Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

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Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

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Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

2018-04-05

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

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Zippelius Annette

2009-06-01

Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. Results To gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT and G93A (SH-SY5YG93A expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. Conclusion In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations

Radioresistant Spodoptera frugiperda 9 insect cells display excessive resistance to 'endoplasmic reticulum' stress and calcium disturbances via pre-emptive activation of unfolded protein response pathway

International Nuclear Information System (INIS)

Guleria, Ayushi; Chandna, Sudhir

2016-01-01

Endoplasmic Reticulum (ER) performs multiple cellular functions such as proper folding of newly synthesized proteins and calcium homeostasis. ER stress triggers unfolded protein response (UPR) that attempts to restore normal ER function and resists damage-induced cell death. Lepidopteran Sf9 insect cells (derived from Spodoptera frugiperda) display 100-200 times higher radioresistance than mammalian cells. We have earlier reported that gamma-radiation doses <1000 Gy fail to trigger increase in cytosolic calcium in Sf9 cells, indicating resilience to calcium/ ER disturbances

Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

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Bading Hilmar

2007-07-01

Full Text Available Abstract Background In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types. Results Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border. Conclusion Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

The effect of organolead and -tin compounds on signal transduction in vitro: Investigations on the cytosolic free calcium concentration; Der Einfluss von organischen Blei- und Zinnverbindungen auf die Signaltransduktion in vitro: Untersuchungen zur Veraenderung der zytosolischen freien Calciumkonzentration

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Ade, T.

1996-03-01

The cellular effects of organolead and -tin compounds are not yet precisely understood. However, on the basis of their immuno- and neurotoxicity it is most likely that these substances interfere with cellular signal transduction. For this reason the effect on cytosolic free calcium concentration was investigated in this study. The organometals used induce a persistent increase in cytosolic free calcium concentration in human leukaemia HL-60 cells as well as in neuroblastoma NG-108-15 cells. Studies of the mechanism of the organometal effect with EGTA and calcium channel blockers revealed that an influx of calcium from the extracellular space is responsible for the organometal-induced calcium elevation in HL-60 cells. The effect of the investigated lead compounds and tributyltin is due to calcium channel opening in the plasma membrane. The same is true for the NG108-15 cells. Activation of distinct receptor-mediated signal transduction is not the reason for channel opening. The regulation of cytosolic free calcium concentration was affected by inhibition of plasmamembrane Ca{sup 2+}-ATPases as well as by disturbance of other ion gradients. A consequence of the organometal effect on the cytosolic calcium concentration is the activation of a cPLA{sub 2} and perhaps the induction of apoptosis. These results contribute towards the understanding of biochemical mechanisms causing the injury of vells by organometals. (orig.) [Deutsch] Die zellulaeren Wirkungsmechanismen organischer Blei- und Zinnverbindungen sind zum grossen Teil nicht verstanden. Die immuno- und neurotoxischen Effekte dieser Xenobiotika lassen jedoch die Beeinflussung der Signalwege in den Zellen vermuten. Daher lag der Schwerpunkt dieser Arbeit in der Untersuchung der Signaluebertragungswege und der damit verbundenen Regulation des Calciums. Sowohl in immunkompetenten Zellen (HL-60) wie auch in neuronalen Zellen (NG108-15) induzierten die untersuchten Organometalle eine persistente Erhoehung der

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[OPEN

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Vincent, Thomas R.; Avramova, Marieta; Canham, James; Higgins, Peter; Bilkey, Natasha; Mugford, Sam T.; Pitino, Marco; Toyota, Masatsugu

2017-01-01

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction. PMID:28559475

Effect of tyrosine kinase blockade on norepinephrine-induced cytosolic calcium response in rat afferent arterioles

DEFF Research Database (Denmark)

Salomonsson, Max; Arendshorst, William J

2004-01-01

We used genistein (Gen) and tyrphostin 23 (Tyr-23) to evaluate the importance of tyrosine phosphorylation in norepinephrine (NE)-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in rat afferent arterioles. [Ca(2+)](i) was measured in microdissected arterioles using...... ratiometric photometry of fura 2 fluorescence. The control [Ca(2+)](i) response to NE (1 microM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 microM, 10 min) caused a slow 40% increase in baseline [Ca(2+)](i...... of nifedipine and Tyr-23 were not additive. Nifedipine had no inhibitory effect after Tyr-23 pretreatment, indicating Tyr-23 inhibition of Ca(2+) entry. Another tyrosine kinase inhibitor, Gen (5 and 50 microM), did not affect baseline [Ca(2+)](i). High-dose Gen inhibited the peak and plateau response to NE...

Calcium efflux systems in stress signalling and adaptation in plants

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Jayakumar eBose

2011-12-01

Full Text Available Transient cytosolic calcium ([Ca2+]cyt elevation is an ubiquitous denominator of the signalling 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 in 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+ signalling. 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 analysed in detail. The spatial and temporal organisation of calcium signalling 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 summarised. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modelled 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, emphasising the crucial role these active efflux systems play in plant adaptive responses to environment.

Cytosolic calcium ions exert a major influence on the firing rate and maintenance of pacemaker activity in guinea-pig sinus node.

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Rebecca Anne Capel

2015-02-01

Full Text Available The sino-atrial node (SAN provides the electrical stimulus to initiate every heart beat. Cellular processes underlying this activity have been debated extensively, especially with regards to the role of intracellular calcium. We have used whole-cell application of 1,2-bis(o-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA, a rapid calcium chelator, to guinea pig isolated SAN myocytes to assess the effect of rapid reduction of intracellular calcium on SAN cell electrical activity. High-dose (10 mM BAPTA induced rapid and complete cessation of rhythmic action potential (AP firing (time to cessation 5.5±1.7 s. Over a range of concentrations, BAPTA induced slowing of action potential firing and disruption of rhythmic activity, which was dose-dependent in its time of onset. Exposure to BAPTA was associated with stereotyped action potential changes similar to those previously reported in the presence of ryanodine, namely depolarisation of the most negative diastolic potential, prolongation of action potentials and a reduction in action potential amplitude. These experiments are consistent with the view that cytosolic calcium is essential to the maintenance of rhythmic pacemaker activity.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

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Takahama, Michihiro; Fukuda, Mitsunori; Ohbayashi, Norihiko; Kozaki, Tatsuya; Misawa, Takuma; Okamoto, Toru; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

2017-09-19

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

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Michihiro Takahama

2017-09-01

Full Text Available Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5, in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING, the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis.

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Waadt, Rainer; Krebs, Melanie; Kudla, Jörg; Schumacher, Karin

2017-10-01

Calcium signals occur in specific spatio-temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium-decoding mechanisms. Simultaneous in vivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and in vivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP. In roots, abscisic acid induced long-term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R-GECO1-mTurquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer-based indicators. Quantitative calcium measurements in Arabidopsis root tips using R-GECO1-mTurquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP. Calcium calibration protocols using R-GECO1-mTurquoise enabled high-resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

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Timmermann, D B; Lund, Trine Meldgaard; Belhage, B

2001-01-01

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise...

PHYSICAL CONTACT BETWEEN HUMAN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS MODULATES CYTOSOLIC AND NUCLEAR CALCIUM HOMEOSTASIS.

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Hassan, Ghada S; Jacques, Danielle; D'Orleans-Juste, Pedro; Magder, Sheldon; Bkaily, Ghassan

2018-05-14

The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is however no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and/or hVSMCs . Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([Ca2+]c) and nuclear calcium ([Ca2+]n) levels via physical contact and/or factors released by both cell types. Quantitative 3D confocal microscopy for [Ca2+]c and [Ca2+]n measurement was performed in cultured hVECs or hVSMCs or in co-culture of hVECs-hVSMCs. Our results show that: 1) physical contact between hVECs-hVECs or hVSMCs-hVSMCs does not affect [Ca2+]c and [Ca2+]n in these two cell types; 2) physical contact between hVECs and hVSMCs induces a significant increase only of [Ca2+]n of hVECs without affecting the level of [Ca2+]c and [Ca2+]n of hVSMCs; and 3) preconditioned culture medium of hVECs or hVSMCs does not affect [Ca2+]c and [Ca2+]n of both types of cells. We concluded that physical contact between hVECs and hVSMCs only modulates [Ca2+]n in hVECs. The increase of [Ca2+]n in hVECs may modulate nuclear functions that are calcium dependent.

Bcl-2 overexpression: effects on transmembrane calcium movement

International Nuclear Information System (INIS)

Rangaswami, Arun A.; Premack, Brett; Walleczek, Jan; Killoran, Pamela; Gardner, Phyllis; Knox, Susan J.

1996-01-01

Purpose/Objective: High levels of expression of the proto-oncogene bcl-2 and its 26 kD protein product Bcl-2 have been correlated with the inhibition of apoptosis and the increased resistance of tumor cells to cytotoxic drugs and ionizing radiation. Unfortunately, the specific mechanism of action of Bcl-2 remains poorly understood. In the studies described here, the role of intracellular calcium fluxes and plasma membrane calcium cycling in the induction of apoptosis, and the effect of Bcl-2 expression on the modulation of transmembrane calcium fluxes following treatment of cells with cytotoxic agents were studied. The relationship between intracellular calcium release, capacitive calcium entry, and the plasma membrane potential were also investigated. Materials and Methods: Human B-cell lymphoma (PW) and human promyelocytic leukemia (HL60) cell lines were transfected with Bcl-2 and a control vector. The Bcl-2 transfectants over expressed the Bcl-2 onco-protein and were more resistant to irradiation than the control cells. Cells were loaded with fluorescent indicators indo-1 and fura-2 AM to quantify the cytosolic calcium concentration and subsequent calcium responses to a variety of cytotoxic stimuli, including the microsomal ATPase inhibitor, thapsigargin, using fluorometric measurements. Comparisons of resting and stimulated cytosolic calcium concentrations were made between the parental, neomycin control, and bcl-2 transfected cells. In order to determine the actual calcium influx rate, cells were loaded with either indo-1 or fura-2 and then exposed to 0.1 mM extracellular manganese, which enters the cells through calcium influx channels and quenches the fluorescent signal in proportion to the calcium influx rate. In order to determine the role of the membrane potential in driving calcium influx, cells were treated with either 0.1 μM Valinomycin or isotonic potassium chloride to either hyper polarize or depolarize the resting membrane potential, and the

Phytoplankton calcification as an effective mechanism to prevent cellular calcium poisoning

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Müller, M. N.; Ramos, J. Barcelos e.; Schulz, K. G.; Riebesell, U.; Kaźmierczak, J.; Gallo, F.; Mackinder, L.; Li, Y.; Nesterenko, P. N.; Trull, T. W.; Hallegraeff, G. M.

2015-08-01

Marine phytoplankton has developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 μmol L-1 in the presence of seawater Ca2+ concentrations of 10 mmol L-1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological time scales. For example, the Cretaceous (145 to 66 Ma ago), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to four times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium-sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to prevent cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations.

Movement of calcium signals and calcium-binding proteins: firewalls, traps and tunnels.

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Barrow, S L; Sherwood, M W; Dolman, N J; Gerasimenko, O V; Voronina, S G; Tepikin, A V

2006-06-01

In the board game 'Snakes and Ladders', placed on the image of a pancreatic acinar cell, calcium ions have to move from release sites in the secretory region to the nucleus. There is another important contraflow - from calcium entry channels in the basal part of the cell to ER (endoplasmic reticulum) terminals in the secretory granule region. Both transport routes are perilous as the messenger can disappear in any place on the game board. It can be grabbed by calcium ATPases of the ER (masquerading as a snake but functioning like a ladder) and tunnelled through its low buffering environment, it can be lured into the whirlpools of mitochondria uniporters and forced to regulate the tricarboxylic acid cycle, and it can be permanently placed inside the matrix of secretory granules and released only outside the cell. The organelles could trade calcium (e.g. from the ER to mitochondria and vice versa) almost depriving this ion the light of the cytosol and noble company of cytosolic calcium buffers. Altogether it is a rich and colourful story.

Use of digitonin permeabilization for characterization of calcium incorporated into. beta. cells in response to glucose

Energy Technology Data Exchange (ETDEWEB)

Wesslen, N [Uppsala Univ. (Sweden)

1985-01-01

..beta.. cell-rich pancreatic islets were used to characterize /sup 45/Ca uptake into pools with different sensitivities to permeabilization with digitonin. The plasma and secretory granule membranes were among the membranes most sensitive to digitonin treatment whereas the mitochondria were more resistant. Most of the /sup 45/Ca incorporated in response to glucose was found in a fraction released after exposure to a high concentration of digitonin. The results suggest that glucose promotes active sequestration of calcium in mitochondria and that the uptake of /sup 45/Ca in the secretory granules in secondary to a raised cytosolic Ca/sup 2 +/ activity.

Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU

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Francesco Chemello

2015-09-01

Full Text Available The mitochondrial calcium uniporter (MCU gene codifies for the inner mitochondrial membrane (IMM channel responsible for mitochondrial Ca2+ uptake. Cytosolic Ca2+ transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2+ regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2+ transients elicit large increases in the [Ca2+] of the mitochondrial matrix ([Ca2+]mt. Mitochondrial Ca2+ uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2+ uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2+ uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection. Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/ (GSE60931.

Phytoplankton calcification as an effective mechanism to alleviate cellular calcium poisoning

Science.gov (United States)

Müller, M. N.; Ramos, J. Barcelos e.; Schulz, K. G.; Riebesell, U.; Kaźmierczak, J.; Gallo, F.; Mackinder, L.; Li, Y.; Nesterenko, P. N.; Trull, T. W.; Hallegraeff, G. M.

2015-11-01

Marine phytoplankton have developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 μmol L-1 in the presence of seawater Ca2+ concentrations of 10 mmol L-1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological timescales. For example, the Cretaceous (145 to 66 Ma), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to 4 times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly ornate physical structures of coccoliths remain elusive.

Transcellular transport of calcium

Energy Technology Data Exchange (ETDEWEB)

Terepka, A R; Coleman, J R; Armbrecht, H J; Gunter, T E

1976-01-01

Studies of two calcium transporting epithelia, embryonic chick chorioallantoic membrane and the small intestine of rat and chick, have strongly suggested that the transfer of calcium across a cell involves processes distinctly different from intracellular calcium ion regulation. In the proposed model, transcellular calcium transport is considered as a specialized process developed only by certain cells in those tissues charged with bulk transfer of calcium. The overall effect of the endocytotic mechanism is bulk calcium movement across a cell, protection of mitochondria from exposure to high concentrations of calcium, and the avoidance of wide and potentially toxic fluctuations in cytosol ionic calcium levels. (MFB)

H2O2 augments cytosolic calcium in nucleus tractus solitarii neurons via multiple voltage-gated calcium channels.

Science.gov (United States)

Ostrowski, Tim D; Dantzler, Heather A; Polo-Parada, Luis; Kline, David D

2017-05-01

Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H 2 O 2 ) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H 2 O 2 on cytosolic Ca 2+ (via fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H 2 O 2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca 2+ concentration ([Ca 2+ ] i ). The H 2 O 2 -mediated increase in [Ca 2+ ] i prevailed during thapsigargin, excluding the endoplasmic reticulum as a Ca 2+ source. The effect, however, was abolished by removal of extracellular Ca 2+ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca 2+ channels (VGCCs) as targets for H 2 O 2 modulation. Recording of the total voltage-dependent Ca 2+ current confirmed H 2 O 2 enhanced Ca 2+ entry. Blocking VGCC L, N, and P/Q subtypes decreased the number of cells and their calcium currents that respond to H 2 O 2 The number of responder cells to H 2 O 2 also decreased in the presence of dithiothreitol, suggesting the actions of H 2 O 2 were dependent on sulfhydryl oxidation. In summary, here, we have shown that H 2 O 2 increases [Ca 2+ ] i and its Ca 2+ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices. Copyright © 2017 the American Physiological Society.

An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells

International Nuclear Information System (INIS)

Borle, A.B.

1990-01-01

An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total call calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca 2+ compartmentalization, but the methods suffer from the possibility of Ca 2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45 Ca uptake or desaturation curves have been used to study the distribution of Ca 2+ among various kinetic pools in living cells and their rate of Ca 2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45 Ca uptake can detect instantaneous changes in calcium influx, while 45 Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

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Xiquan Gao

2014-03-01

Full Text Available An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP, which is called PAMP-triggered immunity (PTI. The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI. Calcium (Ca2+ signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response.

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

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Gao, Xiquan; Cox, Kevin L.; He, Ping

2014-01-01

An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

Altered sarco(endo)plasmic reticulum calcium adenosine triphosphatase 2a content: Targets for heart failure therapy.

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Liu, Gang; Li, Si Qi; Hu, Ping Ping; Tong, Xiao Yong

2018-05-01

Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is responsible for transporting cytosolic calcium into the sarcoplasmic reticulum and endoplasmic reticulum to maintain calcium homeostasis. Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is the dominant isoform expressed in cardiac tissue, which is regulated by endogenous protein inhibitors, post-translational modifications, hormones as well as microRNAs. Dysfunction of sarco(endo)plasmic reticulum calcium adenosine triphosphatase is associated with heart failure, which makes sarco(endo)plasmic reticulum calcium adenosine triphosphatase a promising target for heart failure therapy. This review summarizes current approaches to ameliorate sarco(endo)plasmic reticulum calcium adenosine triphosphatase function and focuses on phospholamban, an endogenous inhibitor of sarco(endo)plasmic reticulum calcium adenosine triphosphatase, pharmacological tools and gene therapies.

Differential calcium sensitivity in NaV 1.5 mixed syndrome mutants.

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Abdelsayed, Mena; Baruteau, Alban-Elouen; Gibbs, Karen; Sanatani, Shubhayan; Krahn, Andrew D; Probst, Vincent; Ruben, Peter C

2017-09-15

SCN5a mutations may express gain-of-function (Long QT Syndrome-3), loss-of-function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium-sensitive regions. Elevated cytosolic calcium attenuates gain-of-function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain-of-function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium-to-calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage-gated sodium channel, Na V 1.5. Mutants in Na V 1.5 result in Brugada Syndrome (BrS1), Long-QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome Na V 1.5 mutants. We used whole-cell patch clamp to assess the biophysical properties of Na V 1.5 wild-type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the Na V 1.5 α subunit (WT or mutants), β1 subunit and enhanced green fluorescent protein. Voltage-dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500�

Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

DEFF Research Database (Denmark)

Müller, B K; Kabos, P; Belhage, B

1996-01-01

Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

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Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

2017-07-01

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium

Magnetically responsive calcium carbonate microcrystals.

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Fakhrullin, Rawil F; Bikmullin, Aidar G; Nurgaliev, Danis K

2009-09-01

Here we report the fabrication of magnetically responsive calcium carbonate microcrystals produced by coprecipitation of calcium carbonate in the presence of citrate-stabilized iron oxide nanoparticles. We demonstrate that the calcite microcrystals obtained possess superparamagnetic properties due to incorporated magnetite nanoparticles and can be manipulated by an external magnetic field. The microcrystals doped with magnetic nanoparticles were utilized as templates for the fabrication of hollow polyelectrolyte microcapsules, which retain the magnetic properties of the sacrificial cores and might be spatially manipulated using a permanent magnet, thus providing the magnetic-field-facilitated delivery and separation of materials templated on magnetically responsive calcite microcrystals.

The Marine Guanidine Alkaloid Crambescidin 816 Induces Calcium Influx and Cytotoxicity in Primary Cultures of Cortical Neurons through Glutamate Receptors.

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Mendez, Aida G; Juncal, Andrea Boente; Silva, Siguara B L; Thomas, Olivier P; Martín Vázquez, Víctor; Alfonso, Amparo; Vieytes, Mercedes R; Vale, Carmen; Botana, Luís M

2017-07-19

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

Probabilistic encoding of stimulus strength in astrocyte global calcium signals.

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Croft, Wayne; Reusch, Katharina; Tilunaite, Agne; Russell, Noah A; Thul, Rüdiger; Bellamy, Tomas C

2016-04-01

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium "puffs" throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites. © 2015 Wiley Periodicals, Inc.

Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri

International Nuclear Information System (INIS)

Perry, S.F.; Flik, G.

1988-01-01

Experiments were performed to determine whether gill transepithelial calcium fluxes in the freshwater trout (Salmo gairdneri) are passive or require active transport and to characterize the mechanism involved. A comparison of the in vivo unidirectional flux ratios with the flux ratios calculated according to the transepithelial electrochemical gradients revealed that calcium uptake from the water requires active transport of Ca 2+ . The inhibition of calcium uptake by external lanthanum, the specific deposition of lanthanum on the apical surface of chloride cells, and the favorable electrochemical gradient for calcium across the apical membrane suggest that the initial step in branchial calcium uptake is the passive entry of calcium into the cytosol of chloride cells through apical channels that are permeable to calcium. The study of gill basolateral plasma membrane vesicles demonstrated the existence of a high-affinity calmodulin-dependent calcium-transporting system. This system actively transports calcium from the cytosol of chloride cells into the plasma against a sizeable electrochemical gradient, thereby completing the transepithelial uptake of calcium. Calcium efflux occurs passively through paracellular pathways between chloride cells and adjacent pavement cells or between neighboring pavement cells

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

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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 H 2 O 2 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. © The Author 2016. Published by Oxford University Press on

Multilayer network representation of membrane potential and cytosolic calcium concentration dynamics in beta cells

International Nuclear Information System (INIS)

Gosak, Marko; Dolenšek, Jurij; Markovič, Rene; Slak Rupnik, Marjan; Marhl, Marko; Stožer, Andraž

2015-01-01

Highlights: • Physiological processes within and among pancreatic beta cells are very complex. • We analyze the simultaneous recordings of membrane potential and calcium dynamics. • We represent the interaction patterns among beta cells as a multilayer network. • The nature of the intracellular dynamics is found to rely on the network structure. - Abstract: Modern theory of networks has been recognized as a very successful methodological concept for the description and analysis of complex systems. However, some complex systems are more complex than others. For instance, several real-life systems are constituted by interdependent subsystems and their elements are subjected to different types of interactions that can also change with time. Recently, the multilayer network formalism has been proposed as a general theoretical framework for the description and analysis of such multi-dimensional complex systems and is acquiring more and more prominence in terms of a new research direction. In the present study, we use this methodology for the description of functional connectivity patterns and signal propagation between pancreatic beta cells in an islet of Langerhans at the levels of membrane potential (MP) and cytosolic calcium concentration ([Ca"2"+]_c) dynamics to study the extent of overlap in the two networks and to clarify whether time lags between the two signals in individual cells are in any way dependent on the role these cells play in the functional networks. The two corresponding network layers are constructed on the basis of signal directions and pairwise correlations, whereas the interlayer connections represent the time lag between both measured signals. Our results confirm our previous finding that both MP and [Ca"2"+]_c change spread across an islet in the form of a depolarization and a [Ca"2"+]_c wave, respectively. Both types of waves follow nearly the same path and the networks in both layers have a similar but not entirely the same structure

VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration.

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Fearnley, Gareth W; Bruns, Alexander F; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2015-04-24

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response. © 2015. Published by The Company of Biologists Ltd.

VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

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Gareth W. Fearnley

2015-07-01

Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

Science.gov (United States)

Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

2017-11-08

The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA

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Yuki Nakaya

2017-07-01

Full Text Available Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs that stimulate the induction of interferons (IFNs and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING. Absent in melanoma 2 (AIM2-like receptors (ALRs have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA.

Calcium dynamics of cortical astrocytic networks in vivo.

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Hajime Hirase

2004-04-01

Full Text Available Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+](i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+](i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+](i activity in individual cells and a robust coordination of [Ca(2+](i signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.

Intracellular calcium modulates basolateral K(+)-permeability in frog skin epithelium

DEFF Research Database (Denmark)

Brodin, Birger; Rytved, K A; Nielsen, R

1994-01-01

Cytosolic calcium ([Ca2+]i) has been suggested as a key modulator in the regulation of active sodium transport across electrically "tight" (high resistance) epithelia. In this study we investigated the effects of calcium on cellular electrophysiological parameters in a classical model tissue, the...

Cytosolic cholesterol ester hydrolase in adrenal cortex

OpenAIRE

Tocher, Douglas R.

1983-01-01

Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

Changes of cytosolic calcium and contractility of young rat vas deferens by acute treatment with amphetamine, fluoxetine or sibutramine.

Science.gov (United States)

Jurkiewicz, Neide Hyppolito; da Silva Júnior, Edilson Dantas; de Souza, Bruno Palmieri; Ferreira Verde, Luciana; Drawanz Pereira, Janaina; Mendes Sobrinho, Cairo; Soubhi Smaili, Soraya; Caricati-Neto, Afonso; Miranda-Ferreira, Regiane; Jurkiewicz, Aron

2012-09-15

Previous studies conducted in our laboratory indicated that administration of amphetamine, fluoxetine or sibutramine affects the sympathetic nervous system of the rat vas deferens. Therefore, our goal was to verify the role of calcium in vasa deferentia from young rats pretreated with a single dose of these drugs. Young 40-day-old male Wistar rats were pretreated with amphetamine 3 mg/kg, fluoxetine 10 mg/kg or sibutramine 6 mg/kg for 4 h before the experiments. CaCl(2) (10 mM) was used to induce contraction through time-effect curves in calcium-free solution to measure phasic and tonic components. We also evaluated the calcium-induced fluorescence of vas deferens cut into thin slices. In rats pretreated with amphetamine, we found an increase of the tonic contraction component which was reduced by verapamil. The phasic and tonic responses were increased in the group treated with fluoxetine, but only the tonic response was more sensitive to the antagonism by verapamil. The group treated with sibutramine showed an increase of phasic response whereas the tonic component was decreased. In this group an increase of the affinity for verapamil antagonism was found. In the calcium fluorescence study it was observed that the group treated with amphetamine, fluoxetine or sibutramine showed higher basal Ca(2+) fluorescence after stimulus with KCl (70 mM), noradrenaline (10(-4)M) or acetylcholine (10(-4)M). In all pretreated groups the calcium fluorescence was diminished by nifedipine 10(-7)M. Therefore, the pretreatment with amphetamine, fluoxetine or sibutramine seems to affect the calcium contractility and homeostasis in young rat vas deferens. Copyright © 2012 Elsevier B.V. All rights reserved.

Calcium-Responsive Liposomes via a Synthetic Lipid Switch.

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Lou, Jinchao; Carr, Adam J; Watson, Alexa J; Mattern-Schain, Samuel I; Best, Michael D

2018-03-07

Liposomal drug delivery would benefit from enhanced control over content release. Here, we report a novel avenue for triggering release driven by chemical composition using liposomes sensitized to calcium-a target chosen due to its key roles in biology and disease. To demonstrate this principle, we synthesized calcium-responsive lipid switch 1, designed to undergo conformational changes upon calcium binding. The conformational change perturbs membrane integrity, thereby promoting cargo release. This was shown through fluorescence-based release assays via dose-dependent response depending on the percentage of 1 in liposomes, with minimal background leakage in controls. DLS experiments indicated dramatic changes in particle size upon treatment of liposomes containing 1 with calcium. In a comparison of ten naturally occurring metal cations, calcium provided the greatest release. Finally, STEM images showed significant changes in liposome morphology upon treatment of liposomes containing 1 with calcium. These results showcase lipid switches driven by molecular recognition principles as an exciting avenue for controlling membrane properties. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

OpenAIRE

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

1981-01-01

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 gluco...

The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol

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Flores Rhonda

2011-04-01

Full Text Available Abstract Background The periplasmic High Temperature Requirement protein A (HtrA plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA in chlamydial pathogenesis is not clear. Results The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor. Conclusion Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

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Pan, Zhi; Avila, Andrew; Gollahon, Lauren

2014-01-01

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis. PMID:24549172

High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes.

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Verkaik, Melissa; Oranje, Maarten; Abdurrachim, Desiree; Goebel, Max; Gam, Zeineb; Prompers, Jeanine J; Helmes, Michiel; Ter Wee, Pieter M; van der Velden, Jolanda; Kuster, Diederik W; Vervloet, Marc G; Eringa, Etto C

2018-04-01

The overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (-13% and -19%, respectively) as was the decay of cytosolic calcium during diastole (-15% and -21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/β-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

Energy Technology Data Exchange (ETDEWEB)

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.

Plasma membrane potential depolarization and cytosolic calcium flux are early events involved in tomato (Solanum lycopersicon) plant-to-plant communication.

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Zebelo, Simon A; Matsui, Kenji; Ozawa, Rika; Maffei, Massimo E

2012-11-01

Tomato plants respond to herbivory by emitting volatile organic compounds (VOCs), which are released into the surrounding atmosphere. We analyzed the tomato herbivore-induced VOCs and tested the ability of tomato receiver plants to detect tomato donor volatiles by analyzing early responses, including plasma membrane potential (V(m)) variations and cytosolic calcium ([Ca²⁺](cyt)) fluxes. Receiver tomato plants responded within seconds to herbivore-induced VOCs with a strong V(m) depolarization, which was only partly recovered by fluxing receiver plants with clean air. Among emitted volatiles, we identified by GC-MS some green leaf volatiles (GLVs) such as (E)-2-hexenal, (Z)-3-hexenal, (Z)-3-hexenyl acetate, the monoterpene α-pinene, and the sesquiterpene β-caryophyllene. GLVs were found to exert the stronger V(m) depolarization, when compared to α-pinene and β-caryophyllene. Furthermore, V(m) depolarization was found to increase with increasing GLVs concentration. GLVs were also found to induce a strong [Ca²⁺](cyt) increase, particularly when (Z)-3-hexenyl acetate was tested both in solution and with a gas. On the other hand, α-pinene and β-caryophyllene, which also induced a significant V(m) depolarization with respect to controls, did not exert any significant effect on [Ca²⁺](cyt) homeostasis. Our results show for the first time that plant perception of volatile cues (especially GLVs) from the surrounding environment is mediated by early events, occurring within seconds and involving the alteration of the plasma membrane potential and the [Ca²⁺](cyt) flux. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Calcium in plant cells

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V. V. Schwartau

2014-04-01

Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

Cytosolic phosphoenolpyruvate carboxykinase is a response gene involved in porcine adipocyte adaptation to heat stress.

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Qu, Huan; Ajuwon, Kolapo M

2018-05-04

Heat stress (HS) leads to increased lipid storage and expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) in pig adipocytes. However, the importance of PCK1 activation and lipid storage in the adaptive response to HS is unknown. Therefore, in vitro experiments were conducted to investigate the effect of PCK1 inhibition with 3-mercaptopicolinic acid (3MPA) on lipid storage and adipocyte response during HS. In vitro culture of adipocytes under HS (41.0 °C) increased (P cultured adipocytes were less able to induce adaptive responses such as upregulation of HSP70 and triglycerides, and this exacerbated ER stress during HS. Thus, PCK1 may function to alleviate ER stress that occurs during HS.

The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

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Yan, Nan; Regalado-Magdos, Ashton D.; Stiggelbout, Bart; Lee-Kirsch, Min Ae; Lieberman, Judy

2010-01-01

Viral infection triggers innate immune sensors to produce type I interferons (IFN). However, HIV infection of T cells and macrophages does not trip these alarms. How HIV avoids activating nucleic acid sensors is unknown. The cytosolic exonuclease TREX1 suppressed IFN triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA interference, cytosolic HIV DNA accumulated, and HIV infection induced type I IFN that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate IFN expression via a TBK1, STING and IRF3 dependent pathway. HIV-stimulated IFN production in cells deficient in TREX1 did not involve known nucleic acid sensors. PMID:20871604

Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

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Yaakov Maman

2011-10-01

Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

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Peoples, Jessica N; Taylor, David G; Katchman, Alexander N; Ebert, Steven N

2018-01-22

Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh -/- ) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca 2+ ] i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca 2+ ] i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5 mM), caffeine (5 mM), and NE (100 nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I Ca,L , in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I Ca,L and that aberrant calcium signaling does not likely contribute

A mathematical model for LH release in response to continuous and pulsatile exposure of gonadotrophs to GnRH

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Reed Michael C

2004-09-01

Full Text Available Abstract In a previous study, a model was developed to investigate the release of luteinizing hormone (LH from pituitary cells in response to a short pulse of gonadotropin-releasing hormone (GnRH. The model included: binding of GnRH to its receptor (R, dimerization and internalization of the hormone receptor complex, interaction with a G protein, production of inositol 1,4,5-trisphosphate (IP3, release of calcium from the endoplasmic reticulum (ER, entrance of calcium into the cytosol via voltage gated membrane channels, pumping of calcium out of the cytosol via membrane and ER pumps, and release of LH. The extended model, presented in this paper, also includes the following physiologically important phenomena: desensitization of calcium channels; internalization of the dimerized receptors and recycling of some of the internalized receptors; an increase in Gq concentration near the plasma membrane in response to receptor dimerization; and basal rates of synthesis and degradation of the receptors. With suitable choices of the parameters, good agreement with a variety of experimental data of the LH release pattern in response to pulses of various durations, repetition rates, and concentrations of GnRH were obtained. The mathematical model allows us to assess the effects of internalization and desensitization on the shapes and time courses of LH response curves.

Multiscale Vision Model Highlights Spontaneous Glial Calcium Waves Recorded by 2-Photon Imaging in Brain Tissue

DEFF Research Database (Denmark)

Brazhe, Alexey; Mathiesen, Claus; Lauritzen, Martin

2013-01-01

Intercellular glial calcium waves constitute a signaling pathway which can be visualized by fluorescence imaging of cytosolic Ca2+ changes. However, there is a lack of procedures for sensitive and reliable detection of calcium waves in noisy multiphoton imaging data. Here we extend multiscale...

Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

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Alex Costa

Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

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Yu J. Shen

2015-04-01

Full Text Available 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.

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

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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: 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. Copyright © 2016 the American Physiological Society.

Blunted neuronal calcium response to hypoxia in naked mole-rat hippocampus.

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Bethany L Peterson

Full Text Available Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6 and older (postnatal day 20 age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals.

Blunted neuronal calcium response to hypoxia in naked mole-rat hippocampus.

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Peterson, Bethany L; Larson, John; Buffenstein, Rochelle; Park, Thomas J; Fall, Christopher P

2012-01-01

Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals.

Crosslink between calcium and sodium signalling.

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Verkhratsky, Alexei; Trebak, Mohamed; Perocchi, Fabiana; Khananshvili, Daniel; Sekler, Israel

2018-02-01

What is the topic of this review? This paper overviews the links between Ca 2+ and Na + signalling in various types of cells. What advances does it highlight? This paper highlights the general importance of ionic signalling and overviews the molecular mechanisms linking Na + and Ca 2+ dynamics. In particular, the narrative focuses on the molecular physiology of plasmalemmal and mitochondrial Na + -Ca 2+ exchangers and plasmalemmal transient receptor potential channels. Functional consequences of Ca 2+ and Na + signalling for co-ordination of neuronal activity with astroglial homeostatic pathways fundamental for synaptic transmission are discussed. Transmembrane ionic gradients, which are an indispensable feature of life, are used for generation of cytosolic ionic signals that regulate a host of cellular functions. Intracellular signalling mediated by Ca 2+ and Na + is tightly linked through several molecular pathways that generate Ca 2+ and Na + fluxes and are in turn regulated by both ions. Transient receptor potential (TRP) channels bridge endoplasmic reticulum Ca 2+ release with generation of Na + and Ca 2+ currents. The plasmalemmal Na + -Ca 2+ exchanger (NCX) flickers between forward and reverse mode to co-ordinate the influx and efflux of both ions with membrane polarization and cytosolic ion concentrations. The mitochondrial calcium uniporter channel (MCU) and mitochondrial Na + -Ca 2+ exchanger (NCLX) mediate Ca 2+ entry into and release from this organelle and couple cytosolic Ca 2+ and Na + fluctuations with cellular energetics. Cellular Ca 2+ and Na + signalling controls numerous functional responses and, in the CNS, provides for fast regulation of astroglial homeostatic cascades that are crucial for maintenance of synaptic transmission. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

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Iurescia, Sandra; Fioretti, Daniela; Rinaldi, Monica

2018-01-01

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8 + T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways

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

Science.gov (United States)

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.

Amino alcohol- (NPS-2143 and quinazolinone-derived calcilytics (ATF936 and AXT914 differentially mitigate excessive signalling of calcium-sensing receptor mutants causing Bartter syndrome Type 5 and autosomal dominant hypocalcemia.

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Saskia Letz

Full Text Available Activating calcium sensing receptor (CaSR mutations cause autosomal dominant hypocalcemia (ADH characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics on activating CaSR mutants.All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o. To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914.All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.

The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism

DEFF Research Database (Denmark)

Madsen, Claus Desler; Lykke, Anne; Rasmussen, Lene Juel

2010-01-01

Several enzymes of the metabolic pathways responsible for metabolism of cytosolic ribonucleotides and deoxyribonucleotides are located in mitochondria. Studies described in this paper suggest dysfunction of the mitochondria to affect these metabolic pathways and limit the available levels...

Localized accumulation of cytosolic calcium near the fused sperm is associated with the calcium- and voltage-dependent block of sperm entry in the sea urchin egg.

Science.gov (United States)

Ivonnet, Pedro I; Mohri, Tatsuma; McCulloh, David H

2017-10-01

Interaction of the sperm and egg depolarizes the egg membrane, allowing the sperm to enter; however, if the egg membrane is not allowed to depolarize from its resting potential (e.g., by voltage-clamp), the sperm will not enter. Previous studies demonstrated that sperm entry into sea urchin eggs that are voltage-clamped at negative membrane potentials is regulated both by the egg's membrane potential and a voltage-dependent influx of calcium into the egg. In these cases, electrical or cytoplasmic continuity (sperm-egg membrane fusion) occurs at negative membrane potentials, but subsequent loss of cytoplasmic continuity results in failure of sperm entry (unfusion). The work presented herein examined where, in relation to the sperm, and when, in relation to the sperm-induced electrophysiological events, the egg's calcium influx occurs, and how these events relate to successful or failed sperm entry. When sperm entered the egg, elevation of intracellular calcium concentration ([Ca 2+ ] i ) began near the fused sperm on average 5.9 s after sperm-egg membrane fusion. Conversely, when sperm failed to enter the egg, [Ca 2+ ] i elevated near the site of sperm-egg fusion on average 0.7 s after sperm-egg membrane fusion, which is significantly earlier than in eggs for which sperm entered. Therefore, the accumulation of calcium near the site of sperm-egg fusion is spatially and temporally consistent with the mechanism that may be responsible for loss of cytoplasmic continuity and failure of sperm entry. © 2017 Wiley Periodicals, Inc.

Spatiotemporal magnetic fields enhance cytosolic Ca.sup.2+./sup. levels and induce actin polymerization via activation of voltage-gated sodium channels in skeletal muscle cells

Czech Academy of Sciences Publication Activity Database

Rubio Ayala, M.; Syrovets, T.; Hafner, S.; Zablotskyy, Vitaliy A.; Dejneka, Alexandr; Simmet, T.

2018-01-01

Roč. 163, May (2018), s. 174-184 ISSN 0142-9612 Institutional support: RVO:68378271 Keywords : alternating magnetic field * skeletal muscle * cytosolic calcium * modeling * eddy current * voltage-gated sodium channels Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 8.402, year: 2016

Comparative study on in vivo response of porous calcium carbonate composite ceramic and biphasic calcium phosphate ceramic

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He, Fupo, E-mail: fphebm@126.com [School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006 (China); Ren, Weiwei [School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006 (China); Tian, Xiumei [Department of Biomedical Engineering, School of Basic Sciences, Guangzhou Medical University, Guangzhou 510182 (China); Liu, Wei; Wu, Shanghua [School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006 (China); Chen, Xiaoming, E-mail: xmchenw@126.com [Department of Biomedical Engineering, School of Basic Sciences, Guangzhou Medical University, Guangzhou 510182 (China)

2016-07-01

In a previous study, robust calcium carbonate composite ceramics (CC/PG) were prepared by using phosphate-based glass (PG) as an additive, which showed good cell response. In the present study the in vivo response of porous CC/PG was compared to that of porous biphasic calcium phosphate ceramics (BCP), using a rabbit femoral critical-size grafting model. The materials degradation and bone formation processes were evaluated by general observation, X-ray radiography, micro-computed tomography, and histological examination. The results demonstrated excellent biocompatibility and osteoconductivity, and progressive degradation of CC/PG and BCP. Although the in vitro degradation rate of CC/PG was distinctly faster than that of BCP, at 4 week post-implantation, the bone generation and material degradation of CC/PG were less than those of BCP. Nevertheless, at postoperative week 8, the increment of bone formation and material degradation of CC/PG was pronouncedly larger than that of BCP. These results show that CC/PG is a potential resorbable bone graft aside from the traditional synthetic ones. - Highlights: • A calcium carbonate composite ceramic (CC/PG) was acquired. • The in vivo response of CC/PG and biphasic calcium phosphate (BCP) was compared. • CC/PG showed faster in vitro degradation rate compared to BCP. • CC/PG showed less in vivo degradation and bone formation than BCP at week 4. • CC/PG had larger increment of degradation and bone formation than BCP at week 8.

Comparative study on in vivo response of porous calcium carbonate composite ceramic and biphasic calcium phosphate ceramic

International Nuclear Information System (INIS)

He, Fupo; Ren, Weiwei; Tian, Xiumei; Liu, Wei; Wu, Shanghua; Chen, Xiaoming

2016-01-01

In a previous study, robust calcium carbonate composite ceramics (CC/PG) were prepared by using phosphate-based glass (PG) as an additive, which showed good cell response. In the present study the in vivo response of porous CC/PG was compared to that of porous biphasic calcium phosphate ceramics (BCP), using a rabbit femoral critical-size grafting model. The materials degradation and bone formation processes were evaluated by general observation, X-ray radiography, micro-computed tomography, and histological examination. The results demonstrated excellent biocompatibility and osteoconductivity, and progressive degradation of CC/PG and BCP. Although the in vitro degradation rate of CC/PG was distinctly faster than that of BCP, at 4 week post-implantation, the bone generation and material degradation of CC/PG were less than those of BCP. Nevertheless, at postoperative week 8, the increment of bone formation and material degradation of CC/PG was pronouncedly larger than that of BCP. These results show that CC/PG is a potential resorbable bone graft aside from the traditional synthetic ones. - Highlights: • A calcium carbonate composite ceramic (CC/PG) was acquired. • The in vivo response of CC/PG and biphasic calcium phosphate (BCP) was compared. • CC/PG showed faster in vitro degradation rate compared to BCP. • CC/PG showed less in vivo degradation and bone formation than BCP at week 4. • CC/PG had larger increment of degradation and bone formation than BCP at week 8.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

Science.gov (United States)

Bartlett, Paula J; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L; Combettes, Laurent; Hoek, Jan B; Gaspers, Lawrence D

2017-05-15

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca 2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca 2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca 2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) production and does not involve changes in the sensitivity of the IP 3 receptor or size of internal Ca 2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca 2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca 2+ ] i ) increases, which can adversely affect mitochondrial Ca 2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca 2+ -mobilizing hormones resulting in more sustained and prolonged [Ca 2+ ] i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca 2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) accumulation and phospholipase C

Cytosolic adenylate changes during exercise in prawn muscle

International Nuclear Information System (INIS)

Thebault, M.T.; Raffin, J.P.; Pichon, R.

1994-01-01

31 P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

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

Science.gov (United States)

Li, Congcong; Bo, Liyan; Liu, Qingqing; Liu, Wei; Chen, Xiangjun; Xu, Dunquan; Jin, Faguang

2016-03-01

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.

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

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Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2013-11-01

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

Serum calcium response following oral zinc oxide administrations in dairy cows

DEFF Research Database (Denmark)

Thilsing-Hansen, T; Jørgensen, R J; Thilsing, Trine

2001-01-01

Six non-pregnant cows were allocated into 3 groups. Group 1 comprised a pair of lactating cows, whereas groups 2 and 3 each comprised a pair of non-lactating cows. The cows in groups 1 and 2 were dosed intraruminally by stomach tube with zinc oxide at 120 mg Zn per kg of bodyweight at weekly...... intervals for a period of 33 days. Each cow received a total of 4 doses of zinc oxide. Group 3 served as non-treated control group. Blood samples were collected from all 6 cows daily. Serum was analysed for concentration of calcium. Within 12-24 h of each zinc oxide administration the serum calcium...... of the hypocalcaemic response decreased with the number of zinc oxide dosings. This effect was explained as a response from the stimulation of the calcium homeostatic mechanisms. In the Zn dosed non-lactating cows responses were similar but less clear. The perspective of these findings is discussed in relation...

Role of calcium in the dopaminergic effect on the proximal convoluted tubule of rat kidney

International Nuclear Information System (INIS)

Chan, Y.L.; Chatsudthipong, V.; Su-Tsai, S.M.; von Riotte, A.

1986-01-01

Microperfusion studies have shown that dopamine inhibits fluid and bicarbonate absorption in the rate proximal tubule. These studies are designed to examine the cellular mechanism underlying the proximal cellular response to dopamine action. In the isolated proximal cells, dopamine, in the concentration of 10 -6 M or less, had no effect on cAMP production. However, dopamine could increase cytosolic calcium concentration (from 90 nM to 210 nM) as measured by fluorospectrometry with fura-2 as a calcium indicator. Ca-45 flux studies have shown that dopamine could increase initial influx but not the steady state uptake of Ca. Dopamine could also increase efflux of Ca. In situ microperfusion of proximal tubule and peritubular capillaries has demonstrated that Ca ionophore, A23187 (10 -6 M), could simulate the inhibitory effects of dopamine on fluid and biocarbonate absorption. There was no additive effect observed when both agents were added together in the capillary perfusate. Removal of calcium from the perfusate could partially blunt the effect of dopamine. These results suggest that intracellular calcium plays a crucial role in the dopaminergic regulation of proximal tubular transport

Cytosolic Calcium Coordinates Mitochondrial Energy Metabolism with Presynaptic Activity

Science.gov (United States)

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

Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels

Czech Academy of Sciences Publication Activity Database

Zemková, Hana; Tomič, M.; Kučka, M.; Aguilera, G.; Stojilkovic, S. S.

2016-01-01

Roč. 157, č. 4 (2016), s. 1576-1589 ISSN 0013-7227 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : action potential * background sodium conductance * bursting activity * cation -conducting channels * cytosolic calcium concentration * resting membrane potential Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.286, year: 2016

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA.

Science.gov (United States)

Nakaya, Yuki; Lilue, Jingtao; Stavrou, Spyridon; Moran, Eileen A; Ross, Susan R

2017-07-05

Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. IMPORTANCE Autoimmune diseases like Aicardi-Goutières syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease. Copyright © 2017 Nakaya et al.

Requirement for nuclear calcium signaling in Drosophila long-term memory.

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Weislogel, Jan-Marek; Bengtson, C Peter; Müller, Michaela K; Hörtzsch, Jan N; Bujard, Martina; Schuster, Christoph M; Bading, Hilmar

2013-05-07

Calcium is used throughout evolution as an intracellular signal transducer. In the mammalian central nervous system, calcium mediates the dialogue between the synapse and the nucleus that is required for transcription-dependent persistent neuronal adaptations. A role for nuclear calcium signaling in similar processes in the invertebrate brain has yet to be investigated. Here, we show by in vivo calcium imaging of adult brain neurons of the fruit fly Drosophila melanogaster, that electrical foot shocks used in olfactory avoidance conditioning evoked transient increases in cytosolic and nuclear calcium concentrations in neurons. These calcium signals were detected in Kenyon cells of the flies' mushroom bodies, which are sites of learning and memory related to smell. Acute blockade of nuclear calcium signaling during conditioning selectively and reversibly abolished the formation of long-term olfactory avoidance memory, whereas short-term, middle-term, or anesthesia-resistant olfactory memory remained unaffected. Thus, nuclear calcium signaling is required in flies for the progression of memories from labile to transcription-dependent long-lasting forms. These results identify nuclear calcium as an evolutionarily conserved signal needed in both invertebrate and vertebrate brains for transcription-dependent memory consolidation.

Early response of plant cell to carbon deprivation: in vivo 31P-NMR spectroscopy shows a quasi-instantaneous disruption on cytosolic sugars, phosphorylated intermediates of energy metabolism, phosphate partitioning, and intracellular pHs.

Science.gov (United States)

Gout, Elisabeth; Bligny, Richard; Douce, Roland; Boisson, Anne-Marie; Rivasseau, Corinne

2011-01-01

• In plant cells, sugar starvation triggers a cascade of effects at the scale of 1-2 days. However, very early metabolic response has not yet been investigated. • Soluble phosphorus (P) compounds and intracellular pHs were analysed each 2.5 min intervals in heterotrophic sycamore (Acer pseudoplatanus) cells using in vivo phosphorus nuclear magnetic resonance ((31)P-NMR). • Upon external-sugar withdrawal, the glucose 6-P concentration dropped in the cytosol, but not in plastids. The released inorganic phosphate (Pi) accumulated transiently in the cytosol before influx into the vacuole; nucleotide triphosphate concentration doubled, intracellular pH increased and cell respiration decreased. It was deduced that the cytosolic free-sugar concentration was low, corresponding to only 0.5 mM sucrose in sugar-supplied cells. • The release of sugar from the vacuole and from plastids is insufficient to fully sustain the cell metabolism during starvation, particularly in the very short term. Similarly to Pi-starvation, the cell's first response to sugar starvation occurs in the cytosol and is of a metabolic nature. Unlike the cytoplasm, cytosolic homeostasis is not maintained during starvation. The important metabolic changes following cytosolic sugar exhaustion deliver early endogenous signals that may contribute to trigger rescue metabolism. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

Kinetic identification of an intracellular calcium compartment sensitive to phosphate and dinitrophenol in intact isolated rabbit aorta

International Nuclear Information System (INIS)

Hai, C.M.; Phair, R.D.

1986-01-01

Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45 Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45 Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment

The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.

Science.gov (United States)

Squires, Paul E; Hills, Claire E; Rogers, Gareth J; Garland, Patrick; Farley, Sophia R; Morgan, Noel G

2004-10-06

beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

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Sasnauskas Kęstutis

2011-05-01

Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

Mechanism and evolution of calcium transport across the plant plasma membrane

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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...

Expression of voltage-activated calcium channels in the early zebrafish embryo.

Science.gov (United States)

Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

2009-05-01

Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane.

Science.gov (United States)

Savino, John A; Evans, Jodi F; Rabinowitz, Dorianne; Auborn, Karen J; Carter, Timothy H

2006-03-01

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Science.gov (United States)

Kobayashi, Shouhei; Koujin, Takako; Kojidani, Tomoko; Osakada, Hiroko; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2015-06-02

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF(+) DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

The role of a cytosolic superoxide dismutase in barley-pathogen interactions

KAUST Repository

Lightfoot, Damien

2016-03-19

Reactive oxygen species (ROS), including superoxide (O2-HO2) and hydrogen peroxide (H2O2), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi-biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of O2-HO2 to H2O2, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi-biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up-regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2-HO2 contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi-biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.

The Arabidopsis cytosolic proteome

DEFF Research Database (Denmark)

Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

2014-01-01

compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

Intracellular Ca2+ Regulation in Calcium Sensitive Phenotype of Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

HERMANSYAH

2010-03-01

Full Text Available Intracellular cytosolic Ca2+ concentration accumulation plays an essential information in Saccharomyces cerevisiae i.e. to explain cellular mechanism of Ca2+ sensitive phenotype. Disruption both S. cerevisiae PPase PTP2 and MSG5 genes showed an inhibited growth in the presence of Ca2+. On the other hand, by using Luminocounter with apoaequorin system, a method based upon luminescent photoprotein aequorin, intracellular Ca2+ concentration was accumulated as a consequence of calcium sensitive phenotype of S. cerevisiae. This fact indicated that PPase ptp2Δ and msg5Δ were involved in intracellular Ca2+ transport in addition their already known pathways i.e Mitogen Activated Protein Kinase cell wall integrity pathway, high osmolarity glycerol (HOG pathway, and pheromone response FUS3 pathway.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture*

Science.gov (United States)

Mauceri, Daniela; Hagenston, Anna M.; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-01-01

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. PMID:26231212

The transition between monostable and bistable states induced by time delay in intracellular calcium oscillation

International Nuclear Information System (INIS)

Duan, Wei-Long

2013-01-01

The revised role of the time delay of active processes with colored noises of transmission of intracellular Ca 2+ in intracellular calcium oscillation (ICO) is investigated by means of a first-order algorithm based on stochastic simulation. The simulation results indicate that time delay induces a double critical phenomenon and a transition between the monostable and bistable states of the ICO system. In addition, as the time delay increases, for a cytosolic Ca 2+ concentration with weak colored noises there appears a calcium burst, and the Ca 2+ concentration of the calcium store shows nonmonotonic variation. (paper)

Painful nerve injury decreases resting cytosolic calcium concentrations in sensory neurons of rats

NARCIS (Netherlands)

Fuchs, Andreas; Lirk, Philipp; Stucky, Cheryl; Abram, Stephen E.; Hogan, Quinn H.

2005-01-01

Neuropathic pain is difficult to treat and poorly understood at the cellular level. Although cytoplasmic calcium ([Ca]c) critically regulates neuronal function, the effects of peripheral nerve injury on resting sensory neuronal [Ca]c are unknown. Resting [Ca]c was determined by microfluorometry in

Circadian Rhythm of Hepatic Cytosolic and Nuclear Estrogen and Androgen Receptors

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FRANCAVILLA, ANTONIO; EAGON, PATRICIA K.; DiLEO, ALFREDO; VAN THIEL, DAVID H.; PANELLA, CARMINE; POLIMENO, LORENZO; AMORUSO, CINZIA; INGROSSO, MARCELLO; AQUILINO, A. MARIA; STARZL, THOMAS E.

2010-01-01

Mammalian liver is a sex steroid-responsive tissue. The effects of these hormones presumably are mediated by hepatic estrogen receptors (ER) and androgen receptors (AR). Serum levels of sex hormones display circadian rhythms. Further, estrogens and androgens are commonly administered; administration of these agents is associated frequently with liver disease. Therefore, we investigated whether the cytosolic and nuclear sex steroid receptors also display a similar circadian rhythm, and whether variations occurred in the distribution of receptors between cytosolic and nuclear compartments. Animals were killed every 4 h from midnight till the following midnight; cytosolic and nuclear levels of both ER and AR were measured. Cytosolic ER reached a maximum level at 4 AM, and a minimum at 8 PM and midnight of both days. Nuclear ER was highest at 8 AM and lowest at 4 PM and 8 PM, a pattern which parallels variations in serum estradiol levels. Cytosolic AR was highest at 8 PM and lowest at midnight and 4 AM. Nuclear AR was highest at 4 AM and lowest at 4 PM and 8 PM. The highest level of nuclear AR does not correspond to the maximum serum testosterone level, which occurred at 4 PM. The total hepatic content of both ER and AR was not constant over the 24-h period, but varied considerably with time of day. These studies suggest that both ER and AR show a distinct circadian rhythm in subcellular compartmentalization, and that total hepatic content of ER and AR varies significantly during a 24-h period. PMID:3710067

Limits to anaerobic energy and cytosolic concentration in the living cell

Science.gov (United States)

Paglietti, A.

2015-11-01

For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

Glycine Receptor Activation Impairs ATP-Induced Calcium Transients in Cultured Cortical Astrocytes

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Tatiana P. Morais

2018-01-01

Full Text Available In central nervous system, glycine receptor (GlyR is mostly expressed in the spinal cord and brainstem, but glycinergic transmission related elements have also been identified in the brain. Astrocytes are active elements at the tripartite synapse, being responsible for the maintenance of brain homeostasis and for the fine-tuning of synaptic activity. These cells communicate, spontaneously or in response to a stimulus, by elevations in their cytosolic calcium (calcium transients, Ca2+T that can be propagated to other cells. How these Ca2+T are negatively modulated is yet poorly understood. In this work, we evaluated GlyR expression and its role on calcium signaling modulation in rat brain astrocytes. We first proved that GlyR, predominantly subunits α2 and β, was expressed in brain astrocytes and its localization was confirmed in the cytoplasm and astrocytic processes by immunohistochemistry assays. Calcium imaging experiments in cultured astrocytes showed that glycine (500 μM, a GlyR agonist, caused a concentration-dependent reduction in ATP-induced Ca2+T, an effect abolished by the GlyR antagonist, strychnine (0.8 μM, as well as by nocodazole (1 μM, known to impair GlyR anchorage to the plasma membrane. This effect was mimicked by activation of GABAAR, another Cl--permeable channel. In summary, we demonstrated that GlyR activation in astrocytes mediates an inhibitory effect upon ATP induced Ca2+T, which most probably involves changes in membrane permeability to Cl- and requires GlyR anchorage at the plasma membrane. GlyR in astrocytes may thus be part of a mechanism to modulate astrocyte-to-neuron communication.

Calcium ions affect the hepatitis B virus core assembly

International Nuclear Information System (INIS)

Choi, Yongwook; Gyoo Park, Sung; Yoo, Jun-hi; Jung, Guhung

2005-01-01

Previous report showed that cytosolic Ca 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 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

Calcium-responsive contractility during fertilization in sea urchin eggs.

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Stack, Christianna; Lucero, Amy J; Shuster, Charles B

2006-04-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. (c) 2006 Wiley-Liss, Inc.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

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Stobart, Jillian L; Ferrari, Kim David; Barrett, Matthew J P; Stobart, Michael J; Looser, Zoe J; Saab, Aiman S; Weber, Bruno

2018-01-01

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

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Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

1991-06-01

RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

The role of a cytosolic superoxide dismutase in barley-pathogen interactions

KAUST Repository

Lightfoot, Damien; Mcgrann, Graham R. D.; Able, Amanda J.

2016-01-01

susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2-HO2 contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

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Mauceri, Daniela; Hagenston, Anna M; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-09-18

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing.

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Seo, Gil Ju; Kim, Charlotte; Shin, Woo-Jin; Sklan, Ella H; Eoh, Hyungjin; Jung, Jae U

2018-02-09

Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity.

A role for barley calcium-dependent protein kinase CPK2a in the response to drought

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Agata Cieśla

2016-10-01

Full Text Available Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L., one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates

International Nuclear Information System (INIS)

Schallreuter, Karin U.; University of Bradford; Elwary, Souna M.; Parkin, Susan M.; Wood, John M.

2007-01-01

The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 x 10 -3 M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. 45 Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H 2 O 2 -mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m 2 surface area with its calcium gradient in the 10 -3 M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue

Redox characteristics of the eukaryotic cytosol

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R

2007-01-01

The eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species...... (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model...... organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist...

Haploinsufficiency of the 22q11.2 microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium.

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Devaraju, P; Yu, J; Eddins, D; Mellado-Lagarde, M M; Earls, L R; Westmoreland, J J; Quarato, G; Green, D R; Zakharenko, S S

2017-09-01

Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity that contributes to working-memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal short-term potentiation (STP), a major form of short-term synaptic plasticity. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

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Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice

International Nuclear Information System (INIS)

Meng Qingyong; Chen Shali; Liu Shuzheng

2003-01-01

Objective: To the effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice. Methods: The expression of proteins was analyzed by gel filtration with Sephadex G-100 and HPLC based on separation of proteins on thymocyte cytosol and nuclei after whole-body irradiation with 75 mGy X-rays and sham-irradiation, and their biological activity was examined by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes. Results: HPLC analysis showed that there was a marked increase in expression of 61.4 kD protein in the extract of thymocyte cytosol and 30.4 kD protein in the extract of thymocyte nuclei in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocyte cytosol and nuclei of the irradiated mice showed both stimulating effect on normal T cell proliferation and protective effect on chromosome damage induced by high dose radiation. Conclusion: These findings might have implications in study of mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

Science.gov (United States)

Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

2018-06-05

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

Calcium response to vitamin D supplementation

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Francisco R. Spivacow

2018-01-01

Full Text Available Several studies show the importance of serum vitamin D sufficient levels to prevent multiple chronic diseases. However, vitamin D supplementation and its effects on urine calcium excretion remain controversial. The objective of this prospective and interventional study was to evaluate urine calcium excretion in women with normal calciuria or hypercalciuria, once serum vitamin D sufficiency was achieved. We studied 63 women with idiopathic hypercalciuria, (9 with renal lithiasis and 50 normocalciuric women. Both groups had serum vitamin D levels low (deficiency or insufficiency. Baseline urine calcium excretion was measured before being supplemented with vitamin D2 or D3 weekly or vitamin D3 100.000 IU monthly. Once serum vitamin D levels were corrected achieving at least 30 ng/ml, a second urine calcium excretion was obtained. Although in the whole sample we did not observe significant changes in urine calcium excretion according to the way of supplementation, some of those with weekly supplementation had significant higher urine calcium excretion, 19% (n = 12 of hypercalciuric women and 12% (n = 6 of the normocalciuric group. Monthly doses, also showed higher urine calcium excretion in 40% of hypercalciuric women (n = 4/10 and in 44% (n = 4/9 of the renal lithiasis hypercalciuric patients. In conclusion, different ways of vitamin D supplementation and adequate serum levels are safe in most patients, although it should be taken into account a subgroup, mainly with monthly loading doses, that could increase the calciuria significantly eventually rising renal lithiasis risk or bone mass loss, if genetically predisposed.

Cytosolic delivery of materials with endosome-disrupting colloids

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

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Pushpika eUdawat

2016-04-01

Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants

Science.gov (United States)

Udawat, Pushpika; Jha, Rajesh K.; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

2016-01-01

The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and O2− radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

Cytosolic PrP Can Participate in Prion-Mediated Toxicity

Science.gov (United States)

Thackray, Alana M.; Zhang, Chang; Arndt, Tina

2014-01-01

ABSTRACT Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCE During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and

Mechanisms of G Protein-Coupled Estrogen Receptor-Mediated Spinal Nociception

DEFF Research Database (Denmark)

Deliu, Elena; Brailoiu, G. Cristina; Arterburn, Jeffrey B.

2012-01-01

. Cytosolic calcium concentration elevates faster and with higher amplitude following G-1 intracellular microinjections compared to extracellular exposure, suggesting subcellular GPER functionality. Thus, GPER activation results in spinal nociception, and the downstream mechanisms involve cytosolic calcium......Human and animal studies suggest that estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pronociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER......) activation. Membrane depolarization and increases in cytosolic calcium and reactive oxygen species (ROS) levels are markers of neuronal activation, underlying pain sensitization in the spinal cord. Using behavioral, electrophysiological, and fluorescent imaging studies, we evaluated GPER involvement...

Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay.

Science.gov (United States)

Kopljar, Ivan; Hermans, An N; Teisman, Ard; Gallacher, David J; Lu, Hua Rong

Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/μCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool. Copyright © 2018 Elsevier Inc. All rights reserved.

Cell response of calcium phosphate based ceramics, a bone substitute material

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Juliana Marchi

2013-01-01

Full Text Available The aim of this study was to characterize calcium phosphate ceramics with different Ca/P ratios and evaluate cell response of these materials for use as a bone substitute. Bioceramics consisting of mixtures of hydroxyapatite (HAp and β-tricalcium phosphate (β-TCP powders in different proportions were pressed and sintered. The physical and chemical properties of these bioceramics were then characterized. Characterization of the biological properties of these materials was based on analysis of cell response using cultured fibroblasts. The number of cells attached to the samples was counted from SEM images of samples exposed to cell culture solution for different periods. These data were compared by analysis of variance (ANOVA complemented by the Tukey's test. The TCP sample had higher surface roughness and lower density. The adherence and growth of FMM1 cells on samples from all groups was studied. Even though the different calcium based ceramics exhibited properties which made them suitable as bone substitutes, those with higher levels of β-TCP revealed improved cell growth on their surfaces. These observations indicated two-phase calcium phosphate based materials with a β-TCP surface layer to be a promising bone substitute.

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

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Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

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

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

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Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

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Charles Godbout

Full Text Available 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.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

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Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

Effect of lowering dietary calcium intake on fractional whole body calcium retention

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Dawson-Hughes, B.; Stern, D.T.; Shipp, C.C.; Rasmussen, H.M.

1988-01-01

Although fractional calcium absorption is known to vary inversely with calcium intake, the extent and timing of individual hormonal and calcium absorption responses to altered calcium intake have not been defined. We measured fractional whole body retention of orally ingested 47 Ca, an index of calcium absorption, in nine normal women after they had eaten a 2000-mg calcium diet for 8 weeks and a 300-mg calcium diet for 1, 2, 4, and 8 weeks. After the diet change, serum intact PTH (32.2% increase; P = 0.005), serum 1,25-dihydroxyvitamin D [1,25-(OH)2D; 43.8% increase; P = 0.003], and fractional whole body calcium retention (42.8% increase; P = 0.004) increased within 1 week. Although the PTH and calcium retention responses remained fairly constant throughout the low calcium intake period, serum 1,25-(OH)2D concentrations declined toward baseline after week 1. Thus, the late increase in calcium retention may have resulted from calcium absorption that was independent of 1,25-(OH)2D stimulation

Calcium oscillations in wounded fibroblast monolayers are spatially regulated through substrate mechanics

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Lembong, Josephine; Sabass, Benedikt; Stone, Howard A.

2017-08-01

The maintenance of tissue integrity is essential for the life of multicellular organisms. Healing of a skin wound is a paradigm for how various cell types localize and repair tissue perturbations in an orchestrated fashion. To investigate biophysical mechanisms associated with wound localization, we focus on a model system consisting of a fibroblast monolayer on an elastic substrate. We find that the creation of an edge in the monolayer causes cytosolic calcium oscillations throughout the monolayer. The oscillation frequency increases with cell density, which shows that wound-induced calcium oscillations occur collectively. Inhibition of myosin II reduces the number of oscillating cells, demonstrating a coupling between actomyosin activity and calcium response. The spatial distribution of oscillating cells depends on the stiffness of the substrate. For soft substrates with a Young’s modulus E ~ 360 Pa, oscillations occur on average within 0.2 mm distance from the wound edge. Increasing substrate stiffness leads to an average localization of oscillations away from the edge (up to ~0.6 mm). In addition, we use traction force microscopy to determine stresses between cells and substrate. We find that an increase of substrate rigidity leads to a higher traction magnitude. For E    ~8 kPa, traction magnitude is on average almost uniform beneath the monolayer. Thus, the spatial occurrence of calcium oscillations correlates with the cell-substrate traction. Overall, the experiments with fibroblasts demonstrate a collective, chemomechanical localization mechanism at the edge of a wound with a potential physiological role.

Adenosine A2A receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

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Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

2013-01-01

Highlights: •A 2A receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A 2A receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A 2A receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A 2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A 2A receptor. A 2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A 2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A 2A receptor-overexpressing HLMVECs. Adenoviral-mediated A 2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A 2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A 2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A 2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A 2A -mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A 2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways

Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

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Aslam, Roohi; Williams, Lorraine E; Bhatti, Muhammad Faraz; Virk, Nasar

2017-10-27

P 2 - type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca 2+ , Mn 2+ and Zn 2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P 2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P 2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. Here we concluded that wheat genome consists of nine P 2B and three P 2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P 2A and P 2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be

PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience.

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Ponsot, Flavien; Shen, Weida; Ashokkumar, Pichandi; Audinat, Etienne; Klymchenko, Andrey S; Collot, Mayeul

2017-11-22

Monitoring calcium concentration in the cytosol is of main importance as this ion drives many biological cascades within the cell. To this end, molecular calcium probes are widely used. Most of them, especially the red emitting probes, suffer from nonspecific interactions with inner membranes due to the hydrophobic nature of their fluorophore. To circumvent this issue, calcium probes conjugated to dextran can be used to enhance the hydrophilicity and reduce the nonspecific interaction and compartmentalization. However, dextran conjugates also feature important drawbacks including lower affinity, lower dynamic range, and slow diffusion. Herein, we combined the advantage of molecular probes and dextran conjugate without their drawbacks by designing a new red emitting turn-on calcium probe based on PET quenching, Rhod-PEG, in which the rhodamine fluorophore bears four PEG 4 units. This modification led to a high affinity calcium probe (K d = 748 nM) with reduced nonspecific interactions, enhanced photostability, two-photon absorbance, and brightness compared to the commercially available Rhod-2. After spectral characterizations, we showed that Rhod-PEG quickly and efficiently diffused through the dendrites of pyramidal neurons with an enhanced sensitivity (ΔF/F 0 ) at shorter time after patching compared to Rhod-2.

Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

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Stephen K Roberts

Full Text Available 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.

Calcium Signaling in Taste Cells

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Medler, Kathryn F.

2014-01-01

The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. PMID:25450977

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

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Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

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

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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

A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

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Christine Dorothee Schmeitz

2013-09-01

Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

Accidental periapical extrusion of non-setting calcium hydroxide: Unusual bone response and management

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Divya S Sharma

2014-01-01

Full Text Available Premixed non-setting calcium hydroxide is frequently used as interim root canal dressing in endodontically involved permanent teeth and as obturating paste in deciduous teeth in pediatric dentistry. Wide apex in both cases makes it more prone for deliberate extrusion especially when applied with pressure delivery systems. Contrary to common belief the mix was not resorbed in two years with the complaint of insufficiency in mastication. Large mass of calcium hydroxide in bone delayed healing process even after its removal. The present case report intends to demonstrate unusual behavior of bone in response to oily non-setting preparation of calcium hydroxide. Therefore its application with pressure syringe should be reconsidered in pediatric dentistry.

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

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

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.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

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Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

[Cognitive Function and Calcium. Structures and functions of Ca2+-permeable channels].

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Kaneko, Shuji

2015-02-01

Calcium is essential for living organisms where the increase in intracellular Ca2+ concentration functions as a second messenger for many cellular processes including synaptic transmission and neural plasticity. The cytosolic concentration of Ca2+ is finely controlled by many Ca2+-permeable ion channels and transporters. The comprehensive view of their expression, function, and regulation will advance our understanding of neural and cognitive functions of Ca2+, which leads to the future drug discovery.

Crambescidin 816 induces calcium influx though glutamate receptors in primary cultures of cortical neurons

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Víctor Martín Vázquez

2014-06-01

In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in cortical neurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

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Moran, Daniel L; Tetteh, Afua O; Goodman, Richard E; Underwood, Mark Y

2014-07-01

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion. Copyright © 2014 Elsevier Inc. All rights reserved.

Buffer regulation of calcium puff sequences

International Nuclear Information System (INIS)

Fraiman, Daniel; Dawson, Silvina Ponce

2014-01-01

Puffs are localized Ca 2+ signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP 3 ). They are the result of the liberation of Ca 2+ from the endoplasmic reticulum through the coordinated opening of IP 3 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 IP 3 R-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. (paper)

Buffer regulation of calcium puff sequences.

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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.

Exogenous Calcium Enhances the Photosystem II Photochemistry Response in Salt Stressed Tall Fescue.

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Wang, Guangyang; Bi, Aoyue; Amombo, Erick; Li, Huiying; Zhang, Liang; Cheng, Cheng; Hu, Tao; Fu, Jinmin

2017-01-01

Calcium enhances turfgrass response to salt stress. However, little is known about PSII photochemical changes when exogenous calcium was applied in salinity-stressed turfgrass. Here, we probe into the rearrangements of PSII electron transport and endogenous ion accumulation in tall fescue ( Festuca arundinacea Schreber) treated with exogenous calcium under salt stress. Three-month-old seedlings of genotype "TF133" were subjected to the control (CK), salinity (S), salinity + calcium nitrate (SC), and salinity + ethylene glycol tetraacetic acid (SE). Calcium nitrate and ethylene glycol tetraacetic acid was used as exogenous calcium donor and calcium chelating agent respectively. At the end of a 5-day duration treatment, samples in SC regime had better photochemistry performance on several parameters than salinity only. Such as the Area (equal to the plastoquinone pool size), N (number of [Formula: see text] redox turnovers until F m is reached), ψE 0 , or δRo (Efficiencdy/probability with which a PSII trapped electron is transferred from Q A to Q B or PSI acceptors), ABS/RC (Absorbed photon flux per RC). All the above suggested that calcium enhanced the electron transfer of PSII (especially beyond [Formula: see text]) and prevented reaction centers from inactivation in salt-stressed tall fescue. Furthermore, both grass shoot and root tissues generally accumulated more C, N, Ca 2+ , and K + in the SC regime than S regime. Interrelated analysis indicated that ψE 0 , δRo, ABS/RC, C, and N content in shoots was highly correlated to each other and significantly positively related to Ca 2+ and K + content in roots. Besides, high salt increased ATP6E and CAMK2 transcription level in shoot at 1 and 5 day, respectively while exogenous calcium relieved it. In root, CAMK2 level was reduced by Salinity at 5 day and exogenous calcium recovered it. These observations involved in electron transport capacity and ion accumulation assist in understanding better the protective role

Exogenous Calcium Enhances the Photosystem II Photochemistry Response in Salt Stressed Tall Fescue

Directory of Open Access Journals (Sweden)

Guangyang Wang

2017-11-01

Full Text Available Calcium enhances turfgrass response to salt stress. However, little is known about PSII photochemical changes when exogenous calcium was applied in salinity-stressed turfgrass. Here, we probe into the rearrangements of PSII electron transport and endogenous ion accumulation in tall fescue (Festuca arundinacea Schreber treated with exogenous calcium under salt stress. Three-month-old seedlings of genotype “TF133” were subjected to the control (CK, salinity (S, salinity + calcium nitrate (SC, and salinity + ethylene glycol tetraacetic acid (SE. Calcium nitrate and ethylene glycol tetraacetic acid was used as exogenous calcium donor and calcium chelating agent respectively. At the end of a 5-day duration treatment, samples in SC regime had better photochemistry performance on several parameters than salinity only. Such as the Area (equal to the plastoquinone pool size, N (number of QA- redox turnovers until Fm is reached, ψE0, or δRo (Efficiencdy/probability with which a PSII trapped electron is transferred from QA to QB or PSI acceptors, ABS/RC (Absorbed photon flux per RC. All the above suggested that calcium enhanced the electron transfer of PSII (especially beyond QA- and prevented reaction centers from inactivation in salt-stressed tall fescue. Furthermore, both grass shoot and root tissues generally accumulated more C, N, Ca2+, and K+ in the SC regime than S regime. Interrelated analysis indicated that ψE0, δRo, ABS/RC, C, and N content in shoots was highly correlated to each other and significantly positively related to Ca2+ and K+ content in roots. Besides, high salt increased ATP6E and CAMK2 transcription level in shoot at 1 and 5 day, respectively while exogenous calcium relieved it. In root, CAMK2 level was reduced by Salinity at 5 day and exogenous calcium recovered it. These observations involved in electron transport capacity and ion accumulation assist in understanding better the protective role of exogenous calcium in tall

PGC-1α accelerates cytosolic Ca2+ clearance without disturbing Ca2+ homeostasis in cardiac myocytes

International Nuclear Information System (INIS)

Chen, Min; Wang, Yanru; Qu, Aijuan

2010-01-01

Energy metabolism and Ca 2+ handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) 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-1α in cardiac Ca 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-1α via adenoviral transduction. Our data shows that overexpressing PGC-1α improved myocyte contractility without increasing the amplitude of Ca 2+ transients, suggesting that myofilament sensitivity to Ca 2+ increased. Interestingly, the decay kinetics of global Ca 2+ transients and Ca 2+ waves accelerated in PGC-1α-expressing cells, but the decay rate of caffeine-elicited Ca 2+ transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), but not Na + /Ca 2+ exchange (NCX) contribute to PGC-1α-induced cytosolic Ca 2+ clearance. Furthermore, PGC-1α induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1α did not disturb cardiac Ca 2+ homeostasis, because SR Ca 2+ load and the propensity for Ca 2+ waves remained unchanged. These data suggest that PGC-1α can ameliorate cardiac Ca 2+ cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1α-calcium handing pathway sheds new light on the role of PGC-1α in the therapy of cardiac diseases.

Inflammation alters AMPA-stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons.

Science.gov (United States)

Winland, Carissa D; Welsh, Nora; Sepulveda-Rodriguez, Alberto; Vicini, Stefano; Maguire-Zeiss, Kathleen A

2017-11-01

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium-permeable versus impermeable AMPARs can result in disruptions of [Ca 2+ ] i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca 2+ ] i and L-type voltage-gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA-stimulated [Ca 2+ ] i through calcium-permeable AMPARs and/or L-type VGCCs in dopamine-2- and dopamine-1-expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA-stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist-induced [Ca 2+ ] i was mediated by calcium-permeable AMPARs because the responses were completely blocked by a selective calcium-permeable AMPAR antagonist. We used isradipine, the highly selective L-type VGCC antagonist to determine the role of L-type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA-stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L-type VGCCs and calcium-permeable AMPARs are important mediators of this effect. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

Science.gov (United States)

Sensusiati, A. D.; Priya, T. K. S.; Dachlan, Y. P.

2017-05-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (pneurons both in quantitatively and qualitatively.

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

Science.gov (United States)

Strižak, Zeljka; Ivanković, Dušica; Pröfrock, Daniel; Helmholz, Heike; Cindrić, Ana-Marija; Erk, Marijana; Prange, Andreas

2014-02-01

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules. © 2013.

Calcium absorption response to cholecalciferol supplementation in hemodialysis.

Science.gov (United States)

Armas, Laura A G; Zena, Mohsen; Lund, Richard; Heaney, Robert P

2013-06-01

Recent understanding of extrarenal production of calcitriol has led to the use of more vitamin D supplementation in CKD populations. This paper reports the effect of cholecalciferol supplementation on calcium absorption. Paired calcium absorption tests were done before and after 12-13 weeks of 20,000 IU weekly cholecalciferol supplementation in 30 participants with stage 5 CKD on hemodialysis. The study was conducted from April to December of 2011. Calcium absorption was tested with a standardized meal containing 300 mg calcium carbonate intrinsically labeled with (45)Ca; 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured. 25-Hydroxyvitamin D rose from 14.2 ng/ml (11.5-18.5) at baseline to 49.3 ng/ml (42.3-58.1) at the end of the study (P<0.001). 1,25-Dihydroxyvitamin D rose from 15.1 (10.5-18.8) pg/ml at baseline to 20.5 (17.0-24.7) pg/ml at the end of the study (P<0.001). The median baseline calcium absorption was 12% (7%-17%) and 12% (7%-16%) at the end of study. Patients with stage 5 CKD on hemodialysis had very low calcium absorption values at baseline, and cholecalciferol supplementation that raised 25(OH)D levels to 50 ng/ml had no effect on calcium absorption.

Calcium sensing in exocytosis

DEFF Research Database (Denmark)

Gustavsson, Natalia; Wu, Bingbing; Han, Weiping

2012-01-01

an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

Cloning and characterization of human liver cytosolic beta-glycosidase

NARCIS (Netherlands)

De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

2001-01-01

Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

Science.gov (United States)

Sergé, Arnauld; Bernard, Anne-Marie; Phélipot, Marie-Claire; Bertaux, Nicolas; Fallet, Mathieu; Grenot, Pierre; Marguet, Didier; He, Hai-Tao; Hamon, Yannick

2013-01-01

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells. PMID:24086124

Complex actions of ionomycin in cultured cerebellar astrocytes affecting both calcium-induced calcium release and store-operated calcium entry

DEFF Research Database (Denmark)

Müller, Margit S; Obel, Linea Lykke Frimodt; Waagepetersen, Helle S

2013-01-01

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca(2+) in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca(2+) into the cytosol, a number of studies have demonstrated that the mechanism of......(2+), consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca(2+), the peak phase persisted. The peak amplitude could be lowered significantly...... by application of dantrolene, demonstrating involvement of Ca(2+)-induced Ca(2+)-release (CICR). The plateau phase was markedly reduced when store-operated Ca(2+)-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca(2+) stores in astrocytes...

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

International Nuclear Information System (INIS)

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

1983-01-01

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

Direct Signaling from Astrocytes to Neurons in Cultures of Mammalian Brain Cells

Science.gov (United States)

Nedergaard, Maiken

1994-03-01

Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy.

Science.gov (United States)

Dreier, Roland Felix; Santos, José Carlos; Broz, Petr

2018-01-01

Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

Science.gov (United States)

Contreras, Laura; Satrústegui, Jorgina

2009-03-13

Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

Mechanistic logic underlying the axonal transport of cytosolic proteins

Science.gov (United States)

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

Thermoluminescent response of calcium di aluminate intrinsic and doped with Cr3+

International Nuclear Information System (INIS)

Ceron R, P. V.; Vallejo H, M. A.; Sosa A, M. A.; Montes R, E.; Diaz G, J. A. I.; Diaz T, L. A.

2017-10-01

In numerous studies the thermoluminescent response (Tl) of aluminum oxide has been presented in which various dopants have been incorporated to increase the number of defects and thus modify their dosimetric properties. In related materials such as aluminates have been doped with trivalent ions and their optical characteristics have been determined. Due to this background, the motivation to study the aluminates as possible thermoluminescent dosimeters arises. In this work the synthesis of intrinsic calcium aluminate powders and doped with Cr 3+ is presented by the combustion method, to elaborate pellets and evaluate their Tl response with different radiation sources. The material obtained was characterized by XRD, X-ray fluorescence and UV-Vis; it was found to belong to the monoclinic phase of calcium di aluminate (CaAl 4 O 7 ) with a crystal size of 55 nm, the presence of Cr 3+ in the crystal lattice was also observed. The samples showed a good response Tl for UV radiation, X-rays from a 70 kV source and for Co-60 gammas. The results found show that CaAl 4 O 7 is a good candidate to study its dosimetric properties. (Author)

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

Science.gov (United States)

Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

International Nuclear Information System (INIS)

Sensusiati, A D; Priya, T K S; Dachlan, Y P

2017-01-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (p<0.05). There was also significant correlation between calcium intensity with the evidence of necrosis and Hsp70 expression at 2 hours after infection. Apoptosis and necrosis were simultaneously shown with calcium contribution in this study. Confocal microscopy can be used to measure calcium changes in infected and uninfected neurons both in quantitatively and qualitatively. (paper)

Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

International Nuclear Information System (INIS)

Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi; Bhatnagar, Rakesh; Banerjee-Bhatnagar, Nirupama

2006-01-01

We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

Glucose acutely reduces cytosolic and mitochondrial H2O2 in rat pancreatic beta-cells.

Science.gov (United States)

Deglasse, Jean-Philippe; Roma, Leticia Prates; Pastor-Flores, Daniel; Gilon, Patrick; Dick, Tobias P; Jonas, Jean-Christophe

2018-05-14

Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion by pancreatic β-cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 and HyPer with its pH-control SypHer, to test the acute effects of glucose, monomethylsuccinate, leucine with glutamine, and α-ketoisocaproate, on β-cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5mM glucose in the cytosol and 10mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15µM) did not affect insulin secretion. By contrast, menadione (1-5µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20mM glucose. Subcellular changes in β-cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in β-cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. The glucose-dependent stimulation of insulin secretion occurs independently of a detectable increase in β-cell cytosolic or mitochondrial H2O2 levels.

Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine

DEFF Research Database (Denmark)

Saxena, S P; McNicol, A; Becker, A B

1992-01-01

of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating...

Prognostic significance of cytosolic pS2 content in ovarian tumors

International Nuclear Information System (INIS)

Raigoso, P.; Allende, T.; Zeidan, N.; Llana, B.; Bernardo, L.; Roiz, C.; Tejuca, S.; Vazquez, J.; Lamelas, M.L.

2002-01-01

Aim: pS2 is an estrogen regulated peptide which has been associated with a good prognosis an with a more favorable response to treatment in breast cancer patients. In ovarian tumors, the expression of pS2 was demonstrated at both mRNA and protein levels. In addition, it has been showed significant association of pS2 with mucinous differentiation or well differentiation grade of the tumors. However, it is little know about the prognostic significance of the pS2 content in ovarian carcinomas. The aims of the present work were to analyze the cytosolic pS2 content in benign and malignant ovarian tumors, its relationship with clinico-pathologic parameters, steroid receptor status, and prognostic significance. Material and Methods: We analysed the cytosolic concentrations of pS2 in 91 specimen ovarian tissues by an immunoradiometric assay (ELSA-pS2, CIS, France). The tissues were 8 normal ovaries, 43 benign tumors and 40 malignant ovarian tumors. The same ovarian tissues processed to pS2 were analyzed to Estrogen (ER) and Progesterone (PgR) Receptor status. These steroid receptors were quantified biochemically following commercial ELISA method (ABBOTT Diagnostics, Germany). The relationship between cytosolic content and clinico-pathologic factors was examined by the Mann-Whitney or Kruskall-Wallis test. Correlation between steroid receptors and pS2 content was calculated with the Spearman test. Survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. Differences were considered significant at 5% probability level. Results: pS2 could be detected in 30 cases (32.9%) with values ranged from 0.04 to 89 ng/mg prt. Only one normal ovary showed detectable levels of pS2 and there were not differences in cytosolic content between benign and malignant ovarian tumors. The pS2 levels were only associated to mucinous differentiation in both benign and malignant ovarian tumors (p=0.029 and p=0.015, respectively). Significantly higher

Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

Science.gov (United States)

Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

2018-01-15

Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

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Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

2018-03-01

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane; Demangel, Caroline; Van Ingen, Jakko; Perez, Jorge; Baldeó n, Lucy R.; Abdallah, Abdallah; Caleechurn, Laxmee; Bottai, Daria; Van Zon, Maaike; De Punder, Karin; Van Der Laan, Tridia; Kant, Arie; Bossers-De Vries, Ruth; Willemsen, Peter Th J; Bitter, Wilbert M.; Van Soolingen, Dick; Brosch, Roland; Van Der Wel, Nicole N.; Peters, Peter J.

2012-01-01

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane

2012-05-08

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells

Science.gov (United States)

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.

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

KAUST Repository

Muleya, Victor

2014-09-23

Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

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Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

2011-01-01

Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata – Tropiduridae strengthen classification in lizard evolution

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Garcia Célia RS

2007-08-01

Full Text Available Abstract Background We have previously reported that a Teiid lizard red blood cells (RBCs such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition. Results Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like. Conclusion T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs, for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs.

Disruption of a Guard Cell–Expressed Protein Phosphatase 2A Regulatory Subunit, RCN1, Confers Abscisic Acid Insensitivity in Arabidopsis

Science.gov (United States)

Kwak, June M.; Moon, Ji-Hye; Murata, Yoshiyuki; Kuchitsu, Kazuyuki; Leonhardt, Nathalie; DeLong, Alison; Schroeder, Julian I.

2002-01-01

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell–expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca2+ increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling. PMID:12417706

Synthesis and characterization of superparamagnetic iron oxide nanoparticles as calcium-responsive MRI contrast agents

International Nuclear Information System (INIS)

Xu, Pengfei; Shen, Zhiwei; Zhang, Baolin; Wang, Jun; Wu, Renhua

2016-01-01

Highlights: • SPIONs were conjugated with EGTA by EDC/sulfo-NHS method. • The presence of Ca"2"+ induced the aggregation of EGTA-SPIONs. • The aggregation of EGTA-SPIONs increased the T2 relaxation time. • EGTA-SPIONs can be used for the calcium imaging with MRI. - Abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) as T2 contrast agents have great potential to sense calcium ion (Ca"2"+) using magnetic resonance imaging (MRI). Here we prepared calcium-responsive SPIONs for MRI, formed by combining poly(ethylene glycol) (PEG) and polyethylenimine (PEI) coated iron oxide nanoparticle (PEI/PEG-SPIONs) contrast agents with the straightforward calcium-sensing compound EGTA (ethylene glycol tetraacetic acid). EGTA was conjugated onto PEI/PEG-SPIONs using EDC/sulfo-NHS method. EGTA-SPIONs were characterized using TEM, XPS, DSL, TGA and SQUIID. DSL results show that the SPIONs aggregate in the presence of Ca"2"+. MRI analyses indicate that the water proton T2 relaxation rates in HEPES suspensions of the EGTA-SPIONs significantly increase with the calcium concentration because the SPIONs aggregate in the presence of Ca"2"+. The T2 values decreased 25% when Ca"2"+ concentration decreased from 1.2 to 0.8 mM. The aggregation of EGTA-SPIONs could be reversed by EDTA. EGTA-SPIONs have potential as smart contrast agents for Ca"2"+-sensitive MRI.

Synthesis and characterization of superparamagnetic iron oxide nanoparticles as calcium-responsive MRI contrast agents

Energy Technology Data Exchange (ETDEWEB)

Xu, Pengfei [State Key Laboratory Breeding Base of Nonferrous Metals and Specific Materials Processing, School of Materials Science and Engineering, Guilin University of Technology, Jian Gan Road 12, Guilin 541004 (China); Shen, Zhiwei [Second Affiliated Hospital of Shantou University Medical College, Dong Xia North Road, Shantou 515041,China (China); Zhang, Baolin, E-mail: baolinzhang@ymail.com [State Key Laboratory Breeding Base of Nonferrous Metals and Specific Materials Processing, School of Materials Science and Engineering, Guilin University of Technology, Jian Gan Road 12, Guilin 541004 (China); Wang, Jun [State Key Laboratory Breeding Base of Nonferrous Metals and Specific Materials Processing, School of Materials Science and Engineering, Guilin University of Technology, Jian Gan Road 12, Guilin 541004 (China); Wu, Renhua, E-mail: rhwu@stu.edu.cn [Second Affiliated Hospital of Shantou University Medical College, Dong Xia North Road, Shantou 515041,China (China)

2016-12-15

Highlights: • SPIONs were conjugated with EGTA by EDC/sulfo-NHS method. • The presence of Ca{sup 2+} induced the aggregation of EGTA-SPIONs. • The aggregation of EGTA-SPIONs increased the T2 relaxation time. • EGTA-SPIONs can be used for the calcium imaging with MRI. - Abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) as T2 contrast agents have great potential to sense calcium ion (Ca{sup 2+}) using magnetic resonance imaging (MRI). Here we prepared calcium-responsive SPIONs for MRI, formed by combining poly(ethylene glycol) (PEG) and polyethylenimine (PEI) coated iron oxide nanoparticle (PEI/PEG-SPIONs) contrast agents with the straightforward calcium-sensing compound EGTA (ethylene glycol tetraacetic acid). EGTA was conjugated onto PEI/PEG-SPIONs using EDC/sulfo-NHS method. EGTA-SPIONs were characterized using TEM, XPS, DSL, TGA and SQUIID. DSL results show that the SPIONs aggregate in the presence of Ca{sup 2+}. MRI analyses indicate that the water proton T2 relaxation rates in HEPES suspensions of the EGTA-SPIONs significantly increase with the calcium concentration because the SPIONs aggregate in the presence of Ca{sup 2+}. The T2 values decreased 25% when Ca{sup 2+} concentration decreased from 1.2 to 0.8 mM. The aggregation of EGTA-SPIONs could be reversed by EDTA. EGTA-SPIONs have potential as smart contrast agents for Ca{sup 2+}-sensitive MRI.

On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases.

Science.gov (United States)

Rasool, Mohammed I; Bairam, Ahsan F; Kurogi, Katsuhisa; Liu, Ming-Cheh

2017-10-01

Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT. The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays. Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media. SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors.

Science.gov (United States)

Deng, Liufu; Liang, Hua; Xu, Meng; Yang, Xuanming; Burnette, Byron; Arina, Ainhoa; Li, Xiao-Dong; Mauceri, Helena; Beckett, Michael; Darga, Thomas; Huang, Xiaona; Gajewski, Thomas F; Chen, Zhijian J; Fu, Yang-Xin; Weichselbaum, Ralph R

2014-11-20

Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-? induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-? treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Low glutathione regulates gene expression and the redox potentials of the nucleus and cytosol in Arabidopsis thaliana.

Science.gov (United States)

Schnaubelt, Daniel; Queval, Guillaume; Dong, Yingping; Diaz-Vivancos, Pedro; Makgopa, Matome Eugene; Howell, Gareth; De Simone, Ambra; Bai, Juan; Hannah, Matthew A; Foyer, Christine H

2015-02-01

Reduced glutathione (GSH) is considered to exert a strong influence on cellular redox homeostasis and to regulate gene expression, but these processes remain poorly characterized. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities. The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 and -320 mV. Growth in the presence of the GSH-synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately -260 mV. GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECATE), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH-synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO. Inhibition of auxin transport had no effect on organ GSH levels, but exogenous auxin decreased the root GSH pool. We conclude that GSH depletion significantly increases the redox potentials of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses, but not oxidative stress. © 2013 John Wiley & Sons Ltd.

A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.

Science.gov (United States)

Wang, Wenjing; Wildes, Craig P; Pattarabanjird, Tanyaporn; Sanchez, Mateo I; Glober, Gordon F; Matthews, Gillian A; Tye, Kay M; Ting, Alice Y

2017-09-01

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

DEFF Research Database (Denmark)

Houy, Sébastien; Groffen, Alexander J; Ziomkiewicz, Iwona

2017-01-01

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated....... We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2......+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping...

Composite mathematical modeling of calcium signaling behind neuronal cell death in Alzheimer's disease.

Science.gov (United States)

Ranjan, Bobby; Chong, Ket Hing; Zheng, Jie

2018-04-11

Alzheimer's disease (AD) is a progressive neurological disorder, recognized as the most common cause of dementia affecting people aged 65 and above. AD is characterized by an increase in amyloid metabolism, and by the misfolding and deposition of β-amyloid oligomers in and around neurons in the brain. These processes remodel the calcium signaling mechanism in neurons, leading to cell death via apoptosis. Despite accumulating knowledge about the biological processes underlying AD, mathematical models to date are restricted to depicting only a small portion of the pathology. Here, we integrated multiple mathematical models to analyze and understand the relationship among amyloid depositions, calcium signaling and mitochondrial permeability transition pore (PTP) related cell apoptosis in AD. The model was used to simulate calcium dynamics in the absence and presence of AD. In the absence of AD, i.e. without β-amyloid deposition, mitochondrial and cytosolic calcium level remains in the low resting concentration. However, our in silico simulation of the presence of AD with the β-amyloid deposition, shows an increase in the entry of calcium ions into the cell and dysregulation of Ca 2+ channel receptors on the Endoplasmic Reticulum. This composite model enabled us to make simulation that is not possible to measure experimentally. Our mathematical model depicting the mechanisms affecting calcium signaling in neurons can help understand AD at the systems level and has potential for diagnostic and therapeutic applications.

Application of Response Surface Methodology to study the effect of different calcium sources in fish muscle-alginate restructured products

Directory of Open Access Journals (Sweden)

Helena María Moreno

2011-03-01

Full Text Available Sodium alginate needs the presence of calcium ions to gelify. For this reason, the contribution of the calcium source in a fish muscle mince added by sodium alginate, makes gelification possible, resulting a restructured fish product. The three different calcium sources considered were: Calcium Chloride (CC; Calcium Caseinate (CCa; and Calcium lactate (CLa. Several physical properties were analyzed, including mechanical properties, colour and cooking loss. Response Surface Methodology (RSM was used to determine the contribution of different calcium sources to a restructured fish muscle. The calcium source that modifies the system the most is CC. A combination of CC and sodium alginate weakened mechanical properties as reflected in the negative linear contribution of sodium alginate. Moreover, CC by itself increased lightness and cooking loss. The mechanical properties of restructured fish muscle elaborated were enhanced by using CCa and sodium alginate, as reflected in the negative linear contribution of sodium alginate. Also, CCa increased cooking loss. The role of CLa combined with sodium alginate was not so pronounced in the system discussed here.

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

Energy Technology Data Exchange (ETDEWEB)

Chen, Min, E-mail: chenminyx@gmail.com [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Yunnan Centers for Diseases Prevention and Control, Kunming 650022 (China); Wang, Yanru [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Qu, Aijuan [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

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.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

Science.gov (United States)

Samigullin, Dmitry; Fatikhov, Nijaz; Khaziev, Eduard; Skorinkin, Andrey; Nikolsky, Eugeny; Bukharaeva, Ellya

2014-01-01

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers-which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal-has hitherto been technically impossible. With the aim of quantifying both Ca(2+) currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

Science.gov (United States)

Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

2015-01-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

Science.gov (United States)

Wen, Xianjie; Xu, Shiyuan; Liu, Hongzhen; Zhang, Quinguo; Liang, Hua; Yang, Chenxiang; Wang, Hanbing

2013-01-01

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. PMID:23658789

Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells.

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Xianjie Wen

Full Text Available There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca(2+ ([Ca2+]i, cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.

Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom.

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Olmedo, Hidekel; Herrera, María; Rojas, Leonardo; Villalta, Mauren; Vargas, Mariángela; Leiguez, Elbio; Teixeira, Catarina; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo; Montero, Mavis L

2014-01-01

The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A2, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts.

A sensor for calcium uptake

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Collins, Sean; Meyer, Tobias

2011-01-01

Mitochondria — the cell’s power plants — increase their energy production in response to calcium signals in the cytoplasm. A regulator of the elusive mitochondrial calcium channel has now been identified. PMID:20844529

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

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Anthony S. Piro

2017-12-01

Full Text Available Dynamin-like guanylate binding proteins (GBPs are gamma interferon (IFN-γ-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6. GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

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Belyavskaya, N. A.

The changes of [Ca^2+]_i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station ``Salyut 6'' /1/. These results: 1) indicate that observed Ca^2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca^2+ influx through membranes. In model presented, I propose that Ca^2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca^2+ into the root cells, causing a rise in cytoplasmic free Ca^2+ levels. The latter, in its turn, may induce the inhibition of a Ca^2+ efflux by Ca^2+-activated ATPases and through a Ca^2+/H^+ antiport. It is possible that increased cytosolic levels of Ca^2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca^2+]_i. Plant cell can response to such a Ca^2+ rise by an enhancement of membranous Ca^2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca^2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca^2+ to plant cell.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

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Dmitry eSamigullin

2015-01-01

Full Text Available At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 рА and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 µM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes

International Nuclear Information System (INIS)

Lim, M.S.; Lim, Priscilla L.K.; Gupta, Rashi; Boelsterli, Urs A.

2006-01-01

Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca 2+ c ) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 μM). At 8 h, diclofenac caused increases in [Ca 2+ ] c (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca 2+ chelator) inhibited cell injury, indicating that Ca 2+ plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, ΔΨ m (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca 2+ ] c . The CYP2C9 inhibitor sulphaphenazole (10 μM) protected from diclofenac-induced cell injury and prevented increases in [Ca 2+ ] c , while it had no effect on the dissipation of the ΔΨ m . Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca 2+ ] c . In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca 2+ ] c , triggering the mPT and precipitating cell death

A new view of the bacterial cytosol environment.

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Benjamin P Cossins

2011-06-01

Full Text Available 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. Mg(2+ 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.

Calcium Phosphate as a Key Material for Socially Responsible Tissue Engineering

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Vuk Uskoković

2016-06-01

Full Text Available Socially responsible technologies are designed while taking into consideration the socioeconomic, geopolitical and environmental limitations of regions in which they will be implemented. In the medical context, this involves making therapeutic platforms more accessible and affordable to patients in poor regions of the world wherein a given disease is endemic. This often necessitates going against the reigning trend of making therapeutic nanoparticles ever more structurally complex and expensive. However, studies aimed at simplifying materials and formulations while maintaining the functionality and therapeutic response of their more complex counterparts seldom provoke a significant interest in the scientific community. In this review we demonstrate that such compositional simplifications are meaningful when it comes to the design of a solution for osteomyelitis, a disease that is in its natural, non-postoperative form particularly prevalent in the underdeveloped parts of the world wherein poverty, poor sanitary conditions, and chronically compromised defense lines of the immune system are the norm. We show that calcium phosphate nanoparticles, which are inexpensive to make, could be chemically designed to possess the same functionality as a hypothetic mixture additionally composed of: (a a bone growth factor; (b an antibiotic for prophylactic or anti-infective purposes; (c a bisphosphonate as an antiresorptive compound; (d a viral vector to enable the intracellular delivery of therapeutics; (e a luminescent dye; (f a radiographic component; (g an imaging contrast agent; (h a magnetic domain; and (i polymers as viscous components enabling the injectability of the material and acting as carriers for the sustained release of a drug. In particular, calcium phosphates could: (a produce tunable drug release profiles; (b take the form of viscous and injectable, self-setting pastes; (c be naturally osteo-inductive and inhibitory for osteoclastogenesis

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin strongylocentrotus droebachiensis

International Nuclear Information System (INIS)

Oberdorf, J.A.

1986-01-01

Two subcellular fractions of the sea urchin egg were studied for their potential role in regulating the transient rise in cytosolic calcium that accompanies fertilization. Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell. This ATP dependent calcium uptake activity, measured in the presence of 5mM Na Azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 μM quercetin and 50 μM vanadate. Cortices preloaded with 45 Ca in the presence of ATP dramatically increased their rate of calcium efflux upon the addition of (1) the calcium ionophore A23187 (10 μM), (2) trifluoperazine (200 μM), (3) concentrations of free calcium that activated cortical granule exocytosis, and (4) the calcium mobilizing agent inositol trisphosphate (IP3). This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum (ER) that remains associated with the cortical region during its isolation. They have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors, however the isolated microsomal vesicles did not show any detectable release of calcium when exposed to IP3. Procedures originally developed for purifying calsequestrin were used to partially purify a 58,000 MW protein from the egg's microsomal vesicles

A Plant Phytosulfokine Peptide Initiates Auxin-Dependent Immunity through Cytosolic Ca2+ Signaling in Tomato.

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Zhang, Huan; Hu, Zhangjian; Lei, Cui; Zheng, Chenfei; Wang, Jiao; Shao, Shujun; Li, Xin; Xia, Xiaojian; Cai, Xinzhong; Zhou, Jie; Zhou, Yanhong; Yu, Jingquan; Foyer, Christine H; Shi, Kai

2018-03-01

Phytosulfokine (PSK) is a disulfated pentapeptide that is an important signaling molecule. Although it has recently been implicated in plant defenses to pathogen infection, the mechanisms involved remain poorly understood. Using surface plasmon resonance and gene silencing approaches, we showed that the tomato ( Solanum lycopersicum ) PSK receptor PSKR1, rather than PSKR2, functioned as the major PSK receptor in immune responses. Silencing of PSK signaling genes rendered tomato more susceptible to infection by the economically important necrotrophic pathogen Botrytis cinerea Analysis of tomato mutants defective in either defense hormone biosynthesis or signaling demonstrated that PSK-induced immunity required auxin biosynthesis and associated defense pathways. Here, using aequorin-expressing tomato plants, we provide evidence that PSK perception by tomato PSKR1 elevated cytosolic [Ca 2+ ], leading to auxin-dependent immune responses via enhanced binding activity between calmodulins and the auxin biosynthetic YUCs. Thus, our data demonstrate that PSK acts as a damage-associated molecular pattern and is perceived mainly by PSKR1, which increases cytosolic [Ca 2+ ] and activates auxin-mediated pathways that enhance immunity of tomato plants to B. cinerea . © 2018 American Society of Plant Biologists. All rights reserved.

Pre-treatment of soybean plants with calcium stimulates ROS responses and mitigates infection by Sclerotinia sclerotiorum.

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Arfaoui, Arbia; El Hadrami, Abdelbasset; Daayf, Fouad

2018-01-01

Considering the high incidence of white mold caused by Sclerotinia sclerotiorum in a variety of field crops and vegetables, different control strategies are needed to keep the disease under economical threshold. This study assessed the effect of foliar application of a calcium formulation on disease symptoms, oxalic acid production, and on the oxidative stress metabolism in soybean plants inoculated with each of two isolates of the pathogen that have contrasting aggressiveness (HA, highly-aggressive versus WA, weakly-aggressive). Changes in reactive oxygen species (ROS) levels in soybean plants inoculated with S. sclerotiorum isolates were assessed at 6, 24, 48 and 72 h post inoculation (hpi). Generation of ROS including hydrogen peroxide (H 2 O 2 ), anion superoxide (O 2 - ) and hydroxyl radical (OH) was evaluated. Inoculation with the WA isolate resulted in more ROS accumulation compared to the HA isolate. Pre-treatment with the calcium formulation restored ROS production in plants inoculated with the HA isolate. We also noted a marked decrease in oxalic acid content in the leaves inoculated with the HA isolate in presence of calcium, which coincided with an increase in plant ROS production. The expression patterns of genes involved in ROS detoxification in response to the calcium treatments and/or inoculation with S. Sclerotiorum isolates were monitored by RT-qPCR. All of the tested genes showed a higher expression in response to inoculation with the WA isolate. The expression of most genes tested peaked at 6 hpi, which preceded ROS accumulation in the soybean leaves. Overall, these data suggest that foliar application of calcium contributes to a decrease in oxalic acid production and disease, arguably via modulation of the ROS metabolism. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A crosstalk between Na⁺ channels, Na⁺/K⁺ pump and mitochondrial Na⁺ transporters controls glucose-dependent cytosolic and mitochondrial Na⁺ signals.

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Nita, Iulia I; Hershfinkel, Michal; Lewis, Eli C; Sekler, Israel

2015-02-01

Glucose-dependent cytosolic Na(+) influx in pancreatic islet β cells is mediated by TTX-sensitive Na(+) channels and is propagated into the mitochondria through the mitochondrial Na(+)/Ca(2+) exchanger, NCLX. Mitochondrial Na(+) transients are also controlled by the mitochondrial Na(+)/H(+) exchanger, NHE, while cytosolic Na(+) changes are governed by Na(+)/K(+) ATPase pump. The functional interaction between the Na(+) channels, Na(+)/K(+) ATPase pump and mitochondrial Na(+) transporters, NCLX and NHE, in mediating Na(+) signaling is poorly understood. Here, we combine fluorescent Na(+) imaging, pharmacological inhibition by TTX, ouabain and EIPA, with molecular control of NCLX expression, so as to investigate the crosstalk between Na(+) transporters on both the plasma membrane and the mitochondria. According to our results, glucose-dependent cytosolic Na(+) response was enhanced by ouabain and was followed by a rise in mitochondrial Na(+) signal. Silencing of NCLX expression using siNCLX, did not affect the glucose- or ouabain-dependent cytosolic rise in Na(+). In contrast, the ouabain-dependent rise in mitochondrial Na(+) was strongly suppressed by siNCLX. Furthermore, mitochondrial Na(+) influx rates were accelerated in cells treated with the Na(+)/H(+) exchanger inhibitor, EIPA or by combination of EIPA and ouabain. Similarly, TTX blocked the cytosolic and mitochondrial Na(+) responses, which were enhanced by ouabain or EIPA, respectively. Our results suggest that Na(+)/K(+) ATPase pump controls cytosolic glucose-dependent Na(+) rise, in a manner that is mediated by TTX-sensitive Na(+) channels and subsequent mitochondrial Na(+) uptake via NCLX. Furthermore, these results indicate that mitochondrial Na(+) influx via NCLX is antagonized by Na(+) efflux, which is mediated by the mitochondrial NHE; thus, the duration of mitochondrial Na(+) transients is set by the interplay between these pivotal transporters. Copyright © 2014 Elsevier Ltd. All rights reserved.

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

DEFF Research Database (Denmark)

Liu, Xiangdong; Martens, Helle; Schulz, Alexander

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

Differential expression of calcium/calmodulin-regulated SlSRs in response to abiotic and biotic stresses in tomato fruit.

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Yang, Tianbao; Peng, Hui; Whitaker, Bruce D; Jurick, Wayne M

2013-07-01

Calcium has been shown to enhance stress tolerance, maintain firmness and reduce decay in fruits. Previously we reported that seven tomato SlSRs encode calcium/calmodulin-regulated proteins, and that their expressions are developmentally regulated during fruit development and ripening, and are also responsive to ethylene. To study their expressions in response to stresses encountered during postharvest handling, tomato fruit at the mature-green stage was subjected to chilling and wounding injuries, infected with Botrytis cinerea and treated with salicylic acid or methyl jasmonate. Gene expression studies revealed that the seven SlSRs differentially respond to different stress signals. SlSR2 was the only gene upregulated by all the treatments. SlSR4 acted as a late pathogen-induced gene; it was upregulated by salicylic acid and methyl jasmonate, but downregulated by cold treatment. SlSR3L was cold- and wound-responsive and was also induced by salicylic acid. SlSR1 and SlSR1L were repressed by cold, wounding and pathogen infection, but were upregulated by salicylic acid and methyl jasmonate. Overall, results of these expression studies indicate that individual SlSRs have distinct roles in responses to the specific stress signals, and SlSRs may act as a coordinator(s) connecting calcium-mediated signaling with other stress signal transduction pathways during fruit ripening and storage. © 2013 Scandinavian Plant Physiology Society.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

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Matthias Hardtke-Wolenski

Full Text Available BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Involvement of plasma membrane calcium influx in bacterial induction of the K+/H+ and hypersensitive responses in tobacco

International Nuclear Information System (INIS)

Atkinson, M.M.; Keppler, L.D.; Orlandi, E.W.; Baker, C.J.; Mischke, C.F.

1990-01-01

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K + /H + response characterized by specific plasma membrane K + efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca 2+ influx of 0.02 to 0.06 micromole per gram per hour as determined from 45 Ca 2+ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K + /H + response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca 2+ influx was prevented by EGTA and calcium channel blockers such as La 3+ , Co 2+ , and Cd 2+ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K + /H + response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increase plasmalemma Ca 2+ influx is required for the K + /H + and hypersensitive responses in tobacco

Leptin's effect on taste bud calcium responses and transmitter secretion.

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Meredith, Tricia L; Corcoran, Alan; Roper, Stephen D

2015-05-01

Leptin, a peptide hormone released by adipose tissue, acts on the hypothalamus to control cravings and appetite. Leptin also acts to decrease taste responses to sweet substances, though there is little detailed information regarding where leptin acts in the taste transduction cascade. The present study examined the effects of leptin on sweet-evoked responses and neuro transmitter release from isolated taste buds. Our results indicate that leptin moderately decreased sweet-evoked calcium mobilization in isolated mouse taste buds. We also employed Chinese hamster ovary biosensor cells to examine taste transmitter release from isolated taste buds. Leptin reduced ATP and increased serotonin release in response to sweet stimulation. However, leptin has no effect on bitter-evoked transmitter release, further showing that the action of leptin is sweet specific. Our results support those of previous studies, which state that leptin acts on taste tissue via the leptin receptor, most likely on Type II (Receptor) cells, but also possibly on Type III (Presynaptic) cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

International Nuclear Information System (INIS)

Petrovic, Uros; Sribar, Jernej; Paris, Alenka; Rupnik, Marjan; Krzan, Mojca; Vardjan, Nina; Gubensek, Franc; Zorec, Robert; Krizaj, Igor

2004-01-01

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A 2 acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment

Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

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Guizzunti, Gianni; Zurzolo, Chiara

2015-01-01

We previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death.

Dietary calcium intake and Renin Angiotensin System polymorphisms alter the blood pressure response to aerobic exercise: a randomized control design

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Tsongalis Gregory J

2007-01-01

Full Text Available Abstract Background Dietary calcium intake and the renin angiotensin system (RAS regulate blood pressure (BP by modulating calcium homeostasis. Despite similar BP regulatory effects, the influence of dietary calcium intake alone and combined with RAS polymorphisms on the BP response following acute aerobic exercise (i.e., postexercise hypotension has not been studied. Thus, we examined the effect of dietary calcium intake and selected RAS polymorphisms on postexercise hypotension. Methods Subjects were men (n = 50, 43.8 ± 1.3 yr with high BP (145.3 ± 1.5/85.9 ± 1.1 mm Hg. They completed three experiments: non-exercise control and two cycle bouts at 40% and 60% of maximal oxygen consumption (VO2max. Subjects provided 3 d food records on five protocol-specific occasions. Dietary calcium intake was averaged and categorized as low (1R A/C were analyzed with molecular methods. Genotypes were reduced from three to two: ACE II/ID and ACE DD; or AT1R AA and AT1R CC/AC. Repeated measure ANCOVA tested if BP differed among experiments, dietary calcium intake level and RAS polymorphisms. Results Systolic BP (SBP decreased 6 mm Hg after 40% and 60% VO2max compared to non-exercise control for 10 h with LowCa (p 2max versus non-exercise control for 10 h among ACE II/ID (6 mm Hg and AT1R AA (8 mm Hg; and by 8 mm Hg after 40% VO2max among ACE DD and AT1R CC/CA (p 2max compared to non-exercise control for 10 h (p 2max (p ≥ 0.05. Conclusion SBP decreased after exercise compared to non-exercise control among men with low but not high dietary calcium intake. Dietary calcium intake interacted with the ACE I/D and AT1R A/C polymorphisms to further modulate postexercise hypotension. Interactions among dietary calcium intake, exercise intensity and RAS polymorphisms account for some of the variability in the BP response to exercise.

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

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Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

2015-09-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

Science.gov (United States)

Schröder, Katrin; Weissmann, Norbert; Brandes, Ralf P

2017-08-01

NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

Science.gov (United States)

Zhang, Xinxin; Tang, Xuexi; Wang, Ming; Zhang, Wei; Zhou, Bin; Wang, You

2017-08-01

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m -2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO 4 3- uptake was more serious compared to NO 3 - uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca 2+ -ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca 2+ -ATPase suppression, and a relation between Ca 2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

Science.gov (United States)

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Calcium dynamics in vascular smooth muscle

OpenAIRE

Amberg, Gregory C.; Navedo, Manuel F.

2013-01-01

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells....

Cerebral blood flow modulation by Basal forebrain or whisker stimulation can occur independently of large cytosolic Ca2+ signaling in astrocytes.

Science.gov (United States)

Takata, Norio; Nagai, Terumi; Ozawa, Katsuya; Oe, Yuki; Mikoshiba, Katsuhiko; Hirase, Hajime

2013-01-01

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.

Serum deprivation induces glucose response and intercellular coupling in human pancreatic adenocarcinoma PANC-1 cells.

Science.gov (United States)

Hiram-Bab, Sahar; Shapira, Yuval; Gershengorn, Marvin C; Oron, Yoram

2012-03-01

This study aimed to investigate whether the previously described differentiating islet-like aggregates of human pancreatic adenocarcinoma cells (PANC-1) develop glucose response and exhibit intercellular communication. Fura 2-loaded PANC-1 cells in serum-free medium were assayed for changes in cytosolic free calcium ([Ca]i) induced by depolarization, tolbutamide inhibition of K(ATP) channels, or glucose. Dye transfer, assayed by confocal microscopy or by FACS, was used to detect intercellular communication. Changes in messenger RNA (mRNA) expression of genes of interest were assessed by quantitative real-time polymerase chain reaction. Proliferation was assayed by the MTT method. Serum-deprived PANC-1 cell aggregates developed [Ca]i response to KCl, tolbutamide, or glucose. These responses were accompanied by 5-fold increase in glucokinase mRNA level and, to a lesser extent, of mRNAs for K(ATP) and L-type calcium channels, as well as increase in mRNA levels of glucagon and somatostatin. Trypsin, a proteinase-activated receptor 2 agonist previously shown to enhance aggregation, modestly improved [Ca]i response to glucose. Glucose-induced coordinated [Ca]i oscillations and dye transfer demonstrated the emergence of intercellular communication. These findings suggest that PANC-1 cells, a pancreatic adenocarcinoma cell line, can be induced to express a differentiated phenotype in which cells exhibit response to glucose and form a functional syncytium similar to those observed in pancreatic islets.

DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

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Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

2013-01-01

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

Science.gov (United States)

Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

2013-02-15

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

Inflammatory cell response to calcium phosphate biomaterial particles: an overview.

Science.gov (United States)

Velard, Frédéric; Braux, Julien; Amedee, Joëlle; Laquerriere, Patrice

2013-02-01

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite (HA) and amorphous calcium phosphate (CaP) crystals deposited in an organic matrix. One objective of bone tissue engineering is to mimic the chemical and structural properties of this complex tissue. CaP ceramics, such as sintered HA and beta-tricalcium phosphate, are widely used as bone substitutes or prosthesis coatings because of their osteoconductive properties. These ceramic interactions with tissues induce a cell response that can be different according to the composition of the material. In this review, we discuss inflammatory cell responses to CaP materials to provide a comprehensive overview of mechanisms governing the integration or loosening of implants, which remains a major concern in tissue engineering. A focus on the effects of the functionalization of CaP biomaterials highlights potential ways to increase tissue integration and limit rejection processes. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction.

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Wolfgang Giese

2018-04-01

Full Text Available The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus.

Synthesis of calcium hydroxyapatite from calcium carbonate and different orthophosphate sources: A comparative study

International Nuclear Information System (INIS)

Pham Minh, Doan; Lyczko, Nathalie; Sebei, Haroun; Nzihou, Ange; Sharrock, Patrick

2012-01-01

Highlights: ► Calcium hydroxyapatite was synthesized from CaCO 3 and four orthophosphates. ► Only H 3 PO 4 led to the complete precipitation of orthophosphate species. ► H 3 PO 4 was also the most efficient for calcium dissolution. ► Reaction pathway was dissolution-precipitation accompanied by agglomeration step. - Abstract: The synthesis of calcium hydroxyapatite (Ca-HA) starting from calcium carbonate and different orthophosphate sources, including orthophosphoric acid, potassium, sodium and ammonium dihydrogen orthophosphates, was investigated under ambient conditions. The reaction started with calcium carbonate dissolution in an acid medium, followed by rapid precipitation of calcium cations with orthophosphate species to form calcium phosphate based particles which were in the size range of 0.4–1 μm. These particles then agglomerated into much larger ones, up to 350 μm in diameter (aggregates). These aggregates possessed an unstable porous structure which was responsible for the porosity of the final products. The highest specific surface area and pore volume were obtained with potassium dihydrogen orthophosphate. On the other hand, orthophosphoric acid led to the highest dissolution of calcium carbonate and the complete precipitation of orthophosphate species. Under ambient conditions, calcium phosphate based solid products of low crystallinity were formed. Different intermediates were identified and a reaction pathway proposed.

AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca(2+) increase.

Science.gov (United States)

Francia, Doriana; Chiltz, Annick; Lo Schiavo, Fiorella; Pugin, Alain; Bonfante, Paola; Cardinale, Francesca

2011-09-01

The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca(2+) in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca(2+)-channel blocker (La(3+)) showed that in these cells, MAPK activation was dependent on the cytosolic Ca(2+) increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca(2+) transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

International Nuclear Information System (INIS)

Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

2007-01-01

The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

In vitro degradation and cell response of calcium carbonate composite ceramic in comparison with other synthetic bone substitute materials

International Nuclear Information System (INIS)

He, Fupo; Zhang, Jing; Yang, Fanwen; Zhu, Jixiang; Tian, Xiumei; Chen, Xiaoming

2015-01-01

The robust calcium carbonate composite ceramics (CC/PG) can be acquired by fast sintering calcium carbonate at a low temperature (650 °C) using a biocompatible, degradable phosphate-based glass (PG) as sintering agent. In the present study, the in vitro degradation and cell response of CC/PG were assessed and compared with 4 synthetic bone substitute materials, calcium carbonate ceramic (CC), PG, hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics. The degradation rates in decreasing order were as follows: PG, CC, CC/PG, β-TCP, and HA. The proliferation of rat bone mesenchymal stem cells (rMSCs) cultured on the CC/PG was comparable with that on CC and PG, but inferior to HA and β-TCP. The alkaline phosphatase (ALP) activity of rMSCs on CC/PG was lower than PG, comparable with β-TCP, but higher than HA. The rMSCs on CC/PG and PG had enhanced gene expression in specific osteogenic markers, respectively. Compared to HA and β-TCP, the rMSCs on the CC/PG expressed relatively lower level of collagen I and runt-related transcription factor 2, but showed more considerable expression of osteopontin. Although CC, PG, HA, and β-TCP possessed impressive performances in some specific aspects, they faced extant intrinsic drawbacks in either degradation rate or mechanical strength. Based on considerable compressive strength, moderate degradation rate, good cell response, and being free of obvious shortcoming, the CC/PG is promising as another choice for bone substitute materials. - Highlights: • A calcium carbonate composite ceramic (CC/PG) was acquired. • The in vitro degradation and cell response of CC/PG were compared to 4 materials. • The CC/PG showed moderate degradation rate. • The CC/PG exhibited good cell response. • The CC/PG was free of obvious drawback compared to other materials

Calcium uptake by sarcoplasmic reticulum isolated from hearts of septic rats

International Nuclear Information System (INIS)

McDonough, K.H.

1988-01-01

Myocardial sarcoplasmic reticulum (SR) plays a critical role in the regulation of the cytosolic calcium fluctuations that occur during the cardiac cycle. One function of the SR is to lower the calcium concentration so that myocardial relaxation and thus ventricular filling can occur. The aim of the present study was to determine if hyperdynamic sepsis induced a decrease in the capacity of SR to take up calcium. This defect would result in decreased ventricular filling and thus decreased cardiac output, as has previously been shown in isolated perfused working hearts removed from septic rats. Therefore, rats were anesthetized with ether, and sepsis was induced by the injection of an aliquot of a fecal homogenate into the peritoneal cavity. Control animals either underwent surgery and received an aliquot of sterilized fecal inoculum (sham) or were untreated (no surgery). On day 2 after surgery, animals were anesthetized with pentobarbital, and hearts were removed, weighted, and SR isolated. The rate of uptake of 45 Ca 2+ by SR from septic rats was not depressed compared to controls but in fact was elevated. Maximum 45 Ca 2+ accumulated by the SR and Ca 2+ -stimulated ATPase activity were similar in SR from control and septic hearts. These results suggest that the contractile dysfunction noted in the myocardium in early sepsis is probably not due to inadequate SR removal of Ca 2+ during diastole

Calcium pumps of plasma membrane and cell interior

DEFF Research Database (Denmark)

Strehler, Emanuel E; Treiman, Marek

2004-01-01

Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco....../endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue......- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping...

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

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Helena H. Chowdhury

2014-10-01

Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

Cytosolic sensing of immuno-stimulatory DNA, the enemy within.

Science.gov (United States)

Dhanwani, Rekha; Takahashi, Mariko; Sharma, Sonia

2018-02-01

In the cytoplasm, DNA is sensed as a universal danger signal by the innate immune system. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor/enzyme that catalyzes formation of 2'-5'-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates the Stimulator of Interferon Genes (STING), resulting in recruitment of Tank Binding Kinase 1 (TBK1), activation of the transcription factor Interferon Regulatory Factor 3 (IRF3), and trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I). Activation of the pro-inflammatory cGAS-STING-IRF3 response is triggered by direct recognition of the DNA genomes of bacteria and viruses, but also during RNA virus infection, neoplastic transformation, tumor immunotherapy and systemic auto-inflammatory diseases. In these circumstances, the source of immuno-stimulatory DNA has often represented a fundamental yet poorly understood aspect of the response. This review focuses on recent findings related to cGAS activation by an array of self-derived DNA substrates, including endogenous retroviral elements, mitochondrial DNA (mtDNA) and micronuclei generated as a result of genotoxic stress and DNA damage. These findings emphasize the role of the cGAS axis as a cell-intrinsic innate immune response to a wide variety of genomic insults. Copyright © 2017. Published by Elsevier Ltd.

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle.

Science.gov (United States)

Manno, Carlo; Sztretye, Monika; Figueroa, Lourdes; Allen, Paul D; Ríos, Eduardo

2013-01-15

The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca(2+)], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca(2+)] were measured integrating Ca(2+) release flux, determined with a cytosolic [Ca(2+)] monitor. Free [Ca(2+)](SR) was measured using the cameleon D4cpv-Casq1. In 34 wild-type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse - when flux was greatest - than at its end, and greater in the ON than in the OFF. In 29 Casq1-null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca(2+)](SR) was tracked with the fast dye fluo-5N. The calcium buffering power, binding capacity and non-linear binding properties of the SR measured here could be accounted for by calsequestrin at the concentration present in mammalian muscle, provided that its properties were substantially different from those found in solution. Its affinity should be higher, or K(D) lower than the conventionally accepted 1 mm; its cooperativity (n in a Hill fit) should be higher and the stoichiometry of binding should be at the higher end of the values derived in solution. The reduction in B during release might reflect changes in calsequestrin conformation upon calcium loss.

Calcium signals in olfactory neurons.

Science.gov (United States)

Tareilus, E; Noé, J; Breer, H

1995-11-09

Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

Directory of Open Access Journals (Sweden)

Jennifer H Hou

2014-09-01

Full Text Available The cardiac action potential (AP and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf. We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 – 102 hours post fertilization (hpf, the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

Calcium signaling in liver.

Science.gov (United States)

Gaspers, Lawrence D; Thomas, Andrew P

2005-01-01

In hepatocytes, hormones linked to the formation of the second messenger inositol 1,4,5-trisphosphate (InsP3) evoke transient increases or spikes in cytosolic free calcium ([Ca2+]i), that increase in frequency with the agonist concentration. These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to down-stream Ca2+-sensitive metabolic processes. We have utilized both confocal and wide field fluorescence microscopy techniques to study the InsP3-dependent signaling pathway at the cellular and subcellular levels in the intact perfused liver. Typically InsP3-dependent [Ca2+]i spikes manifest as Ca2+ waves that propagate throughout the entire cytoplasm and nucleus, and in the intact liver these [Ca2+]i increases are conveyed through gap junctions to encompass entire lobular units. The translobular movement of Ca2+ provides a means to coordinate the function of metabolic zones of the lobule and thus, liver function. In this article, we describe the characteristics of agonist-evoked [Ca2+]i signals in the liver and discuss possible mechanisms to explain the propagation of intercellular Ca2+ waves in the intact organ.

Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds*

Science.gov (United States)

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

Gel chromatography of immunoextracted plasma calcitonin in response to the calcium clamp in healthy males

International Nuclear Information System (INIS)

Bucht, E.; Toerring, O.; Sjoeberg, H.E.

1985-01-01

A radioimmunoassay (RIA) was developed for immunoreactive calcitonin (iCT) in human plasma. Antibodies against synthetic human calcitonin (hCT) coupled to bovine serum albumin (BSA) were raised in rabbits and were directed against the carboxy terminal part of CT. The detection limit of the assay was 8 pg/ml. In 7 males the iCT response to a calcium-clamp was studied. Blood was collected at 0,30 and 60 min after the start of the calcium infusion. iCT was measured directly in plasma and in extracts obtained after purification of plasma iCT by means of immobilized CT antiboides. There was a good correlation between iCT in plasma samples and extracts, r = 0.993, n = 14 (P< 0.001). Dilution curves of extracts and plasma were parallel with the hCT standard curves. Gel chromatography of the extracts on Sephadex G-50 and G-75 disclosed heterogeneity of iCT in normal plasma during basal conditions as well as during calcium stimulation. Thirty min after the start of the calcium clamp all molecular forms, most likely constituting monomeric and dimeric CT and larger forms, were increased, while after 60 min iCT seemed to constitute predominantly forms larger than monomeric TCY. Basal levels of unextracted iCT in healthy males (n = 44, 37 +/- 10 years) were 15 +/- 9 pg-equivalents/ml (mean +/- SD), which was higher than in females (n = 40, 32 +/- 9 years) 11 +/- 4 pg-equivalents/ml (P < 0.05). (author)

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

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Sachie Tanaka

2017-05-01

Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

Presynaptic calcium signalling in cerebellar mossy fibres

DEFF Research Database (Denmark)

Thomsen, Louiza Bohn; Jörntell, Henrik; Midtgaard, Jens

2010-01-01

Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX......)-sensitive fast Na(+) spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers....... Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon...

Mathematical investigation of IP3-dependent calcium dynamics in astrocytes.

Science.gov (United States)

Handy, Gregory; Taheri, Marsa; White, John A; Borisyuk, Alla

2017-06-01

We study evoked calcium dynamics in astrocytes, a major cell type in the mammalian brain. Experimental evidence has shown that such dynamics are highly variable between different trials, cells, and cell subcompartments. Here we present a qualitative analysis of a recent mathematical model of astrocyte calcium responses. We show how the major response types are generated in the model as a result of the underlying bifurcation structure. By varying key channel parameters, mimicking blockers used by experimentalists, we manipulate this underlying bifurcation structure and predict how the distributions of responses can change. We find that store-operated calcium channels, plasma membrane bound channels with little activity during calcium transients, have a surprisingly strong effect, underscoring the importance of considering these channels in both experiments and mathematical settings. Variation in the maximum flow in different calcium channels is also shown to determine the range of stable oscillations, as well as set the range of frequencies of the oscillations. Further, by conducting a randomized search through the parameter space and recording the resulting calcium responses, we create a database that can be used by experimentalists to help estimate the underlying channel distribution of their cells.

Quantifying the Attractive Force Exerted on the Pinned Calcium Spiral Waves by Using the Adventive Field

International Nuclear Information System (INIS)

Qiu Kang; Tang Jun; Luo Jin-Ming; Ma Jun

2013-01-01

The cytosolic calcium system is inhomogenous because of the discrete and random distribution of ion channels on the ER membrane. It is well known that the spiral tip can be pinned by the heterogenous area, and the field can detach the spiral from the heterogeneity. We use the adventive field to counteract the attractive force exerting on the calcium spiral wave by the heterogeneity, then the strength of the adventive field is used to quantify the attractive force indirectly. Two factors determining the attractive force are studied. It is found that: (1) the attractive force sharply increases with size of the heterogeneity for small-size heterogeneity, whereas the force increases to a saturated value for large-size heterogeneity; (2) for large-size heterogeneity, the force almost remains constant unless the level of the heterogeneity vanishes, the force decreases to zero linearly and sharply, and for small-size heterogeneity, the force decreases successively with the level of the heterogeneity. Furthermore, it is found that the forces exist only when the spiral tip is very close to the heterogenous area. Our study may shed some light on the control or suppression of the calcium spiral wave

Enhanced expression of a calcium-dependent protein kinase

Indian Academy of Sciences (India)

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

Science.gov (United States)

Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

2009-04-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

L-Type Calcium Channels Modulation by Estradiol.

Science.gov (United States)

Vega-Vela, Nelson E; Osorio, Daniel; Avila-Rodriguez, Marco; Gonzalez, Janneth; García-Segura, Luis Miguel; Echeverria, Valentina; Barreto, George E

2017-09-01

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

OpenAIRE

Tomokazu, Konishi; Takahiro, Kamoi; Ryuichi, Matsuno; Yukiko, Sasaki; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural College of Agriculture; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Plant Molecular Biology, School of Agricultural Sciences, Nagoya University

1996-01-01

Levels of subunits of two acetyl-coenzyme A carboxylases were high in small leaves of Pisum sativum, decreased with growth, and remained constant in fully expanded leaves. Irradiation of fully expanded leaves induced the cytosolic isozyme only. This result suggests a key role for the cytosolic enzyme in protection against UV-B.

Aequorin-based measurements of intracellular Ca2+-signatures in plant cells

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Mithöfer Axel

2002-01-01

Full Text Available Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing the usefulness of aequorin to study the calcium response to biotic (elicitors and abiotic stimuli (osmotic shocks in various compartments of plant cells such as cytosol and nucleus.

Kokumi Substances, Enhancers of Basic Tastes, Induce Responses in Calcium-Sensing Receptor Expressing Taste Cells

Science.gov (United States)

Maruyama, Yutaka; Yasuda, Reiko; Kuroda, Motonaka; Eto, Yuzuru

2012-01-01

Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami. PMID:22511946

Seasonal Variations in Mercury's Dayside Calcium Exosphere

Science.gov (United States)

Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Merkel, Aimee W.; Vervack, Ronald J., Jr.; Cassidy, Timothy A.; Sarantos, Menelaos

2014-01-01

The Mercury Atmospheric and Surface Composition Spectrometer on the MESSENGER spacecraft has observed calcium emission in Mercury's exosphere on a near-daily basis since March 2011. During MESSENGER's primary and first extended missions (March 2011 - March 2013) the dayside calcium exosphere was measured over eight Mercury years. We have simulated these data with a Monte Carlo model of exospheric source processes to show that (a) there is a persistent source of energetic calcium located in the dawn equatorial region, (b) there is a seasonal dependence in the calcium source rate, and (c) there are no obvious year-to-year variations in the near-surface dayside calcium exosphere. Although the precise mechanism responsible for ejecting the calcium has not yet been determined, the most likely process is the dissociation of Ca-bearing molecules produced in micrometeoroid impact plumes to form energetic, escaping calcium atoms.

The Acid Test: Calcium Signaling in the Skeletogenic Layer of Reef-Building Coral

Science.gov (United States)

Florn, A. M.

2016-02-01

Since the Industrial Revolution, carbon dioxide (CO2) emissions have increased more than 40%. This increased atmospheric CO2 drives ocean acidification and has potentially serious consequences for all marine life, especially calcifying organisms. The specific goal of this study was to examine calcium homeostasis and signaling dynamics within the skeletogenic tissue layers (calicodermal cells) of two coral species (Pavona maldivensis and Porites rus) at three pH treatments corresponding to present-future ocean acidification levels. Confocal microscopy techniques were used to analyze in vivo calcium dynamics of the calicodermal cells in Pavona maldivensis and Porites rus. The results show biological variation between the two reef-building coral species and their response to ocean acidification. Pavona maldivensis showed a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments, but not among the microcolonies. Porites rus did not show a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments or the microcolonies. Upon comparing the calcium response curves, the ionomycin-induced calcium response exhibited by Pavona maldivensis is phenomenologically similar to a calcium response that is commonly found in vertebrates. This well-studied phenomenon in vertebrate biology is known as store-operated calcium entry (SOCE) and is closely associated with the endoplasmic reticulum (ER) and mitochondria-associated endoplasmic reticulum (MAM) calcium stores. This study provides insight into the preliminary steps needed to understand in vivo calcium signaling in the calicodermis of reef-building coral and the associated consequences of ocean acidification.

Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells

International Nuclear Information System (INIS)

Elliott, M.E.; Siegel, F.L.; Hadjokas, N.E.; Goodfriend, T.L.

1985-01-01

We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of 45 Ca+2 into cells and increased efflux of 45 Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of 45 Ca+2 in the presence of EGTA and retarded uptake of 45 Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools

Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

Directory of Open Access Journals (Sweden)

Jiayang Qin

Full Text Available Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans.

Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

Science.gov (United States)

Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

2015-01-01

Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle)' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle)' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans.

Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

International Nuclear Information System (INIS)

Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

1986-01-01

Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase.

Science.gov (United States)

Mittal, Monica; Hasan, Mahmudul; Balagunaseelan, Navisraj; Fauland, Alexander; Wheelock, Craig; Rådmark, Olof; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

2017-08-01

A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca 2+ -dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general. Copyright © 2017 Elsevier B.V. All rights reserved.

Inotropic effect, binding properties, and calcium flux effects of the calcium channel agonist CGP 28392 in intact cultured embryonic chick ventricular cells

International Nuclear Information System (INIS)

Laurent, S.; Kim, D.; Smith, T.W.; Marsh, J.D.

1985-01-01

CGP 28392 is a recently described dihydropyridine derivative with positive inotropic properties. To study the mechanism of action of this putative calcium channel agonist, we have related the effects of CGP 28392 on contraction (measured with an optical video system) and radioactive calcium uptake to ligand-binding studies in cultured, spontaneously beating chick embryo ventricular cells. CGP 28392 produced a concentration-dependent increase in amplitude and velocity of contraction (EC 50 = 2 x 10(-7) M; maximum contractile effect = 85% of the calcium 3.6 mM response). Nifedipine produced a shift to the right of the concentration-effect curve for CGP 28392 without decreasing the maximum contractile response, suggesting competitive antagonism (pA2 = 8.3). Computer analysis of displacement of [ 3 H]nitrendipine binding to intact heart cells by unlabeled CGP 28392 indicated a K /sub D/ = 2.2 +/- 0.95 x 10(-7) M, in good agreement with the EC 50 for the inotropic effect. CGP 28392 increased the rate of radioactive calcium influx (+39% at 10 seconds) without altering beating rate, while nifedipine decreased radioactive calcium influx and antagonized the CGP 28392-induced increase in calcium influx. Our results indicate that, in intact cultured myocytes, CGP 28392 acts as a calcium channel agonist and competes for the dihydropyridine-binding site of the slow calcium channel. In contrast to calcium channel blockers, CGP 28392 increases calcium influx and enhances the contractile state

Calcium paradox and calcium entry blockers

NARCIS (Netherlands)

Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.

1984-01-01

Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture,

Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

Science.gov (United States)

Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

2004-12-01

In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signaling

Directory of Open Access Journals (Sweden)

Zhou eLi

2015-10-01

Full Text Available Endogenous polyamine (PA may play a critical role in tolerance to water stress in plants acting as a signaling molecule activator. Water stress caused increases in endogenous PA content in leaves, including putrescine (Put, spermidine (Spd, and spermine (Spm. Exogenous application of Spd could induce the instantaneous H2O2 burst and accumulation of cytosolic free Ca2+, and activate NADPH oxidase and CDPK gene expression in cells. To a great extent, PA biosynthetic inhibitor reduced the water stress-induced H2O2 accumulation, free cytosolic Ca2+ release, antioxidant enzyme activities and genes expression leading to aggravate water stress-induced oxidative damage, while these suppressing effects were alleviated by the addition of exogenous Spd, indicating PA was involved in water stress-induced H2O2 and cytosolic free Ca2+ production as well as stress tolerance. Dehydrin genes (Y2SK, Y2K, and SK2 were showed to be highly responsive to exogenous Spd. PA-induced antioxidant defense and dehydrin genes expression could be blocked by the scavenger of H2O2 and the inhibitors of H2O2 generation or Ca2+ channels blockers, a calmodulin antagonist, as well as the inhibitor of CDPK. These findings suggested that PA regulated tolerance to water stress in white clover associated with antioxidant defenses and dehydrins via involvement in the calcium messenger system and H2O2 signaling pathways. PA-induced H2O2 production required Ca2+ release, while PA-induced Ca2+ release was also essential for H2O2 production, suggesting an interaction between PA-induced H2O2 and Ca2+ signaling.

Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants.

Science.gov (United States)

Verma, Manish; Callio, Jason; Otero, P Anthony; Sekler, Israel; Wills, Zachary P; Chu, Charleen T

2017-11-15

Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis. SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome.

Science.gov (United States)

Lu, Simin; Kanekura, Kohsuke; Hara, Takashi; Mahadevan, Jana; Spears, Larry D; Oslowski, Christine M; Martinez, Rita; Yamazaki-Inoue, Mayu; Toyoda, Masashi; Neilson, Amber; Blanner, Patrick; Brown, Cris M; Semenkovich, Clay F; Marshall, Bess A; Hershey, Tamara; Umezawa, Akihiro; Greer, Peter A; Urano, Fumihiko

2014-12-09

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

Science.gov (United States)

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

Science.gov (United States)

Lee, Soh-Hyun; Ha, Sun-Ok; Koh, Ho-Jin; Kim, KilSoo; Jeon, Seon-Min; Choi, Myung-Sook; Kwon, Oh-Shin; Huh, Tae-Lin

2010-02-28

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Curry, Merril C.; Peters, Amelia A. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Kenny, Paraic A. [Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Roberts-Thomson, Sarah J. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Monteith, Gregory R., E-mail: gregm@uq.edu.au [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia)

2013-05-10

Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

The effects of irradiation on the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver

International Nuclear Information System (INIS)

Teshima, Teruki; Mori, Masaki; Honke, Yoshifumi

1983-01-01

The effects of irradiation on both the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver were investigated. The liver concentrations of corticosterone and cyclic nucleotides were measured by radioimmunoassay before and after the irradiation of 1,000 rad/l fraction. The glucocorticoid receptor in the liver cytosol was determined by the measurement of the cytosol binding to 3 H-dexamethasone. The cytosol and nuclear corticosterone levels reached a peak 1 day after the irradiation of the rat liver and declined to the control levels after 2 days. The increase in corticosterone levels may be due to the direct stimulation of the right adrenal gland and/ or the stress induced by the irradiation. The binding capacity of the glucocorticoid receptor in rat liver cytosol decreased to the minimum 1 day after the irradiation, and the recovery occurred at 4 days. The Kd value of the glucocorticoid receptor remained unchanged from 1 hour until 4 days but was high at 4 and 7 days. The distinctly increased levels of cyclic GMP in the rat liver were found from 1 hour through 7 days after the irradiation, while cyclic AMP did not change. The inversed relationship between the cytosol glucocorticoid receptor and corticosterone levels in cytosol and the nuclei indicates that the receptor-bound corticosterone in cytosol can be transferred to a nucleus and remain there in the presence of appropriate amounts of corticosterone in cytosol, after which the receptor is released from the nucleus into cytosol. The high Kd values observed 4 -- 7 days after the irradiation may be either due to the direct effect of irradiation or to the replenishment of the receptor with a low affinity. (author)

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum.

Science.gov (United States)

Dixit, Ajay Kumar; Jayabaskaran, Chelliah

2015-05-01

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca(2+) ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 μM and 120 μM indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 μM and 1.7 μM. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a α-helical rich protein. Calcium binding further increased the α-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility.

Directory of Open Access Journals (Sweden)

Jian Sun

2016-05-01

Full Text Available A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.

The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity

DEFF Research Database (Denmark)

Lund, Trine Meldgaard; Ploug, K.B.; Iversen, Anne

2015-01-01

-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown...... an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β...... to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release....

Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.

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Lee, Joo-Yong; Hwang, Jung Jin; Park, Mi-Ha; Koh, Jae-Young

2006-01-01

Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.

Calcium and Bone Metabolism Indices.

Science.gov (United States)

Song, Lu

2017-01-01

Calcium and inorganic phosphate are of critical importance for many body functions, thus the regulations of their plasma concentrations are tightly controlled by the concerted actions of reabsorption/excretion in the kidney, absorption in the intestines, and exchange from bone, the major reservoir for calcium and phosphate in the body. Parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) control calcium homeostasis, whereas PTH, 1,25(OH) 2 D, and bone-derived fibroblast growth factor 23 (FGF 23) control phosphate homeostasis. Hypoparathyroidism can cause hypocalcemia and hyperphosphatemia, whereas deficient vitamin D actions can cause osteomalacia in adults and rickets in children. Hyperparathyroidism, alternatively, can cause hypercalcemia and hypophosphatemia. Laboratory tests of calcium, phosphate, PTH, and 25-hydroxyvitamin D are very useful in the diagnosis of abnormalities associated with calcium and/or phosphate metabolisms. Bone is constantly remodeled throughout life in response to mechanical stress and a need for calcium in extracellular fluids. Metabolic bone diseases such as osteoporosis, osteomalacia in adults or rickets in children, and renal osteodystrophy develop when bone resorption exceeds bone formation. Bone turnover markers (BTM) such as serum N-terminal propeptide of type I procollagen (P1NP) and C-terminal collagen cross-link (CTX) may be useful in predicting future fracture risk or monitoring the response to anti-resorptive therapy. There is a need to standardize sample collection protocols because certain BTMs exhibit large circadian variations and tend to be influenced by food intakes. In the United States, a project to standardize BTM sample collection protocols and to establish the reference intervals for serum P1NP and serum CTX is ongoing. We anticipate the outcome of this project to shine lights on the standardization of BTM assays, sample collection protocols, reference intervals in relation to age, sex, and ethnic

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

NARCIS (Netherlands)

Backx, P. H.; de Tombe, P. P.; van Deen, J. H.; Mulder, B. J.; ter Keurs, H. E.

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

Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

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Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

2010-04-01

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

The interplay between HIF-1 and calcium signalling in cancer.

Science.gov (United States)

Azimi, Iman

2018-04-01

The interplay between hypoxia-inducible factor-1 (HIF-1) and calcium in cancer has begun to be unravelled with recent findings demonstrating the relationships between the two in different cancer types. This is an area of significance considering the crucial roles of both HIF-1 and calcium signalling in cancer progression and metastasis. This review summarises the experimental evidence of the crosstalk between HIF-1 and specific calcium channels, pumps and regulators in the context of cancer. HIF-1 as a master regulator of hypoxic transcriptional responses, mediates transcription of several calcium modulators. On the other hand, specific calcium channels and pumps regulate HIF-1 activity through controlling its transcription, translation, stabilisation, or nuclear translocation. Identifying the interplay between HIF-1 and components of the calcium signal will give new insights into mechanisms underlying cellular responses to physiological and pathophysiological cues, and may provide novel and more efficient therapeutic strategies for the control of cancer progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

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Strong, Averey D; Daniels, Richard L

2017-08-02

The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

Science.gov (United States)

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

Science.gov (United States)

Yao, Zongli; Guo, Wenfei; Lai, Qifang; Shi, Jianquan; Zhou, Kai; Qi, Hongfang; Lin, Tingting; Li, Ziniu; Wang, Hui

2016-01-01

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

Science.gov (United States)

Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

2018-06-09

Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

Exopolysaccharides regulate calcium flow in cariogenic biofilms

Science.gov (United States)

Varenganayil, Muth M.; Decho, Alan W.

2017-01-01

Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries. PMID:29023506

Exopolysaccharides regulate calcium flow in cariogenic biofilms.

Directory of Open Access Journals (Sweden)

Monika Astasov-Frauenhoffer

Full Text Available Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya's agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC. Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

Science.gov (United States)

Hérouart, D; Van Montagu, M; Inzé, D

1994-03-01

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

Effect of dairy calcium or supplementary calcium intake on postprandial fat metabolism, appetite, and subsequent energy intake

DEFF Research Database (Denmark)

Lorenzen, J.K.; Nielsen, S.; Holst, J.J.

2007-01-01

postprandially. Results: Dairy calcium significantly diminished the postprandial lipid response. The baseline adjusted area under the curve for chylomicron triacylglycerol was approximate to 17% lower after the MC meal (P = 0.02) and approximate to 19% lower after the HC meal (P = 0.007) than after the LC meal...... and approximate to 15% lower after the MC meal (P = 0.0495) and approximate to 17% lower after the HC meal (P = 0.02) than after the Suppl meal. No consistent effects of calcium on appetite sensation, or on energy intake at the subsequent meal, or on the postprandial responses of cholecystokinin, glucagon...

Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells

NARCIS (Netherlands)

Gankema, H. S.; Groen, A. K.; Wanders, R. J.; Tager, J. M.

1983-01-01

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins,

Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

DEFF Research Database (Denmark)

Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

2009-01-01

the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

International Nuclear Information System (INIS)

Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

1987-01-01

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

Contracture of Slow Striated Muscle during Calcium Deprivation

Science.gov (United States)

Irwin, Richard L.; Hein, Manfred M.

1963-01-01

When deprived of calcium the slow striated muscle fibers of the frog develop reversible contractures in either hypertonic or isotonic solutions. While calcium deprivation continues because of a flowing calcium-free solution the muscles relax slowly and completely. Restoration of calcium during contracture relaxes the muscle promptly to initial tension. When relaxed during calcium lack the return of calcium does not change tension and the muscle stays relaxed. When contractures are induced by solutions containing small amounts of calcium relaxation does not occur or requires several hours. The rate of tension development depends upon the rate at which calcium moves outward since the contractures develop slower in low concentrations of calcium and are absent or greatly slowed in a stagnant calcium-free solution. Withdrawal of calcium prevents the contractile responses to ACh, KCl, or electrical stimulation through the nerve. Muscles return to their original excitability after calcium is restored. Origin of the contractures is unrelated to nerve activity since they are maximal during transmission failure from calcium lack, occur in denervated muscles, and are not blocked by high concentrations of d-tubocurarine, procaine, or atropine. The experiments also indicate that the contractures do not originate from repetitive activity of muscle membranes. The findings are most simply explained by relating the outward movement of calcium as a link for initiating contraction in slow type striated muscle. PMID:14065284

Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism.

Science.gov (United States)

Spyridopoulos, I; Wischhusen, J; Rabenstein, B; Mayer, P; Axel, D I; Fröhlich, K U; Karsch, K R

2001-03-01

Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with

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

Science.gov (United States)

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

2017-08-01

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

Systemic antibody response to nano-size calcium phospate biocompatible adjuvant adsorbed HEV-71 killed vaccine

OpenAIRE

Saeed, Mohamed Ibrahim; Omar, Abd Rahaman; Hussein, Mohd Zobir; Elkhidir, Isam Mohamed; Sekawi, Zamberi

2015-01-01

Purpose Since 1980s, human enterovirus-71 virus (HEV-71) is one of the common infectious disease in Asian Pacific region since late 1970s without effective commercial antiviral or protective vaccine is unavailable yet. The work examines the role of vaccine adjuvant particle size and the route of administration on postvaccination antibody response towards HEV-71 vaccine adsorbed to calcium phosphate (CaP) adjuvant. Materials and Methods First, CaP nano-particles were compared to a commercial m...

The chaperone BAG6 captures dislocated glycoproteins in the cytosol.

Directory of Open Access Journals (Sweden)

Jasper H L Claessen

Full Text Available Secretory and membrane (glycoproteins are subject to quality control in the endoplasmic reticulum (ER to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle.

Mammary-Specific Ablation of the Calcium-Sensing Receptor During Lactation Alters Maternal Calcium Metabolism, Milk Calcium Transport, and Neonatal Calcium Accrual

Science.gov (United States)

Mamillapalli, Ramanaiah; VanHouten, Joshua; Dann, Pamela; Bikle, Daniel; Chang, Wenhan; Brown, Edward

2013-01-01

To meet the demands for milk calcium, the lactating mother adjusts systemic calcium and bone metabolism by increasing dietary calcium intake, increasing bone resorption, and reducing renal calcium excretion. As part of this adaptation, the lactating mammary gland secretes PTHrP into the maternal circulation to increase bone turnover and mobilize skeletal calcium stores. Previous data have suggested that, during lactation, the breast relies on the calcium-sensing receptor (CaSR) to coordinate PTHrP secretion and milk calcium transport with calcium availability. To test this idea genetically, we bred BLG-Cre mice with CaSR-floxed mice to ablate the CaSR specifically from mammary epithelial cells only at the onset of lactation (CaSR-cKO mice). Loss of the CaSR in the lactating mammary gland did not disrupt alveolar differentiation or milk production. However, it did increase the secretion of PTHrP into milk and decreased the transport of calcium from the circulation into milk. CaSR-cKO mice did not show accelerated bone resorption, but they did have a decrease in bone formation. Loss of the mammary gland CaSR resulted in hypercalcemia, decreased PTH secretion, and increased renal calcium excretion in lactating mothers. Finally, loss of the mammary gland CaSR resulted in decreased calcium accrual by suckling neonates, likely due to the combination of increased milk PTHrP and decreased milk calcium. These results demonstrate that the mammary gland CaSR coordinates maternal bone and calcium metabolism, calcium transport into milk, and neonatal calcium accrual during lactation. PMID:23782944

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Science.gov (United States)

Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

2017-06-15

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

Science.gov (United States)

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.

RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

Science.gov (United States)

Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

2016-05-27

Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates

Directory of Open Access Journals (Sweden)

G.A. Caprara

2014-10-01

Full Text Available Growth-associated protein 43 (GAP43, is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes, and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.

Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

International Nuclear Information System (INIS)

Sherley, J.L.; Kelly, T.J.

1988-01-01

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity

Nitrate Activation of Cytosolic Protein Kinases Diverts Photosynthetic Carbon from Sucrose to Amino Acid Biosynthesis

Science.gov (United States)

Champigny, Marie-Louise; Foyer, Christine

1992-01-01

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis. PMID:16653003

Profiling of cytosolic and mitochondrial H2O2 production using the H2O2-sensitive protein HyPer in LPS-induced microglia cells.

Science.gov (United States)

Park, Junghyung; Lee, Seunghoon; Lee, Hyun-Shik; Lee, Sang-Rae; Lee, Dong-Seok

2017-07-27

Dysregulation of the production of pro-inflammatory mediators in microglia exacerbates the pathologic process of neurodegenerative disease. ROS actively affect microglia activation by regulating transcription factors that control the expression of pro-inflammatory genes. However, accurate information regarding the function of ROS in different subcellular organelles has not yet been established. Here, we analyzed the pattern of cytosolic and mitochondrial H 2 O 2 formation in LPS-activated BV-2 microglia using the H 2 O 2- sensitive protein HyPer targeted to specific subcellular compartments. Our results show that from an early time, cytosolic H 2 O 2 started increasing constantly, whereas mitochondrial H 2 O 2 rapidly increased later. In addition, we found that MAPK affected cytosolic H 2 O 2 , but not mitochondrial H 2 O 2 . Consequently, our study provides the basic information about subcellular H 2 O 2 generation in activated microglia, and a useful tool for investigating molecular targets that can modulate neuroinflammatory responses. Copyright © 2017 Elsevier B.V. All rights reserved.

SH Oxidation Stimulates Calcium Release Channels (Ryanodine Receptors From Excitable Cells

Directory of Open Access Journals (Sweden)

CECILIA HIDALGO

2000-01-01

Full Text Available The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub µM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed

Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

Science.gov (United States)

Hyrc, Krzysztof L; Minta, Akwasi; Escamilla, P Rogelio; Chan, Patrick P L; Meshik, Xenia A; Goldberg, Mark P

2013-10-01

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca(2+)-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (calcium indicators. Copyright © 2013 Elsevier Ltd. All rights reserved.

The relationship between insulin resistance and the change of cytosol free calcium concentration in gestational diabetes mellitus

International Nuclear Information System (INIS)

Li Pu; Zheng Lei; Cen Rongguang; Song Yangxiu

2004-01-01

To investigate the relationship between the insulin resistance (IR) and abnormalities of cellular calcium metabolism in gestational diabetes mellitus, the changes in the [Ca 2+ ]i and the insulin receptor tyrosine kinase activity in circulating erythrocytes of 32 cases gestational diabetes were compared with those of 47 normal pregnant and 43 non pregnant women. The level of [Ca 2+ ]i in circulating erythrocyte in gestational diabetes mellitus women was significantly higher than that in the pregnant and non pregnant women (P 2+ ]i in circulating erythrocytes in gestational diabetes mellitus was positively correlated with fasting blood glucose and fasting insulin, negatively with the insulin receptor tyrosine kisase activity (P 2+ ]i level during the gastational period might be one of the possible factor in the insulin resistance of gestaional diabetes mellitus. (authors)

The effects of in vitro exposure to white spirit on [Ca2+] in synaptosomes from rats exposed prenatally to white spirit

DEFF Research Database (Denmark)

Edelfors, S.; Hass, Ulla; Ravn-Jonsen, A.

1999-01-01

Female rats were exposed to white spirit (400 and 800 ppm for 6 hr/day) at day 7-20 during pregnancy. Thirty-five days after birth all female offspring were sacrificed, the brains removed, and the synaptosomal fractions prepared for in vitro studies. The cytosolic calcium concentration was measured...... using the FURA-2 technique. The results show that cytosolic calcium was increased in synaptosomes from rats exposed to white spirit prenatally compared to synaptosomes from unexposed rats. When synaptosomes were exposed to white spirit in vitro, the cytosolic calcium concentration changes were identical...... in all groups of rats. The membrane leakage measured as FURA-2 leakage from the synaptosomes identical in all three groups of animals. The results suggest that prenatal exposure to white spirit induces long-lasting and possibly irreversible changes in calcium homeostasis in the rat nervous system....

Development of amphiphilic gamma-PGA-nanoparticle based tumor vaccine: potential of the nanoparticulate cytosolic protein delivery carrier.

Science.gov (United States)

Yoshikawa, Tomoaki; Okada, Naoki; Oda, Atsushi; Matsuo, Kazuhiko; Matsuo, Keisuke; Mukai, Yohei; Yoshioka, Yasuo; Akagi, Takami; Akashi, Mitsuru; Nakagawa, Shinsaku

2008-02-08

Nanoscopic therapeutic systems that incorporate biomacromolecules, such as protein and peptides, are emerging as the next generation of nanomedicine aimed at improving the therapeutic efficacy of biomacromolecular drugs. In this study, we report that poly(gamma-glutamic acid)-based nanoparticles (gamma-PGA NPs) are excellent protein delivery carriers for tumor vaccines that delivered antigenic proteins to antigen-presenting cells and elicited potent immune responses. Importantly, gamma-PGA NPs efficiently delivered entrapped antigenic proteins through cytosolic translocation from the endosomes, which is a key process of gamma-PGA NP-mediated anti-tumor immune responses. Our findings suggest that the gamma-PGA NP system is suitable for the intracellular delivery of protein-based drugs as well as tumor vaccines.

[Calcium suppletion for patients who use gastric acid inhibitors: calcium citrate or calcium carbonate?].

NARCIS (Netherlands)

Jonge, H.J. de; Gans, R.O.; Huls, G.A.

2012-01-01

Various calcium supplements are available for patients who have an indication for calcium suppletion. American guidelines and UpToDate recommend prescribing calcium citrate to patients who use antacids The rationale for this advice is that water-insoluble calcium carbonate needs acid for adequate

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

KAUST Repository

Schmöckel, Sandra M.

2015-02-27

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

KAUST Repository

Schmö ckel, Sandra M.; Garcia, Alexandre F.; Berger, Bettina; Tester, Mark A.; Webb, Alex A. R.; Roy, Stuart J.

2015-01-01

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

Science.gov (United States)

Mandal, Amritlal; Delamere, Nicholas A.

2010-01-01

Purpose. Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90RSK and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Methods. Cytoplasmic calcium concentration ([Ca2+]i) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. Results. The increase of HP caused an immediate slow increase in [Ca2+]i. The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 μM) or dantrolene (25 μM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Conclusions. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase. PMID:20071675

Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

Science.gov (United States)

Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

2015-09-01

The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia.

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity.

Science.gov (United States)

Ali, Rashid; Ma, Wei; Lemtiri-Chlieh, Fouad; Tsaltas, Dimitrios; Leng, Qiang; von Bodman, Susannne; Berkowitz, Gerald A

2007-03-01

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.

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

Science.gov (United States)

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.

Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

Energy Technology Data Exchange (ETDEWEB)

Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

2007-08-01

R1 function upon the calcium-dependent activation of CaM. These results indicate that occupancy of the N-terminal domain calcium binding sites in CaM bound to the identified CaM-binding sequence K3619-N3643 is the conformational switch responsible for the CaM-dependent modulation of the RyR1 calcium release channel.

A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

Science.gov (United States)

Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A; Portman, Jonathan L; Sauer, John-Demian

2017-03-21

Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis , are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

DEFF Research Database (Denmark)

Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

2007-01-01

waves sweeping through the cytoplasm when the SR is stimulated to release calcium. A rise in cyclic guanosine monophosphate (cGMP) leads to the experimentally observed transition from waves to whole-cell calcium oscillations. At the same time membrane potential starts to oscillate and the frequency...... approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion. Key words: Vasomotion, Chloride channel, cGMP, Mathematical model, Calcium waves....

21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium pantothenate, calcium chloride double salt... FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.330 Calcium pantothenate, calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may...

Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

Science.gov (United States)

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. © 2015 by the Society for Experimental Biology and Medicine.

Calcium phosphate thin films enhance the response of human mesenchymal stem cells to nanostructured titanium surfaces

Directory of Open Access Journals (Sweden)

Mura M McCafferty

2014-05-01

Full Text Available The development of biomaterial surfaces possessing the topographical cues that can promote mesenchymal stem cell recruitment and, in particular, those capable of subsequently directing osteogenic differentiation is of increasing importance for the advancement of tissue engineering. While it is accepted that it is the interaction with specific nanoscale topography that induces mesenchymal stem cell differentiation, the potential for an attendant bioactive chemistry working in tandem with such nanoscale features to enhance this effect has not been considered to any great extent. This article presents a study of mesenchymal stem cell response to conformal bioactive calcium phosphate thin films sputter deposited onto a polycrystalline titanium nanostructured surface with proven capability to directly induce osteogenic differentiation in human bone marrow–derived mesenchymal stem cells. The sputter deposited surfaces supported high levels of human bone marrow–derived mesenchymal stem cell adherence and proliferation, as determined by DNA quantification. Furthermore, they were also found to be capable of directly promoting significant levels of osteogenic differentiation. Specifically, alkaline phosphatase activity, gene expression and immunocytochemical localisation of key osteogenic markers revealed that the nanostructured titanium surfaces and the bioactive calcium phosphate coatings could direct the differentiation towards an osteogenic lineage. Moreover, the addition of the calcium phosphate chemistry to the topographical profile of the titanium was found to induce increased human bone marrow–derived mesenchymal stem cell differentiation compared to that observed for either the titanium or calcium phosphate coating without an underlying nanostructure. Hence, the results presented here highlight that a clear benefit can be achieved from a surface engineering strategy that combines a defined surface topography with an attendant, conformal

Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo

Science.gov (United States)

Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T

2013-01-01

All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pHcyto) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pHcyto shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pHcyto. Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤4 s) trains of action potentials but did buffer slow (∼60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca2+ increase upon stimulation, and partial inhibition of the plasma membrane Ca2+-ATPase, a Ca2+/H+ exchanger, attenuated pHcyto shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (∼0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pHcyto shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pHcyto shifts cannot be dismissed as artifacts of ex vivo preparations. PMID:23401611

[Optimization of riboflavin sodium phosphate loading to calcium alginate floating microspheres by response surface methodology].

Science.gov (United States)

Zhang, An-yang; Fan, Tian-yuan

2009-12-18

To investigate the preparation, optimization and in vitro properties of riboflavin sodium phosphate floating microspheres. The floating microspheres composed of riboflavin sodium phosphate and calcium alginate were prepared using ion gelatin-oven drying method. The properties of the microspheres were investigated, including the buoyancy, release, appearance and entrapment efficiency. The formulation was optimized by response surface methodology (RSM). The optimized microspheres were round. The entrapment efficiency was 57.49%. All the microspheres could float on the artificial gastric juice over 8 hours. The release of the drug from the microspheres complied with Fick's diffusion.

Inherited Variation in Cytokine, Acute Phase Response, and Calcium Metabolism Genes Affects Susceptibility to Infective Endocarditis

Directory of Open Access Journals (Sweden)

Anastasia V. Ponasenko

2017-01-01

Full Text Available Infective endocarditis (IE is a septic inflammation of the endocardium. Recognition of microbial patterns, cytokine and acute phase responses, hemostasis features, and alterations in plasma lipid and calcium profile all have been reported to affect pathogenesis and clinical course of IE. Having recruited 123 patients with IE and 300 age-, sex-, and ethnicity-matched healthy blood donors, we profiled their genomic DNA for 35 functionally significant polymorphisms within the 22 selected genes involved in the abovementioned pathways, with the further genetic association analysis. We found that the G/A genotype of the rs1143634 polymorphism within the IL1B gene, the G/T genotype of the rs3212227 polymorphism within the IL12B gene, the A/G genotype of the rs1130864 polymorphism within the CRP gene, and the G allele of the rs1801197 polymorphism within the CALCR gene were associated with a decreased risk of IE whereas the T/T genotype of the rs1205 polymorphism within the CRP gene was associated with a higher risk of IE. Furthermore, heterozygous genotypes of the rs1143634 and rs3212227 polymorphisms were associated with the higher plasma levels of IL-1β and IL-12, respectively. Our results indicate that inherited variation in the cytokine, acute phase response, and calcium metabolism pathways may be linked to IE.

Inherited Variation in Cytokine, Acute Phase Response, and Calcium Metabolism Genes Affects Susceptibility to Infective Endocarditis

Science.gov (United States)

Rutkovskaya, Natalia V.; Kondyukova, Natalia V.; Odarenko, Yuri N.; Kazachek, Yana V.; Tsepokina, Anna V.; Barbarash, Leonid S.

2017-01-01

Infective endocarditis (IE) is a septic inflammation of the endocardium. Recognition of microbial patterns, cytokine and acute phase responses, hemostasis features, and alterations in plasma lipid and calcium profile all have been reported to affect pathogenesis and clinical course of IE. Having recruited 123 patients with IE and 300 age-, sex-, and ethnicity-matched healthy blood donors, we profiled their genomic DNA for 35 functionally significant polymorphisms within the 22 selected genes involved in the abovementioned pathways, with the further genetic association analysis. We found that the G/A genotype of the rs1143634 polymorphism within the IL1B gene, the G/T genotype of the rs3212227 polymorphism within the IL12B gene, the A/G genotype of the rs1130864 polymorphism within the CRP gene, and the G allele of the rs1801197 polymorphism within the CALCR gene were associated with a decreased risk of IE whereas the T/T genotype of the rs1205 polymorphism within the CRP gene was associated with a higher risk of IE. Furthermore, heterozygous genotypes of the rs1143634 and rs3212227 polymorphisms were associated with the higher plasma levels of IL-1β and IL-12, respectively. Our results indicate that inherited variation in the cytokine, acute phase response, and calcium metabolism pathways may be linked to IE. PMID:28659664

Determination of percent calcium carbonate in calcium chromate

International Nuclear Information System (INIS)

Middleton, H.W.

1979-01-01

The precision, accuracy and reliability of the macro-combustion method is superior to the Knorr alkalimetric method, and it is faster. It also significantly reduces the calcium chromate waste accrual problem. The macro-combustion method has been adopted as the official method for determination of percent calcium carbonate in thermal battery grade anhydrous calcium chromate and percent calcium carbonate in quicklime used in the production of calcium chromate. The apparatus and procedure can be used to measure the percent carbonate in inorganic materials other than calcium chromate. With simple modifications in the basic apparatus and procedure, the percent carbon and hydrogen can be measured in many organic material, including polymers and polymeric formulations. 5 figures, 5 tables

Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: A 44Ca stable isotope study

International Nuclear Information System (INIS)

Chandra, S.; Fullmer, C.S.; Smith, C.A.; Wasserman, R.H.; Morrison, G.H.

1990-01-01

The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described

Death Don't Have No Mercy and Neither Does Calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and Innate Immunity[W

Science.gov (United States)

Ali, Rashid; Ma, Wei; Lemtiri-Chlieh, Fouad; Tsaltas, Dimitrios; Leng, Qiang; von Bodman, Susannne; Berkowitz, Gerald A.

2007-01-01

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca2+, nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca2+ into cells and provide a model linking this Ca2+ current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca2+ current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen–associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca2+ current, which may be linked to NO generation due to buildup of cytosolic Ca2+/calmodulin. PMID:17384171

Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

Science.gov (United States)

Kojer, Kerstin; Bien, Melanie; Gangel, Heike; Morgan, Bruce; Dick, Tobias P; Riemer, Jan

2012-01-01

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (EGSH) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with EGSH-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of EGSH in the IMS, thus explaining a steady-state EGSH in the IMS which is similar to the cytosol. Moreover, we show that the local EGSH contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells. PMID:22705944

Calcium signalling silencing in atrial fibrillation.

Science.gov (United States)

Greiser, Maura

2017-06-15

Subcellular calcium signalling silencing is a novel and distinct cellular and molecular adaptive response to rapid cardiac activation. Calcium signalling silencing develops during short-term sustained rapid atrial activation as seen clinically during paroxysmal atrial fibrillation (AF). It is the first 'anti-arrhythmic' adaptive response in the setting of AF and appears to counteract the maladaptive changes that lead to intracellular Ca 2+ signalling instability and Ca 2+ -based arrhythmogenicity. Calcium signalling silencing results in a failed propagation of the [Ca 2+ ] i signal to the myocyte centre both in patients with AF and in a rabbit model. This adaptive mechanism leads to a substantial reduction in the expression levels of calcium release channels (ryanodine receptors, RyR2) in the sarcoplasmic reticulum, and the frequency of Ca 2+ sparks and arrhythmogenic Ca 2+ waves remains low. Less Ca 2+ release per [Ca 2+ ] i transient, increased fast Ca 2+ buffering strength, shortened action potentials and reduced L-type Ca 2+ current contribute to a substantial reduction of intracellular [Na + ]. These features of Ca 2+ signalling silencing are distinct and in contrast to the changes attributed to Ca 2+ -based arrhythmogenicity. Some features of Ca 2+ signalling silencing prevail in human AF suggesting that the Ca 2+ signalling 'phenotype' in AF is a sum of Ca 2+ stabilizing (Ca 2+ signalling silencing) and Ca 2+ destabilizing (arrhythmogenic unstable Ca 2+ signalling) factors. Calcium signalling silencing is a part of the mechanisms that contribute to the natural progression of AF and may limit the role of Ca 2+ -based arrhythmogenicity after the onset of AF. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Accumulation of free oligosaccharides and tissue damage in cytosolic α-mannosidase (Man2c1)-deficient mice.

Science.gov (United States)

Paciotti, Silvia; Persichetti, Emanuele; Klein, Katharina; Tasegian, Anna; Duvet, Sandrine; Hartmann, Dieter; Gieselmann, Volkmar; Beccari, Tommaso

2014-04-04

Free Man(7-9)GlcNAc2 is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man5GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man(8-9)GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man(8-9)GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman's capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.

A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

Science.gov (United States)

Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

2006-01-01

Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

DEFF Research Database (Denmark)

Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

2007-01-01

approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... channels on the cell surface stimulating synchronized release of SR-calcium and inducing the shift from waves to whole-cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated...

Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields.

Science.gov (United States)

Hanna, Hanna; Andre, Franck M; Mir, Lluis M

2017-04-20

Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions. One or several electric pulses of 100 μs were used to induce Ca 2+ spikes caused by the penetration of Ca 2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked. According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca 2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability. An easy way to control Ca 2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca 2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca 2+ spikes allowed us to mimic and regulate the Ca 2+ oscillations in these cells. Since microsecond electric pulse delivery

Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes

DEFF Research Database (Denmark)

Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.

2014-01-01

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell...... death and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labelled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining...... a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/ [lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD...

A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei.

OpenAIRE

Guarnieri, C; Flamigni, F; Caldarera, C M

1980-01-01

An alpha-tocopherol-binding macromolecule was isolated from the heart cytosol of rabbits fed for 1 month with an alpha-tocopherol-deficient diet. The amount of [3H]-tocopherol bound to nuclear chromatin was increased when the alpha-tocopherol-deficient heart nuclei were incubated in the presence of [3H]tocopherol-cytosol complex. In this condition, large amounts of [3H]tocopherol were associated with a subnuclear fraction that contained non-histone acidic proteins.

Detection of photospheric calcium in a DBA white dwarf

International Nuclear Information System (INIS)

Kenyon, S.J.; Shipman, H.L.; Sion, E.M.; Aannestad, P.A.

1988-01-01

The detection of photospheric calcium absorption lines in the white dwarf star G200-39 (DBAZ4) is reported. The abundance of calcium relative to that of hydrogen is approximately solar, a result which lends support to the hypothesis that accretion of interstellar matter is responsible for hybrid composition white dwarfs. 21 references

Evolution of the Calcium Paradigm: The Relation between Vitamin D, Serum Calcium and Calcium Absorption

Directory of Open Access Journals (Sweden)

Borje E. Christopher Nordin

2010-09-01

Full Text Available Osteoporosis is the index disease for calcium deficiency, just as rickets/osteomalacia is the index disease for vitamin D deficiency, but there is considerable overlap between them. The common explanation for this overlap is that hypovitaminosis D causes malabsorption of calcium which then causes secondary hyperparathyroidism and is effectively the same thing as calcium deficiency. This paradigm is incorrect. Hypovitaminosis D causes secondary hyperparathyroidism at serum calcidiol levels lower than 60 nmol/L long before it causes malabsorption of calcium because serum calcitriol (which controls calcium absorption is maintained until serum calcidiol falls below 20 nmol/L. This secondary hyperparathyroidism, probably due to loss of a “calcaemic” action of vitamin D on bone first described in 1957, destroys bone and explains why vitamin D insufficiency is a risk factor for osteoporosis. Vitamin D thus plays a central role in the maintenance of the serum (ionised calcium, which is more important to the organism than the preservation of the skeleton. Bone is sacrificed when absorbed dietary calcium does not match excretion through the skin, kidneys and bowel which is why calcium deficiency causes osteoporosis in experimental animals and, by implication, in humans.

Calcium absorption

International Nuclear Information System (INIS)

Carlmark, B.; Reizenstein, P.; Dudley, R.A.

1976-01-01

The methods most commonly used to measure the absorption and retention of orally administered calcium are reviewed. Nearly all make use of calcium radioisotopes. The magnitude of calcium absorption and retention depends upon the chemical form and amount of calcium administered, and the clinical and nutritional status of the subject; these influences are briefly surveyed. (author)

Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

Science.gov (United States)

Baldissera, Matheus D; Souza, Carine F; Júnior, Guerino B; Verdi, Camila Marina; Moreira, Karen L S; da Rocha, Maria Izabel U M; da Veiga, Marcelo L; Santos, Roberto C V; Vizzotto, Bruno S; Baldisserotto, Bernardo

2017-09-01

Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na + , K + -ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na + , K + -ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8+ T cell responses in mice

Science.gov (United States)

Zhou, Weibin; Moguche, Albanus; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-01-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration “cold chain”. Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8+ T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8+ T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. PMID:24275478

Studies of the activity of cytosol on the mixed disulfide bond formed by proteins and radioprotector mercaptoethylguanidine

Energy Technology Data Exchange (ETDEWEB)

Horvath, M [National Inst. of Oncology, Budapest (Hungary); Holland, J [Orszagos Onkologiai Intezet, Budapest (Hungary)

1979-01-01

The cytoplasm of normal and tumorous rat liver cells contains a heat-resistant compound with reducing ability to break the mixed disulfide bond of albumin-/sup 14/C-mercaptoethylguanidine. The reducing activity of cytosol is destoryed by 1000 krd /sup 60/Co-gamma-ray doses in diluted solution. In vivo supralethal of rats does not affect the activity of cytosol prepared from liver cells.

Calcium-deficiency assessment and biomarker identification by an integrated urinary metabonomics analysis

Science.gov (United States)

2013-01-01

Background Calcium deficiency is a global public-health problem. Although the initial stage of calcium deficiency can lead to metabolic alterations or potential pathological changes, calcium deficiency is difficult to diagnose accurately. Moreover, the details of the molecular mechanism of calcium deficiency remain somewhat elusive. To accurately assess and provide appropriate nutritional intervention, we carried out a global analysis of metabolic alterations in response to calcium deficiency. Methods The metabolic alterations associated with calcium deficiency were first investigated in a rat model, using urinary metabonomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and multivariate statistical analysis. Correlations between dietary calcium intake and the biomarkers identified from the rat model were further analyzed to confirm the potential application of these biomarkers in humans. Results Urinary metabolic-profiling analysis could preliminarily distinguish between calcium-deficient and non-deficient rats after a 2-week low-calcium diet. We established an integrated metabonomics strategy for identifying reliable biomarkers of calcium deficiency using a time-course analysis of discriminating metabolites in a low-calcium diet experiment, repeating the low-calcium diet experiment and performing a calcium-supplement experiment. In total, 27 biomarkers were identified, including glycine, oxoglutaric acid, pyrophosphoric acid, sebacic acid, pseudouridine, indoxyl sulfate, taurine, and phenylacetylglycine. The integrated urinary metabonomics analysis, which combined biomarkers with regular trends of change (types A, B, and C), could accurately assess calcium-deficient rats at different stages and clarify the dynamic pathophysiological changes and molecular mechanism of calcium deficiency in detail. Significant correlations between calcium intake and two biomarkers, pseudouridine (Pearson

Association of estrogen receptor-alpha and vitamin D receptor genotypes with therapeutic response to calcium in postmenopausal Chinese women

Institute of Scientific and Technical Information of China (English)

Zhen-lin ZHANG; Yue-juan QIN; Qi-ren HUANG; Jin-wei HE; Miao LI; Qi ZHOU; Yun-qiu HU; Yu-juan LIU

2004-01-01

AIM: To investigate the correlation between calcium treatment in postmenopausal women and estrogen receptoralpha (ER-alpha) Xba Ⅰ and Pvu Ⅱ genotype and vitamin D receptor (VDR) Apa Ⅰ genotype. METHODS: One hundred fifteen postmenopausal Chinese women of Han population were enrolled and treated with calcichew-D3(1000 mg calcium and 400 U vitamin D3) daily for 1 year. At entry and after 1 year treatment, the bone mineral density (BMD), serum and urinary bone turnover biochemical markers were evaluated. ER-alpha and VDR genotype were analyzed using PCR-restriction fragment length polymorphism. RESULTS: After 1 year of calcium supplementation, a significant increase of BMD and a marked reduction in serum ALP and PTH levels, and a significant increase of serum 25-(OH) vitamin D level were observed (P＜0.01 or P＜0.05). At entry and after 1 year of treatment, no significant association was found between Xba Ⅰ, Pvu Ⅱ, and Apa Ⅰ genotypes and BMD in L1-4,Neck, and Troch, and all bone turnover marker levels. However, the percentage of change (median, QR) in Neck BMD was significantly different in homozygous XX [-4.14 (from -6.54 to -1.34)] in comparison with Xx [1.72(from -1.12 to 3.20)] (P＜0.001) or xx [1.22 (from -1.74 to 3.06)] Xba Ⅰ ER-alpha genotype (P=0.001).CONCLUSION: Women with ER-α Xba Ⅰ genotype XX may have a higher risk of relatively fast bone mass loss in femoral neck after menopause and that they may have a poor responsiveness to calcium supplementation. The changes in BMD are not associated with ER-alpha Pvu Ⅱ genotype and VDR Apa Ⅰ genotype after 1 year of calcium supplementation.

Radioisotope 45Ca labeling four calcium chemical compounds and tracing calcium bioavailability

International Nuclear Information System (INIS)

Zheng Hui; Zhen Rong; Niu Huisheng; Li Huaifen

2004-01-01

Objective: To build up a new method of the radioisotope 45 Ca labeling four calcium chemical compounds, observe and tracing bioavailability change of calcium labeled with radioisotope 45 Ca. Methods: The calcium gluconate (Ca-Glu), calcium citrate (Ca-Cit), calcium carbonate (Ca-Car) and calcium L-threonate (Ca-Thr)were labeled by radioisotope 45 Ca. Four calcium chemical compounds of 45 Ca labeling were used of calcium content 200 mg/kg in the rats and measure the absorption content and bioavailability of calcium in tissue of heart, lever spleen, stomach, kidney, brain, intestine, whole blood, urine, faeces. Results: 1) Radioisotope 45 Ca labeling calcium chemical compound has high radio intensity, more steady standard curve and recover rate. 2) The absorption of organic calcium chemical compounds is higher than the inorganic calcium chemical compound in the study of calcium bioavailability. Conclusion: The method of tracing with radioisotope 45 Ca labeling calcium chemical compounds has the characteristic of the sensitive, objective, accurate and steady in the study of calcium bioavailability

The impact of calcium assay change on a local adjusted calcium equation.

Science.gov (United States)

Davies, Sarah L; Hill, Charlotte; Bailey, Lisa M; Davison, Andrew S; Milan, Anna M

2016-03-01

Deriving and validating local adjusted calcium equations is important for ensuring appropriate calcium status classification. We investigated the impact on our local adjusted calcium equation of a change in calcium method by the manufacturer from cresolphthalein complexone to NM-BAPTA. Calcium and albumin results from general practice requests were extracted from the Laboratory Information Management system for a three-month period. Results for which there was evidence of disturbance in calcium homeostasis were excluded leaving 13,482 sets of results for analysis. The adjusted calcium equation was derived following least squares regression analysis of total calcium on albumin and normalized to the mean calcium concentration of the data-set. The revised equation (NM-BAPTA calcium method) was compared with the previous equation (cresolphthalein complexone calcium method). The switch in calcium assay resulted in a small change in the adjusted calcium equation but was not considered to be clinically significant. The calcium reference interval differed from that proposed by Pathology Harmony in the UK. Local adjusted calcium equations should be re-assessed following changes in the calcium method. A locally derived reference interval may differ from the consensus harmonized reference interval. © The Author(s) 2015.

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

Science.gov (United States)

Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

2017-06-15

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

Directory of Open Access Journals (Sweden)

Shilpee Srivastava

Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

Activity-Dependent Calcium, Oxygen, and Vascular Responses in a Mouse Model of Familial Hemiplegic Migraine Type 1

DEFF Research Database (Denmark)

Khennouf, Lila; Gesslein, Bodil; Lind, Barbara Lykke

2016-01-01

it with assessment of local field potentials by electrophysiological recordings, cerebral blood flow by laser Doppler flowmetry, and oxygen consumption with measurement of the oxygen tissue tension. Results: During spreading depression, the evoked increase in cytosolic Ca2+ was larger and faster in FHM1 mice than...... wild-type (WT) mice. It was accompanied by larger increases in oxygen consumption in FHM1 mice, leading to tissue anoxia, but moderate hypoxia, in WT mice. In comparison, before CSD, Ca2+ and hemodynamic responses to somatosensory stimulations were smaller in FHM1 mice than WT mice and almost abolished...... after CSD. The CSD-induced Ca2+ changes were mitigated by the CaV2.1 gating modifier, tert-butyl dihydroquinone. Interpretation: Our findings suggest that tissue anoxia might be a mechanism for prolonged aura in FHM1. Reduced Ca2+ signals during normal network activity in FHM1 as compared to WT mice may...

Calcium supplements

Science.gov (United States)

... this page: //medlineplus.gov/ency/article/007477.htm Calcium supplements To use the sharing features on this page, please enable JavaScript. WHO SHOULD TAKE CALCIUM SUPPLEMENTS? Calcium is an important mineral for the ...

Purification and properties of a 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs.

OpenAIRE

Penning, T M; Mukharji, I; Barrows, S; Talalay, P

1984-01-01

An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley...

Altered calcium pump and secondary deficiency of γ-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from δ-sarcoglycan knockout mice

Science.gov (United States)

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

2016-01-01

Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and δ-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of δ-SG isoforms in TT and SR results in a secondary deficiency of γ-SG and µSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the δ-SG isoforms may stabilize the expression of γ-SG and µSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. PMID:20638123

Calcium waves.

Science.gov (United States)

Jaffe, Lionel F

2008-04-12

Waves through living systems are best characterized by their speeds at 20 degrees C. These speeds vary from those of calcium action potentials to those of ultraslow ones which move at 1-10 and/or 10-20 nm s(-1). All such waves are known or inferred to be calcium waves. The two classes of calcium waves which include ones with important morphogenetic effects are slow waves that move at 0.2-2 microm s(-1) and ultraslow ones. Both may be propagated by cycles in which the entry of calcium through the plasma membrane induces subsurface contraction. This contraction opens nearby stretch-sensitive calcium channels. Calcium entry through these channels propagates the calcium wave. Many slow waves are seen as waves of indentation. Some are considered to act via cellular peristalsis; for example, those which seem to drive the germ plasm to the vegetal pole of the Xenopus egg. Other good examples of morphogenetic slow waves are ones through fertilizing maize eggs, through developing barnacle eggs and through axolotl embryos during neural induction. Good examples of ultraslow morphogenetic waves are ones during inversion in developing Volvox embryos and across developing Drosophila eye discs. Morphogenetic waves may be best pursued by imaging their calcium with aequorins.

Calcium and Nuclear Signaling in Prostate Cancer

Directory of Open Access Journals (Sweden)

Ivan V. Maly

2018-04-01

Full Text Available Recently, there have been a number of developments in the fields of calcium and nuclear signaling that point to new avenues for a more effective diagnosis and treatment of prostate cancer. An example is the discovery of new classes of molecules involved in calcium-regulated nuclear import and nuclear calcium signaling, from the G protein-coupled receptor (GPCR and myosin families. This review surveys the new state of the calcium and nuclear signaling fields with the aim of identifying the unifying themes that hold out promise in the context of the problems presented by prostate cancer. Genomic perturbations, kinase cascades, developmental pathways, and channels and transporters are covered, with an emphasis on nuclear transport and functions. Special attention is paid to the molecular mechanisms behind prostate cancer progression to the malignant forms and the unfavorable response to anti-androgen treatment. The survey leads to some new hypotheses that connect heretofore disparate results and may present a translational interest.

Absorbability of calcium from calcium-bound phosphoryl oligosaccharides in comparison with that from various calcium compounds in the rat ligated jejunum loop.

Science.gov (United States)

To-o, Kenji; Kamasaka, Hiroshi; Nishimura, Takahisa; Kuriki, Takashi; Saeki, Shigeru; Nakabou, Yukihiro

2003-08-01

Calcium-bound phosphoryl oligosaccharides (POs-Ca) were prepared from potato starch. Their solubility and in situ absorbability as a calcium source were investigated by comparing with the soluble calcium compounds, calcium chloride and calcium lactate, or insoluble calcium compounds, calcium carbonate and dibasic calcium phosphate. The solubility of POs-Ca was as high as that of calcium chloride and about 3-fold higher than that of calcium lactate. An in situ experiment showed that the intestinal calcium absorption rate of POs-Ca was almost comparable with that of the soluble calcium compounds, and was significantly higher (pcalcium groups. Moreover, the total absorption rate of a 1:1 mixture of the calcium from POs-Ca and a whey mineral complex (WMC) was significantly higher (psoluble calcium source with relatively high absorption in the intestinal tract.

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

Science.gov (United States)

Lenzi, M; Manotti, P; Muratori, L; Cataleta, M; Ballardini, G; Cassani, F; Bianchi, F B

1995-01-01

Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7797126

Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.

Science.gov (United States)

Chen, Qi; Sun, Lijun; Chen, Zhijian J

2016-09-20

The recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor STING. The cGAS-STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. Thus, the cGAS pathway must be properly regulated. Here we review the recent advances in understanding of the cGAS-STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease.

Protein abundance profiling of the Escherichia coli cytosol

DEFF Research Database (Denmark)

Ishihama, Y.; Schmidt, T.; Rappsilber, J.

2008-01-01

sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI...... approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal...

Vitamin D treatment in calcium-deficiency rickets: a randomised controlled trial.

Science.gov (United States)

Thacher, Tom D; Fischer, Philip R; Pettifor, John M

2014-09-01

To determine whether children with calcium-deficiency rickets have a better response to treatment with vitamin D and calcium than with calcium alone. Randomised controlled trial. Jos University Teaching Hospital, Jos, Nigeria. Nigerian children with active rickets treated with calcium carbonate as limestone (approximately 938 mg elemental calcium twice daily) were, in addition, randomised to receive either oral vitamin D2 50,000 IU (Ca+D, n=44) or placebo (Ca, n=28) monthly for 24 weeks. Achievement of a 10-point radiographic severity score ≤1.5 and serum alkaline phosphatase ≤350 U/L. The median (range) age of enrolled children was 46 (15-102) months, and baseline characteristics were similar in the two groups. Mean (±SD) 25-hydroxyvitamin D (25(OH)D) was 30.2±13.2 nmol/L at baseline, and 29 (43%) had values rickets, there is a trend for vitamin D to improve the response to treatment with calcium carbonate as limestone, independent of baseline 25(OH)D concentrations. ClinicalTrials.gov NCT00949832. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Chloride concentrations in human hepatic cytosol and mitochondria are a function of age.

Science.gov (United States)

Jahn, Stephan C; Rowland-Faux, Laura; Stacpoole, Peter W; James, Margaret O

2015-04-10

We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n = 97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2 ± 62.4 mM; range: 24.7-365.7 mM) were strikingly higher than those in mitochondria (4.2 ± 3.8 mM; range 0.9-22.2 mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology. Copyright © 2015 Elsevier Inc. All rights reserved.

Get Enough Calcium

Science.gov (United States)

... Calcium Print This Topic En español Get Enough Calcium Browse Sections The Basics Overview Foods and Vitamins ... women, don't get enough calcium. How much calcium do I need every day? Women: If you ...

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8(+) T cell responses in mice.

Science.gov (United States)

Zhou, Weibin; Moguche, Albanus O; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-04-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration "cold chain". Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8(+) T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8(+) T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. This paper reports that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize into a biocompatible adjuvant in a single step, enabling distributed and on-demand vaccine production and eliminating the need for refrigeration of vaccines. The findings highlight the possibility of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytosolic glutamine synthetase in barley

DEFF Research Database (Denmark)

Thomsen, Hanne Cecilie

remobilisation from ageing plant parts. Thus, GS is highly involved in determining crop yield and NUE. The major objective of this PhD project was to investigate the NUE properties of transgenic barley designed to constitutively overexpress a GS1 isogene (HvGS1.1). These transgenic lines exhibited an increased...... for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root...... fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

The Effects of Dietary Calcium and/or Iron Deficiency upon Murine Intestinal Calcium Binding Protein Activity and Calcium Absorption

OpenAIRE

McDonald, Catherine M.

1980-01-01

Iron deficiency has been shown to impair calcium absorption, leading to decreased bone mass. Vitamin D3-dependent calcium binding protein (CaBP) has been demonstrated to be necessary for the active transport of calcium in the intestine of numerous species. Iron deficiency might affect the activity of the calcium binding protein. Four experimental diets were formulated as follows: Diet 1, iron adequate, calcium adequate; Diet 2, iron deficient, calcium adequate; Diet 3, iron adequate, calci...

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

Science.gov (United States)

Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

2013-07-01

GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs

International Nuclear Information System (INIS)

Yasuda, Shin; Idell, Steven; Fu Jian; Carter, Glendora; Snow, Rhodora; Liu, M.-C.

2007-01-01

The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [ 35 S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [ 35 S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [ 35 S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previously prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17β-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC 50 values determined for the sulfation of 17β-estradiol by SULT1A1 were 11.8 μM, 28.2 μM, and 500 μM, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17β-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [ 35 S]sulfated 17β-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17β-estradiol and other endogenous

Effects of diphosphonate on kidney calcium content and duodenal absorption of 45calcium

International Nuclear Information System (INIS)

Goulding, A.; Cameron, V.

1978-01-01

In rats the relationships between EHDP-induced changes in serum calcium concentration, kidney calcium content and duodenal transport of 45 calcium were studied. Body weights and kidney weights were similar in all groups. EHDP administration was associated with an increase in serum calcium concentration and kidney calcium content, and a decrease in duodenal 45 calcium transport. In the EHDP-treated rats, there was a significant negative correlation between kidney calcium concentration and duodenal 45 calcium transport but no correlation between either kidney calcium content and serum calcium concentration (r = 0.116) or between serum calcium concentration and duodenal 45 calcium transport (r = 0.02). Further experiments will be needed to determine whether the demonstrated increase in kidney calcium content induced by EHDP administration was the cause of, or was secondary to, inhibition of 1, 25(OH) 2 D 3 synthesis. (orig./AJ) [de

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

Science.gov (United States)

Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

2007-11-01

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

The alpha hemolysin of Escherichia Coli power the metabolism oxidative of neutrophils human beings in response to the peptide chemotactic FMLP: comparison with the ionophore of calcium A23187

International Nuclear Information System (INIS)

Garcia, J.

2000-01-01

The calcium ionophore ionomycin primes polymorphonuclear leukocytes (PMN) for increased superoxide production upon stimulation with the chemotactic peptide FMLP (Helman Finkel, T. et al J Biol Chem 1987; 262: 12589-12596) In this investigation we assessed the effect of PMN priming with either alpha hemolysin (AH) or the calcium ionophore A23187, both of which increase intracellular calcium, on the oxidative metabolism of PMN (as measured by chemiluminescence) in response to secondary stimulation with FMLP. Both A23187 and AH priming increased, the luminol-enhanced chemiluminescence in response to secondary stimulation with FMLP, indicating overstimulation of PMLP oxidative metabolism. Additional experiments using lucigenin as chemiluminescence enhancer showed that A23187, but not AH priming of PMN, increased superoxide release in a manner similar to that reported for ionomycin. These results are discussed in reference to infectious processes involving hemolytic E. coli (Author) [es

Loose excitation-secretion coupling in astrocytes.

Science.gov (United States)

Vardjan, Nina; Parpura, Vladimir; Zorec, Robert

2016-05-01

Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing. © 2015 Wiley Periodicals, Inc.

Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

Science.gov (United States)

Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

1992-11-01

The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

Physiological studies in heterozygous calcium sensing receptor (CaSR gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo

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Kovacs Christopher S

2004-04-01

Full Text Available Abstract Background The calcium sensing receptor (CaSR regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium. To demonstrate regulatory effects of CaSR on calcitonin release, we studied calcitonin responsiveness to calcium in normal and CaSR heterozygous-ablated (Casr+/- mice. Casr+/- mice have hypercalcemia and hypocalciuria, and live normal life spans. Each mouse received either 500 μl of normal saline or one of two doses of elemental calcium (500 μmol/kg or 5 mmol/kg by intraperitoneal injection. Ionized calcium was measured at baseline and 10 minutes, and serum calcitonin was measured on the 10 minute sample. Results At baseline, Casr+/- mice had a higher blood calcium, and in response to the two doses of elemental calcium, had greater increments and peak levels of ionized calcium than their wild type littermates. Despite significantly higher ionized calcium levels, the calcitonin levels of Casr+/- mice were consistently lower than wild type at any ionized calcium level, indicating that the dose-response curve of calcitonin to increases in ionized calcium had been significantly blunted or shifted to the right in Casr+/- mice. Conclusions These results confirm that the CaSR is a physiological regulator of calcitonin; therefore, in response to increases in ionized calcium, the CaSR inhibits parathyroid hormone secretion and stimulates calcitonin secretion.

A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial-mesenchymal transition.

Science.gov (United States)

Stewart, Teneale A; Azimi, Iman; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

2015-03-13

Epithelial-mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies.

Science.gov (United States)

Korzeniewski, Bernard; Zoladz, Jerzy A

2002-07-01

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

Relationship between NaCl- and H2O2-induced cytosolic Ca2+ increases in response to stress in Arabidopsis.

Directory of Open Access Journals (Sweden)

Zhonghao Jiang

Full Text Available Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca(2+ concentration ([Ca(2+]i via Ca(2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS. It is well established that ROS also triggers increases in [Ca(2+]i. However, the relationship and interaction between salinity stress-induced [Ca(2+]i increases and ROS-induced [Ca(2+]i increases remain poorly understood. Using an aequorin-based Ca(2+ imaging assay we have analyzed [Ca(2+]i changes in response to NaCl and H2O2 treatments in Arabidopsis thaliana. We found that NaCl and H2O2 together induced larger increases in [Ca(2+]i in Arabidopsis seedlings than either NaCl or H2O2 alone, suggesting an additive effect on [Ca(2+]i increases. Following a pre-treatment with either NaCl or H2O2, the subsequent elevation of [Ca(2+]i in response to a second treatment with either NaCl or H2O2 was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca(2+]i seen with a second NaCl treatment more than that seen with a second treatment of H2O2. A similar response was seen when the initial treatment was with H2O2; subsequent addition of H2O2 led to less of an increase in [Ca(2+]i than did addition of NaCl. These results imply that NaCl-gated Ca(2+ channels and H2O2-gated Ca(2+ channels may differ, and also suggest that NaCl- and H2O2-evoked [Ca(2+]i may reduce the potency of both NaCl and H2O2 in triggering [Ca(2+]i increases, highlighting a feedback mechanism. Alternatively, NaCl and H2O2 may activate the same Ca(2+ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.

Calcium hydroxide isotope effect in calcium isotope enrichment by ion exchange

International Nuclear Information System (INIS)

Jepson, B.E.; Shockey, G.C.

1984-01-01

The enrichment of calcium isotopes has been observed in ion-exchange chromatography with an aqueous phase of calcium hydroxide and a solid phase of sulfonic acid resin. The band front was exceedingly sharp as a result of the acid-base reaction occuring at the front of the band. Single-stage separation coefficients were found to be epsilon( 44 Ca/ 40 Ca) = 11 x 10 -4 and epsilon( 48 Ca/ 40 Ca) = 18 x 10 -4 . The maximum column separation factors achieved were 1.05 for calcium-44 and 1.09 for calcium-48 with the heavy isotopes enriching in the fluid phase. The calcium isotope effect between fully hydrated aqueous calcium ions and undissociated aqueous calcium hydroxide was estimated. For the calcium-44/40 isotope pair the separation coefficient was 13 x 10 -4 . 20 references, 2 figures

Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

KAUST Repository

Huerlimann, Roger

2015-07-01

The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

Single molecular image of cytosolic free Ca2+ of skeletal muscle cells in rats pre- and post-exercise-induced fatigue

Science.gov (United States)

Liu, Yi; Zhang, Heming; Zhao, Yanping; Liu, Zhiming

2009-08-01

A growing body of literature indicated the cytosolic free Ca2+ concentration of skeletal muscle cells changes significantly during exercise-induced fatigue. But it is confusing whether cytosolic free Ca2+ concentration increase or decrease. Furthermore, current researches mainly adopt muscle tissue homogenate as experiment material, but the studies based on cellular and subcellular level is seldom. This study is aimed to establish rat skeletal muscle cell model of exercise-induced fatigue, and confirm the change of cytosolic free Ca2+ concentration of skeletal muscle cells in rats preand post- exercise-induced fatigue. In this research, six male Wistar rats were randomly divided into two groups: control group (n=3) and exercise-induced fatigue group (n=3). The former group were allowed to freely move and the latter were forced to loaded swimming to exhaustive. Three days later, all the rats were sacrificed, the muscle tissue from the same site of skeletal muscle were taken out and digested to cells. After primary culture of the two kinds of skeletal muscle cells from tissue, a fluorescent dye-Fluo-3 AM was used to label the cytosolic free Ca2+. The fluorescent of Ca2+ was recorded by confocal laser scanning microscopy. The results indicated that, the Ca2+ fluorescence intensity of cells from the rat of exercise-induced fatigue group was significantly higher than those in control group. In conclusion, cytosolic free Ca2+ concentration of skeletal muscle cells has a close relation with exercise-induced fatigue, and the increase of cytosolic free Ca2+ concentration may be one of the important factors of exercise-induced fatigue.

Calcium

Science.gov (United States)

... You can get decent amounts of calcium from baked beans, navy beans, white beans, and others. Canned fish. You're in luck if you like sardines and canned salmon with bones. Almond milk. Working Calcium Into Your ...

Influence of dietary calcium on bone calcium utilization

International Nuclear Information System (INIS)

Farmer, M.; Roland, D.A. Sr.; Clark, A.J.

1986-01-01

In Experiment 1, 10 microCi 45 Ca/day were administered to 125 hens for 10 days. Hens were then allocated to five treatments with calcium levels ranging from .08 to 3.75% of the diet. In Experiment 2, hens with morning oviposition times were randomly allocated to 11 treatments that were periods of time postoviposition ranging from 6 hr to 24 hr, in 2-hr increments (Experiment 2). At the end of each 2-hr period, eggs from 25 hens were removed from the uterus. The 18-, 20-, and 22-hr treatments were replicated three times. In Experiment 3, hens were fed either ad libitum or feed was withheld the last 5 or 6 hr before oviposition. In Experiment 4, hens were fed 10 microCi of 45 Ca for 15 days to label skeletal calcium. Hens were divided into two groups and fed a .08 or 3.75% calcium diet for 2 days. On the second day, 25 hens fed the 3.75% calcium diet were intubated with 7 g of the same diet containing .5 g calcium at 1700, 2100, 0100, 0500, and 0700 hr. The measurements used were egg weight, shell weight, and 45 Ca content of the egg shell. Results indicated a significant linear or quadratic regression of dietary calcium levels on 45 Ca accumulation in eggshells and eggshell weight (Experiment 1). As the calcium level of the diet increased, eggshell weight increased and 45 Ca recovery decreased. Utilization of skeletal calcium for shell formation ranged from 28 to 96%. In Experiment 2, the rate of shell calcification was not constant throughout the calcification process but varied significantly

LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

Energy Technology Data Exchange (ETDEWEB)

Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

2015-05-08

Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

Calcium D-saccharate

DEFF Research Database (Denmark)

Garcia, André Castilho; Hedegaard, Martina Vavrusova; Skibsted, Leif Horsfelt

2016-01-01

Molar conductivity of saturated aqueous solutions of calcium d-saccharate, used as a stabilizer of beverages fortified with calcium d-gluconate, increases strongly upon dilution, indicating complex formation between calcium and d-saccharate ions, for which, at 25 °C, Kassoc = 1032 ± 80, ΔHassoc......° = -34 ± 6 kJ mol-1, and ΔSassoc° = -55 ± 9 J mol-1 K-1, were determined electrochemically. Calcium d-saccharate is sparingly soluble, with a solubility product, Ksp, of (6.17 ± 0.32) × 10-7 at 25 °C, only moderately increasing with the temperature: ΔHsol° = 48 ± 2 kJ mol-1, and ΔSassoc° = 42 ± 7 J mol-1...... K-1. Equilibria in supersaturated solutions of calcium d-saccharate seem only to adjust slowly, as seen from calcium activity measurements in calcium d-saccharate solutions made supersaturated by cooling. Solutions formed by isothermal dissolution of calcium d-gluconate in aqueous potassium d...

CBL-CIPK network for calcium signaling in higher plants

Science.gov (United States)

Luan, Sheng

Plants sense their environment by signaling mechanisms involving calcium. Calcium signals are encoded by a complex set of parameters and decoded by a large number of proteins including the more recently discovered CBL-CIPK network. The calcium-binding CBL proteins specifi-cally interact with a family of protein kinases CIPKs and regulate the activity and subcellular localization of these kinases, leading to the modification of kinase substrates. This represents a paradigm shift as compared to a calcium signaling mechanism from yeast and animals. One example of CBL-CIPK signaling pathways is the low-potassium response of Arabidopsis roots. When grown in low-K medium, plants develop stronger K-uptake capacity adapting to the low-K condition. Recent studies show that the increased K-uptake is caused by activation of a specific K-channel by the CBL-CIPK network. A working model for this regulatory pathway will be discussed in the context of calcium coding and decoding processes.

Dynamic Changes in Cytosolic ATP Levels in Cultured Glutamatergic Neurons During NMDA-Induced Synaptic Activity Supported by Glucose or Lactate.

Science.gov (United States)

Lange, Sofie C; Winkler, Ulrike; Andresen, Lars; Byhrø, Mathilde; Waagepetersen, Helle S; Hirrlinger, Johannes; Bak, Lasse K

2015-12-01

We have previously shown that synaptic transmission fails in cultured neurons in the presence of lactate as the sole substrate. Thus, to test the hypothesis that the failure of synaptic transmission is a consequence of insufficient energy supply, ATP levels were monitored employing the ATP biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30 s pulses of NMDA (30 µM) with a 4 min interval, changes in relative ATP levels were measured in the presence of lactate (1 mM), glucose (2.5 mM) or the combination of the two. ATP levels reversibly declined following NMDA-induced neurotransmission activity, as indicated by a reversible 10-20 % decrease in the response of the biosensor. The responses were absent when the NMDA receptor antagonist memantine was present. In the presence of lactate alone, the ATP response dropped significantly more than in the presence of glucose following the 2nd pulse of NMDA (approx. 10 vs. 20 %). Further, cytosolic Ca(2+) homeostasis during NMDA-induced synaptic transmission is partially inhibited by verapamil indicating that voltage-gated Ca(2+) channels are activated. Lastly, we showed that cytosolic Ca(2+) homeostasis is supported equally well by both glucose and lactate, and that a pulse of NMDA causes accumulation of Ca(2+) in the mitochondrial matrix. In summary, we have shown that ATP homeostasis during neurotransmission activity in cultured neurons is supported by both glucose and lactate. However, ATP homeostasis seems to be negatively affected by the presence of lactate alone, suggesting that glucose is needed to support neuronal energy metabolism during activation.

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye

2005-01-01

different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other...... to osteoclasts as well. We demonstrated that paracrine action of ATP was responsible for the wave propagation, but now the purinergic P2X7 receptor was involved. Thus, the studies demonstrate that calcium signals can be propagated not only among osteoblasts, but also between osteoblasts and osteoclasts...

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz)

OpenAIRE

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-01-01

Background Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Results Transgenic cassava plants that co-express cytosolic superoxide dismu...

Yolk-Shell Porous Microspheres of Calcium Phosphate Prepared by Using Calcium L-Lactate and Adenosine 5'-Triphosphate Disodium Salt: Application in Protein/Drug Delivery.

Science.gov (United States)

Ding, Guan-Jun; Zhu, Ying-Jie; Qi, Chao; Sun, Tuan-Wei; Wu, Jin; Chen, Feng

2015-06-26

A facile and environmentally friendly approach has been developed to prepare yolk-shell porous microspheres of calcium phosphate by using calcium L-lactate pentahydrate (CL) as the calcium source and adenosine 5'-triphosphate disodium salt (ATP) as the phosphate source through the microwave-assisted hydrothermal method. The effects of the concentration of CL, the microwave hydrothermal temperature, and the time on the morphology and crystal phase of the product are investigated. The possible formation mechanism of yolk-shell porous microspheres of calcium phosphate is proposed. Hemoglobin from bovine red cells (Hb) and ibuprofen (IBU) are used to explore the application potential of yolk-shell porous microspheres of calcium phosphate in protein/drug loading and delivery. The experimental results indicate that the as-prepared yolk-shell porous microspheres of calcium phosphate have relatively high protein/drug loading capacity, sustained protein/drug release, favorable pH-responsive release behavior, and a high biocompatibility in the cytotoxicity test. Therefore, the yolk-shell porous microspheres of calcium phosphate have promising applications in various biomedical fields such as protein/drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NMDAR-mediated calcium transients elicited by glutamate co-release at developing inhibitory synapses

Directory of Open Access Journals (Sweden)

Abigail Kalmbach

2010-07-01

Full Text Available Before hearing onset, the topographic organization of the inhibitory sound localization pathway from the medial nucleus of the trapezoid body (MNTB to the lateral superior olive (LSO is refined by means of synaptic silencing and strengthening. During this refinement period MNTB-LSO synapses not only release GABA and glycine but also release glutamate. This co-released glutamate can elicit postsynaptic currents that are predominantly mediated by NMDA receptors (NMDARs. To gain a better understanding of how glutamate contributes to synaptic signaling at developing MNTB-LSO inhibitory synapse, we investigated to what degree and under what conditions NMDARs contribute to postsynaptic calcium responses. Our results demonstrate that MNTB-LSO synapses can elicit compartmentalized calcium responses along aspiny LSO dendrites. These responses are significantly attenuated by the NMDARs antagonist APV. APV, however, has no effect on somatically recorded electrical postsynaptic responses, indicating little, if any, contribution of NMDARs to spike generation. Small NMDAR-mediated calcium responses were also observed under physiological levels of extracellular magnesium concentrations indicating that MNTB-LSO synapses activate magnesium sensitive NMDAR on immature LSO dendrites. In Fura-2 AM loaded neurons, blocking GABAA and glycine receptors decreased NMDAR contribution to somatic calcium responses suggesting that GABA and glycine, perhaps by shunting backpropagating action potentials, decrease the level of NMDAR activation under strong stimulus conditions.

Calcium carbonate production response to future ocean warming and acidification

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A. J. Pinsonneault

2012-06-01

Full Text Available Anthropogenic carbon dioxide (CO₂ emissions are acidifying the ocean, affecting calcification rates in pelagic organisms, and thereby modifying the oceanic carbon and alkalinity cycles. However, the responses of pelagic calcifying organisms to acidification vary widely between species, contributing uncertainty to predictions of atmospheric CO₂ and the resulting climate change. At the same time, ocean warming caused by rising CO₂ is expected to drive increased growth rates of all pelagic organisms, including calcifiers. It thus remains unclear whether anthropogenic CO₂ emissions will ultimately increase or decrease pelagic calcification rates. Here, we assess the importance of this uncertainty by introducing a dependence of calcium carbonate (CaCO₃ production on calcite saturation state (Ω_CaCO3 in an intermediate complexity coupled carbon-climate model. In a series of model simulations, we examine the impact of several variants of this dependence on global ocean carbon cycling between 1800 and 3500 under two different CO₂ emissions scenarios. Introducing a calcification-saturation state dependence has a significant effect on the vertical and surface horizontal alkalinity gradients, as well as on the removal of alkalinity from the ocean through CaCO₃ burial. These changes result in an additional oceanic uptake of carbon when calcification depends on Ω_CaCO3 (of up to 270 Pg C, compared to the case where calcification does not depend on acidification. In turn, this response causes a reduction of global surface air temperature of up to 0.4 °C in year 3500. Different versions of the model produced varying results, and narrowing this range of uncertainty will require better understanding of both temperature and acidification effects on pelagic calcifiers. Nevertheless, our results suggest that alkalinity observations can be used

Research of calcium oxide hydration in calcium nitrate solutions

Directory of Open Access Journals (Sweden)

M.A. Oliynyk

2016-09-01

Full Text Available Mineral fertilizers are one of the important factors of agriculture intensification and increasing of food products quantity. The volume of fertilizers production and its domestic consumption in Ukraine indicate that nitrogen fertilizer using only comes nearer to the required number of science-based. One of the most widespread artificial fertilizers is the calcium nitrate. Aim: The aim is to study and theoretically substantiate the processes occurring in the preparation of suspensions of calcium hydroxide Са(ОН2 in solution of calcium nitrate Ca(NО32. Materials and Methods: The technical calcium oxide (quicklime DSTU BV.2.7-90-99, solutions of calcium nitrate of 15, 20, 25, 30, 35 and 40% Ca(NО32 concentrations were used in the work. The content of lime in the preparation of a suspension in the solution changed (in terms of calcium oxide CaO from 150 g/dm3 to the maximum possible. Each of these solutions saturated at 40°С in lime to maximum concentration. Suitable for use in these experiments and in the technology of calcium nitrate obtaining are considered the solutions (suspensions that within 12 hours did not lose their mobility (transportability. Results: The experimental results show that increasing of the concentration of calcium nitrate in solution within the range 15...40%, the amount of lime that you can put into the solution without loss of transportability decreases. Further increasing of lime quantity in solutions concentrations causes to its solidifying, loss of mobility (transportability. Calculations showed that in the presence of calcium nitrate the solubility of Са(ОН2 is reduced nearly by order that can lead to the formation of calcium oxide CaO the solid phase Са(ОН2 on the surface, which also can form hydrogen bonds with the components of the solution. As the probability of formation of hydrogen bonds in solutions is high, there is a possibility of formation of clusters.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

Science.gov (United States)

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellular uptake of 99mTcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

International Nuclear Information System (INIS)

Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel; Fontaine, Eric; Pasqualini, Roberto

2006-01-01

A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ( 99m TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether 99m TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of 99m TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of 99m TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG 2 M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of 99m TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of 99m TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG 2 M. The uptake of 99m TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that 99m TcN-NOET might be a marker of cell proliferation. (orig.)

Production of precipitated calcium carbonate from calcium silicates and carbon dioxide

International Nuclear Information System (INIS)

Teir, Sebastian; Eloneva, Sanni; Zevenhoven, Ron

2005-01-01

The possibilities for reducing carbon dioxide emissions from the pulp and paper industry by calcium carbonation are presented. The current precipitated calcium carbonate (PCC) production uses mined, crushed calcium carbonate as raw materials. If calcium silicates were used instead, carbon dioxide emissions from the calcination of carbonates would be eliminated. In Finland, there could, thus, be a potential for eliminating 200 kt of carbon dioxide emissions per year, considering only the PCC used in the pulp and paper industry. A preliminary investigation of the feasibility to produce PCC from calcium silicates and the potential to replace calcium carbonate as the raw material was made. Calcium carbonate can be manufactured from calcium silicates by various methods, but only a few have been experimentally verified. The possibility and feasibility of these methods as a replacement for the current PCC production process was studied by thermodynamic equilibrium calculations using HSC software and process modelling using Aspen Plus[reg]. The results from the process modelling showed that a process that uses acetic acid for extraction of the calcium ions is a high potential option for sequestering carbon dioxide by mineral carbonation. The main obstacle seems to be the limited availability and relatively high price of wollastonite, which is a mineral with high calcium silicate content. An alternative is to use the more common, but also more complex, basalt rock instead

Calcium electroporation in three cell lines; a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

2013-01-01

offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium...

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

Science.gov (United States)

O'Brien, Darragh P; Perez, Ana Cristina Sotomayor; Karst, Johanna; Cannella, Sara E; Enguéné, Véronique Yvette Ntsogo; Hessel, Audrey; Raoux-Barbot, Dorothée; Voegele, Alexis; Subrini, Orso; Davi, Marilyne; Guijarro, J Inaki; Raynal, Bertrand; Baron, Bruno; England, Patrick; Hernandez, Belen; Ghomi, Mahmoud; Hourdel, Véronique; Malosse, Christian; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Brier, Sébastien; Ladant, Daniel; Chenal, Alexandre

2018-01-12

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the

Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death

Science.gov (United States)

Zhu, Xiaohong; Caplan, Jeffrey; Mamillapalli, Padmavathi; Czymmek, Kirk; Dinesh-Kumar, Savithramma P

2010-01-01

Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca2+-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca2+-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response. PMID:20075858

Calcium hydroxide poisoning

Science.gov (United States)

Hydrate - calcium; Lime milk; Slaked lime ... Calcium hydroxide ... These products contain calcium hydroxide: Cement Limewater Many industrial solvents and cleaners (hundreds to thousands of construction products, flooring strippers, brick cleaners, cement ...

Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

African Journals Online (AJOL)

Yomi

2010-12-27

Dec 27, 2010 ... The neutrophil cytosol factor 1 (NCF1) gene consists of 11 exons and is found in two forms; one is wild ... granulomatous disease, multiple sclerosis, arthritis and parasitic infection. ... TCR, T cell receptor; AhR, aryl hydrocarbon receptor; RA, .... During malaria, ROS production can contribute to both.

Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

2012-01-01

in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP......, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment...

Kinetics of calcium sulfoaluminate formation from tricalcium aluminate, calcium sulfate and calcium oxide

International Nuclear Information System (INIS)

Li, Xuerun; Zhang, Yu; Shen, Xiaodong; Wang, Qianqian; Pan, Zhigang

2014-01-01

The formation kinetics of tricalcium aluminate (C 3 A) and calcium sulfate yielding calcium sulfoaluminate (C 4 A 3 $) and the decomposition kinetics of calcium sulfoaluminate were investigated by sintering a mixture of synthetic C 3 A and gypsum. The quantitative analysis of the phase composition was performed by X-ray powder diffraction analysis using the Rietveld method. The results showed that the formation reaction 3Ca 3 Al 2 O 6 + CaSO 4 → Ca 4 Al 6 O 12 (SO 4 ) + 6CaO was the primary reaction 4 Al 6 O 12 (SO 4 ) + 10CaO → 6Ca 3 Al 2 O 6 + 2SO 2 ↑ + O 2 ↑ primarily occurred beyond 1350 °C with an activation energy of 792 ± 64 kJ/mol. The optimal formation region for C 4 A 3 $ was from 1150 °C to 1350 °C and from 6 h to 1 h, which could provide useful information on the formation of C 4 A 3 $ containing clinkers. The Jander diffusion model was feasible for the formation and decomposition of calcium sulfoaluminate. Ca 2+ and SO 4 2− were the diffusive species in both the formation and decomposition reactions. -- Highlights: •Formation and decomposition of calcium sulphoaluminate were studied. •Decomposition of calcium sulphoaluminate combined CaO and yielded C 3 A. •Activation energy for formation was 231 ± 42 kJ/mol. •Activation energy for decomposition was 792 ± 64 kJ/mol. •Both the formation and decomposition were controlled by diffusion

Why Calcium? How Calcium Became the Best Communicator*

Science.gov (United States)

Carafoli, Ernesto; Krebs, Joachim

2016-01-01

Calcium carries messages to virtually all important functions of cells. Although it was already active in unicellular organisms, its role became universally important after the transition to multicellular life. In this Minireview, we explore how calcium ended up in this privileged position. Most likely its unique coordination chemistry was a decisive factor as it makes its binding by complex molecules particularly easy even in the presence of large excesses of other cations, e.g. magnesium. Its free concentration within cells can thus be maintained at the very low levels demanded by the signaling function. A large cadre of proteins has evolved to bind or transport calcium. They all contribute to buffer it within cells, but a number of them also decode its message for the benefit of the target. The most important of these “calcium sensors” are the EF-hand proteins. Calcium is an ambivalent messenger. Although essential to the correct functioning of cell processes, if not carefully controlled spatially and temporally within cells, it generates variously severe cell dysfunctions, and even cell death. PMID:27462077

Codissolution of calcium hydrogenphosphate and sodium hydrogencitrate in water. Spontaneous supersaturation of calcium citrate increasing calcium bioavailability

DEFF Research Database (Denmark)

Hedegaard, Martina Vavrusova; Danielsen, Bente Pia; Garcia, André Castilho

2018-01-01

The sparingly soluble calcium hydrogenphosphate dihydrate, co-dissolving in water during dissolution of freely soluble sodium hydrogencitrate sesquihydrate as caused by proton transfer from hydrogencitrate to hydrogenphosphate, was found to form homogenous solutions supersaturated by a factor up...... to 8 in calcium citrate tetrahydrate. A critical hydrogencitrate concentration for formation of homogeneous solutions was found to depend linearly on dissolved calcium hydrogenphosphate: [HCitr2-] = 14[CaHPO4] - 0.05 at 25 °C. The lag phase for precipitation of calcium citrate tetrahydrate......, as identified from FT-IR spectra, from these spontaneously formed supersaturated solutions was several hours, and the time to reach solubility equilibrium was several days. Initial calcium ion activity was found to be almost independent of the degree of supersaturation as determined electrochemically...

Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response.

Science.gov (United States)

Giretova, Maria; Medvecky, Lubomir; Stulajterova, Radoslava; Sopcak, Tibor; Briancin, Jaroslav; Tatarkova, Monika

2016-12-01

Polyhydroxybutyrate/chitosan/calcium phosphate composites are interesting biomaterials for utilization in regenerative medicine and they may by applied in reconstruction of deeper subchondral defects. Insufficient informations were found in recent papers about the influence of lysozyme degradation of chitosan in calcium phosphate/chitosan based composites on in vitro cytotoxicity and proliferation activity of osteoblasts. The effect of enzymatic chitosan degradation on osteoblasts proliferation was studied on composite films in which the porosity of origin 3D scaffolds was eliminated and the surface texture was modified. The significantly enhanced proliferation activity with faster population growth of osteoblasts were found on enzymatically degraded biopolymer composite films with α-tricalcium phosphate and nanohydroxyapatite. No cytotoxicity of composite films prepared from lysozyme degraded scaffolds containing a large fraction of low molecular weight chitosans (LMWC), was revealed after 10 days of cultivation. Contrary to above in the higher cytotoxicity origin untreated nanohydroxyapatite films and porous composite scaffolds. The results showed that the synergistic effect of surface distribution, morphology of nanohydroxyapatite particles, microtopography and the presence of LMWC due to chitosan degradation in composite films were responsible for compensation of the cytotoxicity of nanohydroxyapatite composite films or porous composite scaffolds.

Calcium blood test

Science.gov (United States)

... page: //medlineplus.gov/ency/article/003477.htm Calcium blood test To use the sharing features on this page, please enable JavaScript. The calcium blood test measures the level of calcium in the blood. ...

A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

Directory of Open Access Journals (Sweden)

Julie Adam

2013-05-01

Full Text Available The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH, predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Calcium carbonate overdose

Science.gov (United States)

Tums overdose; Calcium overdose ... Calcium carbonate can be dangerous in large amounts. ... Products that contain calcium carbonate are certain: Antacids (Tums, Chooz) Mineral supplements Hand lotions Vitamin and mineral supplements Other products may also contain ...

Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

Science.gov (United States)

Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

2017-05-15

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

Science.gov (United States)

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis