.6 GHz. The circuit is built as a multi-chip module (MCM), since it makes use of integrated circuit technologies in silicon and lithium niobate as well as discrete passive components. This report describes the development of an MCM-based version of the complete StaLO, fabricated on an alumina thick film hybrid substrate.

  9. Oscillations of complex networks

    NASA Astrophysics Data System (ADS)

    Wang, Xingang; Lai, Ying-Cheng; Lai, Choy Heng

    2006-12-01

    A complex network processing information or physical flows is usually characterized by a number of macroscopic quantities such as the diameter and the betweenness centrality. An issue of significant theoretical and practical interest is how such quantities respond to sudden changes caused by attacks or disturbances in recoverable networks, i.e., functions of the affected nodes are only temporarily disabled or partially limited. By introducing a model to address this issue, we find that, for a finite-capacity network, perturbations can cause the network to oscillate persistently in the sense that the characterizing quantities vary periodically or randomly with time. We provide a theoretical estimate of the critical capacity-parameter value for the onset of the network oscillation. The finding is expected to have broad implications as it suggests that complex networks may be structurally highly dynamic.

  10. THz Local Oscillator Technology

    NASA Technical Reports Server (NTRS)

    Mehdi, Imran

    2004-01-01

    The last decade has seen a number of technological advancements that have now made it possible to implement fully solid state local oscillator chains up to 2 THz. These chains are composed of cascaded planar multiplier stages that are pumped with W-band high power sources. The high power W-band sources are achieved by power combining MMIC amplifiers and can provide in access of 150 mW with about 10% bandwidth. Planar diode technology has also enabled novel circuit topologies that can take advantage of the high input power and demonstrate significant efficiencies well into the THz range. Cascaded chains to 1.9 THz have now been demonstrated with enough output power to successfully pump hot-electron bolometer mixers in this frequency range. An overview of the current State-of-the-Art of the local oscillator technology will be presented along with highlighting future trends and challenges.

  11. Voltage-dependent membrane potential oscillations of rat striatal fast-spiking interneurons

    PubMed Central

    Bracci, Enrico; Centonze, Diego; Bernardi, Giorgio; Calabresi, Paolo

    2003-01-01

    We used whole-cell recordings to investigate subthreshold membrane potential oscillations and their relationship with intermittent firing in striatal fast-spiking inter