Sample records for calcium induced genes

  1. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    Meulen, R.M. van der; Visser, K.; Wang, M.


    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  2. The role of calcium in hypoxia-induced signal transduction and gene expression.

    Seta, Karen A; Yuan, Yong; Spicer, Zachary; Lu, Gang; Bedard, James; Ferguson, Tsuneo K; Pathrose, Peterson; Cole-Strauss, Allyson; Kaufhold, Alexa; Millhorn, David E


    Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors. PMID:15261489


    Multiple molecular targets for pyrethroid insecticides have been evaluated in in vitro preparations, including but not limited to voltage-sensitive sodium channels (VSSCs), voltage-sensitive calcium channels (VSCCs), GABAergic receptors, ATPases and mitochondrial respiratory chai...

  4. Exogenous calcium induces tolerance to atrazine stress in Pennisetum seedlings and promotes photosynthetic activity, antioxidant enzymes and psbA gene transcripts.

    Erinle, Kehinde Olajide; Jiang, Zhao; Ma, Bingbing; Li, Jinmei; Chen, Yukun; Ur-Rehman, Khalil; Shahla, Andleeb; Zhang, Ying


    Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented. PMID:27391035


    The molecular details deciphering the spontaneous calcium-induced protein aggregation process in the calcium caseinate system remain obscure. Understanding this complex process could lead to potential new applications of this important food ingredient. In this work, we studied calcium-induced supra...

  6. Possible role of calcium dependent protein phosphorylation in the modulation of wound induced HRGP gene activation in potatoes after gamma irradiation

    Hydroxyproline rich glycoprotein (HRGP) gene is induced in both control and gamma irradiated potato tubers after wounding. The enhanced RNA synthesis in response to wounding correlated well with the accumulation of both HRGP gene transcripts and protein. Initially, the level of HRGP gene expression in gamma irradiated potatoes in response to wounding was 30% more than the corresponding controls. After post irradiation storage of 3-5 weeks, HRGP gene expression in response to wounding was significantly lower than the unirradiated samples. This low level of HRGP gene expression in irradiated potatoes was partially retrieved by 5 mM Ca2+ treatment. Prior treatment with trifluoperazine, a calcium channel blocker resulted in 35% reduction in wound induced HRGP gene expression in control potatoes, further providing evidence for the involvement of Ca2+ dependency for HRGP gene activation. A comparative study on in vivo protein phosphorylation induced by wounding in control and irradiated potatoes exhibited significant differences. A good correlation was observed in the modulation of phosphorylation and HRGP gene expression by Ca2+ in irradiated potatoes. Wound induced signal transduction system and subsequent Ca2+ dependent protein phosphorylation for the activation of HRGP gene is affected in potatoes after gamma irradiation, thus impairing the wound healing process adversely. (author). 25 refs., 5 figs

  7. IGF-1 induces IP3 -dependent calcium signal involved in the regulation of myostatin gene expression mediated by NFAT during myoblast differentiation.

    Valdés, Juan A; Flores, Sylvia; Fuentes, Eduardo N; Osorio-Fuentealba, Cesar; Jaimovich, Enrique; Molina, Alfredo


    Skeletal muscle differentiation is a complex and highly regulated process characterized by cell cycle arrest, which is associated with morphological changes including myoblast alignment, elongation, and fusion into multinucleated myotubes. This is a balanced process dynamically coordinated by positive and negative signals such as the insulin-like growth factor I (IGF-1) and myostatin (MSTN), respectively. In this study, we report that the stimulation of skeletal myoblasts during differentiation with IGF-1 induces a rapid and transient calcium increase from intracellular stores, which are principally mediated through the phospholipase C gamma (PLC γ)/inositol 1,4,5-triphosphate (IP3 )-dependent signaling pathways. This response was completely blocked when myoblasts were incubated with LY294002 or transfected with the dominant-negative p110 gamma, suggesting a fundamental role of phosphatidylinositol 3-kinase (PI3K) in PLCγ activation. Additionally, we show that calcium released via IP3 and induced by IGF-1 stimulates NFAT-dependent gene transcription and nuclear translocation of the GFP-labeled NFATc3 isoform. This activation was independent of extracellular calcium influx and calcium release mediated by ryanodine receptor (RyR). Finally, we examined mstn mRNA levels and mstn promoter activity in myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents and in reporter activity, which was inhibited by cyclosporin A, 11R-VIVIT, and by inhibitors of the PI3Kγ, PLCγ, and IP3 receptor. Our results strongly suggest that IGF-1 regulates myostatin transcription through the activation of the NFAT transcription factor in an IP3 /calcium-dependent manner. This is the first study to demonstrate a role of calcium-dependent signaling pathways in the mRNA expression of myostatin. PMID:23255067

  8. Inhibitory gene expression of the Cav3.1 T-type calcium channel to improve neuronal injury induced by lidocaine hydrochloride.

    Wen, Xianjie; Xu, Shiyuan; Zhang, Qingguo; Li, Xiaohong; Liang, Hua; Yang, Chenxiang; Wang, Hanbing; Liu, Hongzhen


    Cav3.1 is a low-voltage-activated (LVA) calcium channel that plays a key role in regulating intracellular calcium ion levels. In this study, we observed the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y cells that silenced Cav3.1 mRNA by RNA interference, and investigated the roles of p38 MAPK in these effects. We constructed the pNC-puro-CACNA1G-SH-SY5Y cells and pshRNA-CACNA1G -SH-SY5Y cells by the RNA interference. All the cells were cultured with or without 10mM lidocaine hydrochloride for 24h. The cell morphology, cell viability, Cav3.1 and p38 protein expression, cell apoptosis rate and intracellular calcium ion concentration were detected. We found that all cells treated with 10mM lidocaine hydrochloride for 24h showed cellular rounding, axonal regression, and cellular floating. Compared with the cells in SH-SY5Y+Lido group and NC+Lido group, those in the RNAi+Lido group showed similar changes, but of smaller magnitude. Additionally, following lidocaine hydrochloride all cells displayed increased Cav3.1 and p38 MAPK protein, apoptosis rate, and intracellular calcium ion levels; however,these changes in the RNAi+Lido group were less pronounced than in the SH-SY5Y+Lido and NC+Lido groups. The cell viability decreased following lidocaine hydrochloride treatment, but viability of the cells in the RNAi+Lido group was higher than in the SH-SY5Y+Lido and NC+Lido groups. The results showed that Cav3.1 may be involved in neuronal injury induced by lidocaine hydrochloride and that p38 MAPK phosphorylation was reduced upon Cav3.1 gene silencing. PMID:26852957

  9. A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists

    Sheth, Heeral; Gorey, Colleen; Roush, Nicole; Smallman, Shelly; Collantes, Elizabeth; Santoro, Maxine; Olson, Barbara; Fitzgerald, Laura; Paul H. Lee; Shen, Xiqiang John


    Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the ...

  10. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

    Liang, Willmann


    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  11. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Yubo Sun


    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  12. Calcium control of gene regulation in rat hippocampal neuronal cultures.

    Pinato, Giulietta; Pegoraro, Silvia; Iacono, Giovanni; Ruaro, Maria Elisabetta; Torre, Vincent


    Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20 microM gabazine applied for 30 min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs. PMID:19441076

  13. Idiopathic Calcium Nephrolithiasis And Hypercalciuria: The Role Of Genes

    Gambaro, Giovanni; Abaterusso, Cataldo


    Idiopathic calcium nephrolithiasis and hypercalciuria are multifactorial disease conditions, the pathogenesis of which involves the interaction of environmental and individual factors. Data support a strong role of genes in the pathogenesis of these two conditions. Findings obtained in monogenic disorders characterized by renal calcium stones, and/or hypercalciuria, and/or nephrocalcinosis have proposed a number of genes as candidate genes in the pathogenesis of the common idiopathic calcium nephrolithiasis and hypercalciuria. The physiological role of these genes, and findings in monogenic disorders and idiopathic, multifactorial disorders will be presented.

  14. A calcium-induced calcium release mechanism mediated by calsequestrin.

    Lee, Young-Seon; Keener, James P


    Calcium (Ca(2+))-induced Ca(2+) release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca(2+) with ryanodine receptors (RyRs) and inducing Ca(2+) release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca(2+) may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca(2+) buffer. We investigate how SR Ca(2+) release via RyR is regulated by Ca(2+) and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca(2+) activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P(o)) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca(2+). Both peak and steady-state P(o) effectively increase as SR lumenal Ca(2+) increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca(2+) loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca(2+) release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca(2+) release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca(2+) release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes. PMID:18538346

  15. Calcium signaling in UV-induced damage

    Sun, Dan; Zhang, Su-juan; Li, Yuan-yuan; Qu, Ying; Ren, Zhao-Yu


    Hepa1-6 cells were irradiated with UV and incubated for varying periods of time. [Ca 2+] i (intracellular calcium concentration) of UV-irradiated cell was measured by ratio fluorescence imaging system. The comet assay was used to determine DNA damage. During the UVB-irradiation, [Ca 2+] i had an ascending tendency from 0.88 J/m2 to 92.4J/m2. Comet assay instant test indicated that when the irradiation dosage was above 0.88J/m2, DNA damage was observed. Even after approximate 2 h of incubation, DNA damage was still not detected by 0.88J/m2 of UVB irradiation. During UVA-irradiation, the elevation of [Ca 2+] i was not dose-dependent in a range of 1200 J/m2-6000J/m2 and DNA damage was not observed by comet assay. These results suggested that several intracellular UV receptors might induce [Ca 2+] i rising by absorption of the UV energy. Just [Ca 2+] i rising can't induce DNA damage certainly, it is very likely that the breakdown of calcium steady state induces DNA damage.u

  16. Calcium waves induced by large voltage pulses in fish keratocytes.

    Brust-Mascher, I; Webb, W W


    Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, whic...

  17. Glutamate (mGluR-5 gene expression in brain regions of streptozotocin induced diabetic rats as a function of age: role in regulation of calcium release from the pancreatic islets in vitro

    Paulose CS


    Full Text Available Abstract Metabotrophic glutamate receptors (mGluRs modulate cellular activities involved in the processes of differentiation and degeneration. In this study, we have analysed the expression pattern of group-I metabotropic glutamate receptor (mGlu-5 in cerebral cortex, corpus striatum, brainstem and hippocampus of streptozotocin induced and insulin treated diabetic rats (D+I as a function of age. Also, the functional role of glutamate receptors in intra cellular calcium release from the pancreatic islets was studied in vitro. The gene expression studies showed that mGlu-5 mRNA in the cerebral cortex increased siginficantly in 7 weeks old diabetic rats whereas decreased expression was observed in brainstem, corpus striatum and hippocampus when compared to control. 90 weeks old diabetic rats showed decreased expression in cerebral cortex, corpus striatum and hippocampus whereas in brainstem the expression increased significantly compared to their respective controls. In 7 weeks old D+I group, mGlu-5 mRNA expression was significantly decreased in cerebral cortex and corpus striatum whereas the expression increased significantly in brainstem and hippocampus. 90 weeks old D+I group showed an increased expression in cerebral cortex, while it was decreased significantly in corpus striatum, brainstem and hippocampus compared to their respective controls. In vitro studies showed that glutamate at lower concentration (10-7 M stimulated calcium release from the pancreatic islets. Our results suggest that mGlu-5 receptors have differential expression in brain regions of diabetes and D+I groups as a function of age. This will have clinical significance in management of degeneration in brain function and memory enhancement through glutamate receptors. Also, the regulatory role of glutamate receptors in calcium release has immense therapeutic application in insulin secretion and function.

  18. The pressure-induced calcium deposition on crosslinked polyurethanes.

    Shunmugakumar, N; Jayabalan, M


    The pressure-induced calcium deposition in crosslinked polyurethane was studied. Two polyurethane systems, IPDI-PTMG/PPG-TMP and SMDI-PTMG/PPG-TMP were subjected to calcification under induced pressure. Calcium deposition in IPDI polymers was linear with the increase of soft segment (PTMG) content whereas in SMDI polymers the reverse trend was observed. Decreased phase mixing and hydrophilicity in the polymer (SMDI based) having increased soft segment content was attributed to the decreased calcification. The enhanced amount of calcium deposition under pressure indicates the possible influence of pressure on calcification. PMID:10078255

  19. Sulfhydryl oxidation overrides Mg(2+) inhibition of calcium-induced calcium release in skeletal muscle triads.

    Donoso, P; Aracena, P; Hidalgo, C.


    We studied the effect of oxidation of sulfhydryl (SH) residues on the inhibition by Mg(2+) of calcium-induced calcium release (CICR) in triad-enriched sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. Vesicles were either passively or actively loaded with calcium before eliciting CICR by dilution at pCa 4.6-4.4 in the presence of 1.2 mM free [ATP] and variable free [Mg(2+)]. Native triads exhibited a significant inhibition of CICR by Mg(2+), with a K(0.5) approximately 50 ...

  20. Rickets induced by calcium or phosphate depletion.

    Abugassa, S.; Svensson, O.


    We studied the effects of calciopenia and phosphopenia on longitudinal growth, skeletal mineralization, and development of rickets in young Sprague-Dawley rats. At an age of 21 days, two experimental groups were given diets containing 0.02% calcium or 0.02% phosphorus; otherwise the diets were nutritionally adequate. After 7, 14, and 21 days, five animals from each group were randomly chosen. The animals were anaesthetized and blood samples were drawn for analysis of calcium, phosphorus, and ...

  1. Gravity, stress, calcium and gene expression.

    Davies, E; Shimps, B; Brown, K; Stankovic, B


    Cytoplasmic calcium is a major regulator of plant metabolism and its levels are under strict control, but it increases rapidly and transiently after stress treatments such as cold and touch (Trewavas 1999). Gravity is also thought to affect Ca2+ levels, although one report, using fluorescence microscopy of Ca(2+)-binding dyes, showed no changes (Legue et al. 1997). However, in these studies Ca2+ could not be visualized for at least 1 min after gravistimulation and thus changes could have occurred more rapidly. In order to circumvent problems associated with the delay in taking readings imposed by the fluorescence microscopy techniques, we chose a different method using plants transgenic for the Ca(2+)-binding, light-emitting jellyfish protein aequorin (Knight & Knight 1995). We subjected Arabidopsis and tomato plants to heat-wounding, vibration and gravity stimulation and measured cytoplasmic Ca2+ levels. We also measured the levels of several transcripts after heat-wound and gravity stimulation to determine whether both treatments evoked the same changes. PMID:11543010

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

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S


    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling. PMID:26901094

  3. Light-induced ejection of calcium atoms from polymer surfaces

    Mango, F.; Maccioni, E.


    Laser-induced fluorescence (LIF) of calcium atoms at room temperature has been observed in a polydimethylsiloxane (PDMS) coated cell when the walls are illuminated with non resonant visible light. Ca atomic density in the gas phase, monitored by the LIF, is much higher than normal room-temperature vapour pressure of calcium. In past years photon-stimulated desorption (PSD) was observed for several alkali metals that adsorbed to solid films of PDMS polymers. High yields of photo-desorbed atoms (and molecules in the case of sodium) can be induced, at room temperature and below, by weak intensity radiation. The desorption is characterised by a frequency threshold, whereas any power threshold is undetectable. The calcium photo-ejection is characterised both by a frequency threshold (about 18 500 cm-1) and by an observable power threshold (whose value becomes lower when the photo-ejecting light wavelength decreases).

  4. Calcium ionophore A23187 as a regulator of gene expression in mammalian cells


    The calcium ionophore A23187 can reversibly induce the expression of two glucose-regulated genes, p3C5 and p4A3. This induction requires a continuous presence of the ionophore for over 2 h. Although extracellular Ca2+ is important for the optimal effect of A23187, it is not necessary for the induction, since a similar response with a lower magnitude can be triggered in cells cultured in low Ca2+ medium buffered with EGTA. Both the basal and induced levels of p3C5 and p4A3 transcripts can be m...

  5. Cyclodextrin induces calcium-dependent lysosomal exocytosis.

    Fannie W Chen

    Full Text Available Cyclodextrins (CDs have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.

  6. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

    Freitas, Hercules R; Ferraz, Gabriel; Ferreira, Gustavo C; Ribeiro-Resende, Victor T; Chiarini, Luciana B; do Nascimento, José Luiz M; Matos Oliveira, Karen Renata H; Pereira, Tiago de Lima; Ferreira, Leonardo G B; Kubrusly, Regina C; Faria, Robson X; Herculano, Anderson Manoel; Reis, Ricardo A de Melo


    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  7. Vesicular demyelination induced by raised intracellular calcium.

    Smith, K J; Hall, S M; Schauf, C L


    Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 microM, 10 micrograms/ml) or low (1-1.5 microM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15-20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Ni2+, Sr2+) could not substitute for Ca2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn2+ (greater than or equal to 1 microM), Ni2+ (greater than or equal to 1-10 microM), Co2+ (greater than or equal to 100 microM) or Mn2+ (greater than or equal to 100 microM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (greater than or equal to 1 microM), an ionophore with a greater selectivity for Ca2+ than A23187, also induced a prompt Ca2+-dependent

  8. Laser-Induced Damage of Calcium Fluoride

    Radiation damage of materials has long been of fundamental interest, especially since the growth of laser technology. One such source of damage comes from UV laser light. Laser systems continue to move into shorter wavelength ranges, but unfortunately are limited by the damage threshold of their optical components. For example, semiconductor lithography is making its way into the 157nm range and requires a material that can not only transmit this light (air cannot), but also withstand the highly energetic photons present at this shorter wavelength. CaF2, an alkaline earth halide, is the chosen material for vacuum UV 157 nm excimer radiation. It can transmit light down to 120 nm and is relatively inexpensive. Although it is readily available through natural and synthetic sources, it is often times difficult to find in high purity. Impurities in the crystal can result in occupied states in the band gap that induce photon absorption [2] and ultimately lead to the degradation of the material. In order to predict how well CaF2 will perform under irradiation of short wavelength laser light, one must understand the mechanisms for laser-induced damage. Laser damage is often a two-step process: initial photons create new defects in the lattice and subsequent photons excite these defects. When laser light is incident on a solid surface there is an initial production of electron-hole (e-h) pairs, a heating of free electrons and a generation of local heating around optically absorbing centers [3]. Once this initial excitation converts to the driving energy for nuclear motion, the result is an ejection of atoms, ions and molecules from the surface, known as desorption or ablation [3]. Secondary processes further driving desorption are photoabsorption, successive excitations of self-trapped excitons (STE's) and defects, and ionization of neutrals by incident laser light [3]. The combination of laser-induced desorption and the alterations to the electronic and geometrical

  9. Calcium-ATPases: Gene disorders and dysregulation in cancer.

    Dang, Donna; Rao, Rajini


    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26608610

  10. Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

    Saraiva R.M.


    Full Text Available Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

  11. Role for voltage gated calcium channels in calcitonin gene-related peptide release in the rat trigeminovascular system

    Amrutkar, D V; Ploug, K B; Olesen, J;


    Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons and are...... potassium releases CGRP, and the release is regulated by Ca2+ ions and voltage-gated calcium channels....... potassium induced CGRP release. In the absence of calcium ions (Ca2+) and in the presence of a cocktail of blockers, the stimulated CGRP release from dura mater was reduced almost to the same level as basal CGRP release. In the TG ω-conotoxin GVIA inhibited the potassium induced CGRP release significantly...

  12. Role for voltage gated calcium channels in calcitonin gene-related peptide release in the rat trigeminovascular system

    Amrutkar, D V; Ploug, K B; Olesen, J;


    Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons and are...... potassium releases CGRP, and the release is regulated by Ca2+ ions and voltage-gated calcium channels....... potassium induced CGRP release. In the absence of calcium ions (Ca2+) and in the presence of a cocktail of blockers, the stimulated CGRP release from dura mater was reduced almost to the same level as basal CGRP release. In the TG ¿-conotoxin GVIA inhibited the potassium induced CGRP release significantly...

  13. SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

    Chami, M; Gozuacik, D; Lagorce, D; Brini, M; Falson, P; Peaucellier, G; Pinton, P; Lecoeur, H; Gougeon, M L; le Maire, M; Rizzuto, R; Bréchot, C; Paterlini-Bréchot, P


    By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis. PMID:11402072

  14. Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

    Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)


    Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

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

    Zhi Pan


    Full Text Available 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.

  16. Amyloid β-peptide directly induces spontaneous calcium transients, delayed intercellular calcium waves and gliosis in rat cortical astrocytes

    Marius Buibas


    Full Text Available The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein/PS1 (presenilin 1 Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.

  17. Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

    Xiang Yang HU; Steven J NEILL; Wei Ming CAI; Zhang Cheng TANG


    Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca2+]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H2O2. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca2+]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.

  18. Acidosis and Urinary Calcium Excretion

    Alexander, R Todd; Cordat, Emmanuelle; Chambrey, Régine;


    Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and...... renal tubule and then discuss why not all gene defects that cause renal tubular acidosis are associated with hypercalciuria and nephrocalcinosis....

  19. Association of Vitamin D Receptor Gene Polymorphisms with Calcium Oxalate Calcul us Disease

    王少刚; 刘继红; 胡少群; 叶章群


    To study the relationship between polymorphism of vitamin D receptor (VDR) allele with formation of calcium oxalate calculus and find the predisposing genes of calcium oxalate calculus, we screened out 150 patients who suffered from calcium oxalate calculus. 36 of them had idiopathic hypercalciuria according to analysis of calculus component and assay of urine calcium. The polymorphisms of VDR gene Taq1, Apa1 and Fok1 were detected using PCR-RFLP technique and the correlation were analyzed between the polymorphism and urinary calculus or between the polymorphism and hypercalciuria. The difference in each genotypic frequency of the allele of promoter Fok1 between calculus group and healthy group or between idiopathic hypercalciuria calculus group and health group was significant. The content of 24-h urine calcium of those who had genotype ff was obviously higher than that of those who have other genotypes in the same group. There was no significant difference in the polymorphism of gene Apa1 and Taq1 between each two groups. It is concluded that hypercalciuria and calcium oxalate calculus were related to the polymorphism of VDR gene's promoter Fok1 allele, but it had nothing to do with the polymorphism of gene Apa1 and Taq1. The genotype ff was a candidate heredity marker of calcium calculus disease.

  20. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease

    Teresa Schacht; Jacek Kuznicki


    Huntington's disease (HD) is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome). Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in ...

  1. Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene

    Jyoti K Jaiswal; Vidyanand Nanjundiah


    In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution of GlnRS and AsnRS in eukaryotes.

  2. Polyethyleneimine-modified calcium carbonate nanoparticles for p53 gene delivery.

    Chen, Cen; Han, Huafeng; Yang, Wei; Ren, Xiaoyuan; Kong, Xiangdong


    In this study, calcium carbonate (CaCO3) nanoparticles with spherical structure were regulated by arginine and successfully synthesized via a facile co-precipitation method. The average particle size of as-prepared CaCO3 was about 900 nm. The properties of nanostructured CaCO3 particles were characterized by scanning electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction and size distribution. After modified with polyethyleneimine (PEI), the ability of PEI-CaCO3 nanoparticles to carry GFP-marked p53 gene (pEGFP-C1-p53) into cancer cells to express P53 protein were studied. Meanwhile, the cytotoxicity, transfection efficiency, cells growth inhibition and the ability to induce apoptosis by expressed P53 protein were conducted to evaluate the performances of PEI-CaCO3 nanoparticles. The results show that prepared PEI-CaCO3 nanoparticles had good biocompatibility and low cytotoxicity in a certain concentration range. PEI-CaCO3 effectively transfected pEGFP-C1 gene into epithelial-like cancer cells. And with the expression of GFP-P53 fusion protein, pEGFP-C1-p53-gene-loaded PEI-CaCO3 particles significantly reduced the proliferation of cancer cells. These findings indicate that our PEI-modified CaCO3 nanoparticles are potential to be successfully used as carriers for gene therapy. PMID:26816656

  3. Biofilm-induced calcium carbonate precipitation: application in the subsurface

    Phillips, A. J.; Eldring, J.; Lauchnor, E.; Hiebert, R.; Gerlach, R.; Mitchell, A. C.; Esposito, R.; Cunningham, A. B.; Spangler, L.


    We have investigated mitigation strategies for sealing high permeability regions, like fractures, in the subsurface. This technology has the potential to, for example, improve the long-term security of geologically-stored carbon dioxide (CO2) by sealing fractures in cap rocks or to mitigate leakage pathways to prevent contamination of overlying aquifers from hydraulic fracturing fluids. Sealing technologies using low-viscosity fluids are advantageous since they potentially reduce the necessary injection pressures and increase the radius of influence around injection wells. In this technology, aqueous solutions and suspensions are used to promote microbially-induced mineral precipitation which can be applied in subsurface environments. To this end, a strategy was developed to twice seal a hydraulically fractured, 74 cm (2.4') diameter Boyles Sandstone core, collected in North-Central Alabama, with biofilm-induced calcium carbonate (CaCO3) precipitates under ambient pressures. Sporosarcina pasteurii biofilms were established and calcium and urea containing reagents were injected to promote saturation conditions favorable for CaCO3 precipitation followed by growth reagents to resuscitate the biofilm's ureolytic activity. Then, in order to evaluate this process at relevant deep subsurface pressures, a novel high pressure test vessel was developed to house the 74 cm diameter core under pressures as high as 96 bar (1,400 psi). After determining that no impact to the fracture permeability occurred due to increasing overburden pressure, the fractured core was sealed under subsurface relevant pressures relating to 457 meters (1,500 feet) below ground surface (44 bar (650 psi) overburden pressure). After fracture sealing under both ambient and subsurface relevant pressure conditions, the sandstone core withstood three times higher well bore pressure than during the initial fracturing event, which occurred prior to biofilm-induced CaCO3 mineralization. These studies suggest

  4. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCα-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis

  5. Reevaluation of the effect of calcium ions on auxin-induced elongation

    Cleland, R.E. (Univ. of Washington, Seattle); Rayle, D.L.


    The mechanism by which calcium ions inhibit cell elongation has been reinvestigated. Growth-inhibiting levels of calcium, when applied to isolated walls (in vitro treatment) do not decrease cell wall extensibility as measured by the Instron technique. Thus, the hypothesis that calcium inhibits growth by forming wall-stiffening calcium bridges must be abandoned. Treatment of living auxin-treated sections with calcium (in vivo treatment) does cause a decrease in the subsequently measured wall extensibility, but this decline appears to be simply a consequence of the growth inhibition rather that its cause. Growth-inhibiting levels of calcium do not appreciably reduce the rate of auxin-enhanced H+ excretion. Pretreatment with calcium does not reduce the capacity of walls to undergo acid-activated wall loosening in the absence of calcium. High concentrations of CaC1/sub 2/ (0.02 M) cause an initial elastic shrinkage of Avena sections comparable to that caused by the same osmolarity of mannitol, but the subsequent growth inhibition is to great to be explained by an osmotic inhibition.Calcium ions do inhibit H+-induced extension of frozen-thawed sections under tension. The growth-inhibitory effects of calcium, then, may be ascribed to a direct inhibition exerted by calcium ions on the H+-induced wall-loosening process.

  6. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

    Brinton Roberta


    Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that APα-induced

  7. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    Song, Lei; Santos-Sacchi, Joseph


    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  8. Lipid-coated nano-calcium-phosphate (LNCP) for gene delivery

    Zhou, Chenguang; Yu, Bo; Yang, Xiaojuan; Huo, Tianyao; Lee, L. James; Barth, Rolf F; Lee, Robert J.


    While calcium-phosphate has been used to deliver plasmid DNA (pDNA) for decades, the method is typically characterized by low and irreproducible transfection efficiency relative to the other non-viral approaches, such as liposomes and polymers. Here we report a novel gene transfer vector comprising lipid-coated nano-calcium-phosphate (LNCP) that provides consistently efficient and satisfactory pDNA delivery. It is based on core-shell nanoparticles comprising a calcium-phosphate core and a cat...

  9. Prophylactic and therapeutic functions of T-type calcium blockers against noise-induced hearing loss

    Shen, Haiyan; Zhang, BaoPing; Shin, June-Ho; Lei, Debin; Du, Yafei; Gao, Xiang; Wang, Qiuju; Ohlemiller, Kevin K.; Piccirillo, Jay; Bao, Jianxin


    Cochlear noise injury is the second most frequent cause of sensorineural hearing loss, after aging. Because calcium dysregulation is a widely recognized contributor to noise injury, we examined the potential of calcium channel blockers to reduce noise-induced hearing loss (NIHL) in mice. We focused on two T-type calcium blockers, trimethadione and ethosuximide, which are anti-epileptics approved by the Food and Drug Administration. Young C57BL/6 mice of either gender were divided into three g...

  10. Mean field strategies induce unrealistic nonlinearities in calcium puffs



    Full Text Available Mean field models are often useful approximations to biological systems, but sometimes, they can yield misleading results. In this work, we compare mean field approaches with stochastic models of intracellular calcium release. In particular, we concentrate on calcium signals generated by the concerted opening of several clustered channels (calcium puffs. To this end we simulate calcium puffs numerically and then try to reproduce features of the resulting calcium distribution using mean field models were all the channels open and close simultaneously. We show that an unrealistic nonlinear relationship between the current and the number of open channels is needed to reproduce the simulated puffs. Furthermore, a single channel current which is five times smaller than the one of the stochastic simulations is also needed. Our study sheds light on the importance of the stochastic kinetics of the calcium release channel activity to estimate the release fluxes.

  11. Phagocytosis-induced 45calcium efflux in polymorphonuclear leucocytes

    The role of calcium ions in regulating the structure and function of non-muscle cells is a subject of intense study. Several lines of evidence that calcium may be essential in the function of polymorphonuclear leucocytes (PMNL) and an important control element in the process of phagocytosis. Direct studies of calcium distribution and fluxes have only recently been undertaken. To our knowledge, no report of calcium movements during normal phagocytosis has been published. In the context of an overall study of calcium dynamics in the PMNL, we report here initial studies on 45Ca efflux in prelabelled guinea pig PMNL. The results demonstrate the energy-dependence of resting calcium efflux and an increased efflux upon addition of phagocytic particles which is not dependent on particle internalization

  12. Fluid flow induced calcium response in osteoblasts: mathematical modeling.

    Su, J H; Xu, F; Lu, X L; Lu, T J


    Fluid flow in the bone lacuno-canalicular network can induce dynamic fluctuation of intracellular calcium concentration ([Ca(2+)](i)) in osteoblasts, which plays an important role in bone remodeling. There has been limited progress in the mathematical modeling of this process probably due to its complexity, which is controlled by various factors such as Ca(2+) channels and extracellular messengers. In this study we developed a mathematical model to describe [Ca(2+)](i) response induced by fluid shear stress (SS) by integrating the major factors involved and analyzed the effects of different experimental setups (e.g. [Ca(2+)](i) baseline, pretreatment with ATP). In this model we considered the ATP release process and the activities of multiple ion channels and purinergic receptors. The model was further verified quantitatively by comparing the simulation results with experimental data reported in literature. The results showed that: (i) extracellular ATP concentration has more significant effect on [Ca(2+)](i) baseline (73% increase in [Ca(2+)](i) with extracellular ATP concentration varying between 0 and 10 μM), as compared to that induced by SS (25% variation in [Ca(2+)](i) with SS varying from 0 to 3.5 Pa); (ii) Pretreatment with ATP-medium results in different [Ca(2+)](i) response as compared to the control group (ATP-free medium) under SS; (iii) Relative [Ca(2+)](i) fluctuation over baseline is more reliable to show the [Ca(2+)](i) response process than the absolute [Ca(2+)](i) response peak. The developed model may improve the experimental design and facilitate our understanding of the mechanotransduction process in osteoblasts. PMID:21665208

  13. Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

    XIU Meihong; PENG Jianxin; HONG Huazhu


    Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (△Ψm) began to decrease in SL-1 cells at 4 h post infection and △Ψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was down- regulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrion-mediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.

  14. Pressure induced reactions amongst calcium aluminate hydrate phases

    Moon, Ju-hyuk


    The compressibilities of two AFm phases (strätlingite and calcium hemicarboaluminate hydrate) and hydrogarnet were obtained up to 5 GPa by using synchrotron high-pressure X-ray powder diffraction with a diamond anvil cell. The AFm phases show abrupt volume contraction regardless of the molecular size of the pressure-transmitting media. This volume discontinuity could be associated to a structural transition or to the movement of the weakly bound interlayer water molecules in the AFm structure. The experimental results seem to indicate that the pressure-induced dehydration is the dominant mechanism especially with hygroscopic pressure medium. The Birch-Murnaghan equation of state was used to compute the bulk modulus of the minerals. Due to the discontinuity in the pressure-volume diagram, a two stage bulk modulus of each AFm phase was calculated. The abnormal volume compressibility for the AFm phases caused a significant change to their bulk modulus. The reliability of this experiment is verified by comparing the bulk modulus of hydrogarnet with previous studies. © 2011 Elsevier Ltd. All rights reserved.

  15. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco


    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage. PMID:27305947

  16. Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

    Rosana L. Pagano


    Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

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

    Zhi Pan; Andrew Avila; Lauren Gollahon


    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...

  18. Nano-sized calcium phosphate (CaP) carriers for non-viral gene deilvery

    Highlights: ► Nanostructured calcium phosphates (NanoCaPs): comprehensive review. ► Non viral gene delivery mechanisms: detailed mechanisms are outlined. ► Barriers to non-viral gene delivery: detailed barriers are discussed. - Abstract: Gene therapy has garnered much interest due to the potential for curing multiple inherited and/or increases in the acquired diseases. As a result, there has been intense activity from multiple research groups for developing effective delivery methods and carriers, which is a critical step in advancing gene delivery technologies. In order for the carriers to effectively deliver the genetic payloads, multiple extracellular and intracellular barriers need to be overcome. Although overcoming these challenges to improve the effectiveness is critical, the development of safe gene delivery agents is even more vital to assure its use in clinical applications. The development of safe and effective strategies has therefore been a major challenge impeding gene therapy progress. In this regard, calcium phosphate (CaP) based nano-particles has been considered as one of the candidate non-viral gene delivery vehicles, but has been plagued by inconsistent and low transfection efficiencies limiting its progress. There has been major research effort to improve the consistency and effectiveness of CaP based vectors. Currently, it is therefore thought that by controlling the various synthesis factors such as Ca/P ratio, mode of mixing, and type of calcium phosphate phase, such variability and inefficiency could be modulated. This review attempts to provide a comprehensive analysis of the current research activity in the development of CaP based ceramic and polymer-ceramic hybrid systems for non-viral gene delivery. Preliminary transfection results of hydroxyapatite (HA or NanoCaPs), amorphous calcium phosphate (ACP) and brushite phases are also compared to assess the effect of various CaP phases, and correspondingly, changes in the dissolution

  19. Genome-wide meta-analysis for serum calcium identifies significantly associated SNPs near the calcium-sensing receptor (CASR gene.

    Karen Kapur


    Full Text Available Calcium has a pivotal role in biological functions, and serum calcium levels have been associated with numerous disorders of bone and mineral metabolism, as well as with cardiovascular mortality. Here we report results from a genome-wide association study of serum calcium, integrating data from four independent cohorts including a total of 12,865 individuals of European and Indian Asian descent. Our meta-analysis shows that serum calcium is associated with SNPs in or near the calcium-sensing receptor (CASR gene on 3q13. The top hit with a p-value of 6.3 x 10(-37 is rs1801725, a missense variant, explaining 1.26% of the variance in serum calcium. This SNP had the strongest association in individuals of European descent, while for individuals of Indian Asian descent the top hit was rs17251221 (p = 1.1 x 10(-21, a SNP in strong linkage disequilibrium with rs1801725. The strongest locus in CASR was shown to replicate in an independent Icelandic cohort of 4,126 individuals (p = 1.02 x 10(-4. This genome-wide meta-analysis shows that common CASR variants modulate serum calcium levels in the adult general population, which confirms previous results in some candidate gene studies of the CASR locus. This study highlights the key role of CASR in calcium regulation.

  20. Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

    Zhong Yao

    Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

  1. Effects of calcium channel on 3-morpholinosydnonimine-induced rat hippocampal neuronal apoptosis

    Quanzhong Chang; Shuling Zhang; Yuanyin Zheng; Lijuan Xu; Jinbao Yin; Shining Cai


    Previous studies have demonstrated that increased chloride channel activity plays a role in nitric oxide-induced neuronal apoptosis in the rat hippocampus.The present study investigated the effects of the broad-spectrum calcium channel blocker CdC12 on survival rate, percentage of apoptosis, and morphological changes in hippocampal neurons cultured in vitro, as well as the effects of calcium channels on neuronal apoptosis.The chloride channel blockers 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS) or 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) increased the survival rate of 3-morpholinosydnonimine (SIN-1)-treated neurons and suppressed SIN-1-induced neuronal apoptosis.The calcium channel blocker CdC12 did not increase the survival rate of neurons and did not affect SIN-1-induced apoptosis or SITS- or DIDS-suppressed neuronal apoptosis.Results demonstrated that calcium channels did not significantly affect neuronal apoptosis.

  2. Salmonella induces prominent gene expression in the rat colon

    Roosing Susanne


    Full Text Available Abstract Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point. As fructo-oligosaccharides (FOS affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase, antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2, inflammation (e.g. calprotectin, oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2 and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9. Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap, showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in

  3. Ca2+ signals induced from calcium stores in pancreatic islet β cells


    In single rat pancreatic β cells,using fura-2 microfluorometry to measure [Ca2+]i response upon different stimuli,the ways of calcium regulation have been studied.When the extracellular calcium concentration was 2.5 mmol/L,either 60 mmol/L KCl,20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]i.Such increase in [Ca2+]i was absent when the same stimuli were applied under zero extracellular calcium.These results indicate that the increase of [Ca2+]i is induced by the activation of voltage-dependent calcium channels in β cells.The manifold forms of [Ca2+]i change induced by glucose imply that the effects of glucose are complex.5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+]i ,which is independent of the external calcium,suggesting that [Ca2+]i can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in β cells.The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s).It is concluded that there are multiple calcium stores in rat pancreatic β cells.

  4. Fenvalerate-induced Alterations in Calcium Homeostasis in Rat Ovary


    Objective To observe the effects of fenvalerate on calcium homeostasis in rat ovary. Methods Female SpragueDawley rats were orally given fenvalerate at daily doses of 0.00, 1.91, 9.55, and 31.80 mg/kg for four weeks. The ovary ultrastucture was observed by electron microscopy. Serum free calcium concentration was measured by atomic absorption spectrophotometry. The activities of phosphorylase a in rat ovary were evaluated by the chromatometry. The total content of calmodulin in ovary was estimated by ELISA at each stage of estrous cycle. Radioimmunoassay (RIA) was used to evaluate the level of serum progesterone. Results Histopathologically, damages of ovarian corpus luteum cells were observed. An increase in serum free calcium concentration was observed in rats treated with 31.80mg/kg fenvalerate. The activities of phosphorylase a enhanced in all treated groups, and fenvalerate increased the total content of calmodulin significantly in estrus period. Serum progesterone levels declined in fenvalerate exposed rats in diestrus. Conclusion Fenvalerate interferes with calcium homeostasis in rat ovary. Also, the inhibitory effects of fenvalerate on serum progesterone levels may be mediated partly through calcium signals.

  5. Ethanol induces calcium influx via the Cch1-Mid1 transporter in Saccharomyces cerevisiae.

    Courchesne, William E; Vlasek, Christopher; Klukovich, Rachel; Coffee, Sara


    Yeast suffers from a variety of environmental stresses, such as osmotic pressure and ethanol produced during fermentation. Since calcium ions are protective for high concentrations of ethanol, we investigated whether Ca(2+) flux occurs in response to ethanol stress. We find that exposure of yeast to ethanol induces a rise in the cytoplasmic concentration of Ca(2+). The response is enhanced in cells shifted to high-osmotic media containing proline, galactose, sorbitol, or mannitol. Suspension of cells in proline and galactose-containing media increases the Ca(2+) levels in the cytoplasm independent of ethanol exposure. The enhanced ability for ethanol to induce Ca(2+) flux after the hypertonic shift is transient, decreasing rapidly over a period of seconds to minutes. There is partial recovery of the response after zymolyase treatment, suggesting that cell wall integrity affects the ethanol-induced Ca(2+) flux. Acetate inhibits the Ca(2+) accumulation elicited by the ethanol/osmotic stress. The Ca(2+) flux is primarily via the Cch1 Ca(2+) influx channel because strains carrying deletions of the cch1 and mid1 genes show greater than 90% reduction in Ca(2+) flux. Furthermore, a functional Cch1 channel reduced growth inhibition by ethanol. PMID:21259000

  6. UV-A induces two calcium waves in Physcomitrella patens

    Our understanding of the role of Ca(2+) in blue/UV-A photoreceptor signaling in a single cell is limited. Insight into calcium signaling has now been attained in Physcomitrella patens and its cryptochrome and phototropin knock-outs. Physcomitrella patens caulonemal filaments grow in the dark by apical extension and their apical cells are highly polarized. Fura-2-dextran ratio images of the apical cell from wild type (WT), Ppcry1a/1b and PpphotA2/B1/B2 were obtained immediately following UV-A exposure (30 mu W cm(-2) at 340 nm for 1,000 ms plus 30 mu W cm(-2) at 380 nm for 1,000 ms) [abbreviated as 1,000 ms (340/ 380 nm)] and demonstrated two intracellular waves: a Ca(2+) wave from the growing apical tip through the apical cap, and a wave from the junction of the neighboring cell through the vacuolar, nuclear and plastid regions. In WT, the UV-A-induced tip wave increase had a magnitude of 454.0 +- 40 nM, traveled at a rate of 3.4 +- 0.7 mu m s(-1) and was complete within 26.6 +- 2.3 s, while the basal vacuolar wave had a magnitude of 596.8 +- 110 nM, a rate of 8.4 +- 0.8 mu m s(-1) and duration of 25.3 +- 4.9 s. Subsequent Ca(2+) spikes of similar magnitude followed these waves. The amplitude of the Ca(2+) waves in the apical cap and basal vacuolar regions of Ppcry1a/1b were higher than those in the WT, while the duration of those in PpphotA2/B1/B2 was longer. Subsequent Ca(2+) spikes occurred in WT and Ppcry1a/1b but not in PpphotA2/B1/B2. When Mn(2+) was added to the culture medium, the [Ca(2+)]cyt increase was delayed, did not move as a wave and lasted longer. The results indicate that plants respond to blue light and UV-A radiation by generating a wave of changes in the [Ca(2+)]cyt. The characteristics of these Ca(2+) waves were dependent upon cryptochrome and phototropin. Blue/UV-A signaling in P patens appears to differ from that in Arabidopsis

  7. Gene networks and haloperidol-induced catalepsy

    Iancu, O. D.; Darakjian, P.; Malmanger, B.; Walter, N. A. R.; McWeeney, S.; Hitzemann, R


    The current study examined the changes in striatal gene network structure induced by short-term selective breeding from a heterogeneous stock for haloperidol response. Brain (striatum) gene expression data were obtained using the Illumina WG 8.2 array, and the datasets from responding and non-responding selected lines were independently interrogated using a weighted gene coexpression network analysis (WGCNA). We detected several gene modules (groups of coexpressed genes) in each dataset; the ...

  8. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte


    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts. PMID:19397697

  9. A facile approach to construct hybrid multi-shell calcium phosphate gene particles*

    Xu, Zhi-xue; Zhang, Ran; Wang, You-xiang; Hu, Qiao-ling


    The calcium phosphate (CaP) particles have attracted much attention in gene therapy. How to construct stable gene particles was the determining factor. In this study, hybrid multi-shell CaP gene particles were successfully constructed. First, CaP nanoparticles served as a core and were coated with DNA for colloidal stabilization. The ξ-potential of DNA-coated CaP nanoparticles was −15 mV. Then polyethylenimine (PEI) was added and adsorbed outside of the DNA layer due to the electrostatic attr...

  10. Dietary Fructose Inhibits Intestinal Calcium Absorption and Induces Vitamin D Insufficiency in CKD

    Douard, Veronique; Asgerally, Abbas; Sabbagh, Yves; Sugiura, Shozo; SHAPSES, SUE A.; Casirola, Donatella; Ferraris, Ronaldo P.


    Renal disease leads to perturbations in calcium and phosphate homeostasis and vitamin D metabolism. Dietary fructose aggravates chronic kidney disease (CKD), but whether it also worsens CKD-induced derangements in calcium and phosphate homeostasis is unknown. Here, we fed rats diets containing 60% glucose or fructose for 1 mo beginning 6 wk after 5/6 nephrectomy or sham operation. Nephrectomized rats had markedly greater kidney weight, blood urea nitrogen, and serum levels of creatinine, phos...

  11. Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons


    Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2...

  12. Plasma cytokine concentration changes induced by the antitumor agents dipterinyl calcium pentahydrate (DCP) and related calcium pterins.

    Moheno, Phillip; Pfleiderer, Wolfgang; Fuchs, Dietmar


    Analysis of plasma cytokine concentration changes determined that oral dosing with the antitumor agent (1:4 mol:mol) calcium pterin (CaPterin) increased plasma IL-10, decreased plasma IL-6, and decreased plasma IFN-gamma concentrations in nude mice with MDA-MB-231 xenograph tumors [Moheno, P., Pfleiderer, W., Dipasquale, A.G., Rheingold, A.L., Fuchs, D., 2008. Cytokine and IDO metabolite changes effected by calcium pterin during inhibition of MDA-MB-231 xenograph tumors in nude mice. Int. J. Pharm. 355, 238-248]. A further analysis, reported here, of plasma cytokine concentration changes in nude mice with the same tumor xenographs treated with dipterinyl calcium pentahydrate (DCP), (1:2 mol:mol) calcium pterin, and CaCl(2).2H(2)O has been carried out. The measured cytokines included: IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. The major preliminary findings from the analyses of these data are that (1) the overall relative tumor volumes for the treatments correlated significantly with a full study antitumor plasma cytokine pattern (fsAPCP), a composite measure consisting of decreased plasma IL-6 and increased IL-4 concentrations, and (2) DCP induces a significant threshold antitumor response strongly correlated to a derived DCP antitumor plasma cytokine pattern (DCP/APCP) consisting of plasma IL-12, IL-6, and IL-4 concentration changes. This DCP/APCP composite measure identifies plasma IL-12 concentration increases, plasma IL-6 concentration decreases, and plasma IL-4 concentration increases correlated to relative tumor volume decreases caused by DCP dosing. The finding that the novel calcium pterins and CaCl(2).2H(2)O treatments decrease plasma IL-6 concentrations corroborates the previous finding that CaPterin dosing decreases plasma IL-6 concentrations in this mouse/tumor system [Moheno, P., Pfleiderer, W., Dipasquale, A.G., Rheingold, A.L., Fuchs, D., 2008. Cytokine and IDO metabolite changes effected by calcium pterin during inhibition

  13. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.


    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  14. Radiation-induced gene responses

    In the process of identifying genes that are differentially regulated in cells exposed to ultraviolet radiation (UV), we identified a transcript that was repressed following the exposure of cells to a combination of UV and salicylate, a known inhibitor of NF-kappaB. Sequencing this band determined that it has identify to lactate dehydrogenase, and Northern blots confirmed the initial expression pattern. Analysis of the sequence of the LDH 5' region established the presence of NF-kappaB, Sp1, and two Ap-2 elements; two partial AP- 1; one partial RE, and two halves of E-UV elements were also found. Electromobility shift assays were then performed for the AP-1, NF- kappaB, and E-UV elements. These experiments revealed that binding to NF-kappaB was induced by UV but repressed with salicylic acid; UV did not affect AP-1 binding, but salicylic acid inhibited it alone or following UV exposure; and E-UV binding was repressed by UV, and salicylic acid had little effect. Since the binding of no single element correlated with the expression pattern of LDH, it is likely that multiple elements govern UV/salicylate-mediated expression

  15. The study of serum calcium and serum magnesium in pregnancy induced hypertension and normal pregnancy

    Jagannath Pairu


    Full Text Available Background: Preeclampsia along with its complications is one of the major causes of maternal and fetal mortality and morbidity. Association of calcium and magnesium with pregnancy induced hypertension is known since decades. Evidence of decreased serum calcium and decreased serum magnesium has been observed in patients with pregnancy induced hypertension and has been implicated in the etiopathogenesis of preeclampsia. Methods: The present study was undertaken in 100 pregnant women. Data for the study was collected from 50 normotensive pregnant women with more than 20 weeks of gestational age (control group and 50 pregnancy induced hypertension patients (study group attending for the antenatal care in department of obstetrics and gynaecology in Vanivilas hospital, Bowring and Lady Curzon hospital attached to Bangalore medical college and research institute. Cases and controls were matched. Serum calcium and serum magnesium levels were estimated by spectrophotometry method. Results: The mean serum calcium is significantly lower in pregnancy induced hypertension group (8.15 +/- 0.37 mg/dl compared to normal pregnancy (9.16 +/- 0.82 mg/dl. The mean serum magnesium is lower in pregnancy induced hypertension group (1.78 +/- 0.70 mEq/L than normal pregnancy (2.08 +/- 0.46 mEq/L which is moderately significant. Conclusions: The serum calcium and serum magnesium levels are decreased in pregnancy induced hypertension patients compared to normotensive normal pregnant women, suggesting the possible role of calcium and magnesium in etiopathophysiology of pregnancy induced hypertension. [Int J Reprod Contracept Obstet Gynecol 2015; 4(1.000: 30-34

  16. A genetic polymorphism (rs17251221 in the calcium-sensing receptor gene (CASR is associated with stone multiplicity in calcium nephrolithiasis.

    Yii-Her Chou

    Full Text Available Calcium nephrolithiasis is one of the most common causes of renal stones. While the prevalence of this disease has increased steadily over the last 3 decades, its pathogenesis is still unclear. Previous studies have indicated that a genetic polymorphism (rs17251221 in the calcium-sensing receptor gene (CASR is associated with the total serum calcium levels. In this study, we collected DNA samples from 480 Taiwanese subjects (189 calcium nephrolithiasis patients and 291 controls for genotyping the CASR gene. Our results indicated no significant association between the CASR polymorphism (rs17251221 and the susceptibility of calcium nephrolithiasis. However, we found a significant association between rs17251221 and stone multiplicity. The risk of stone multiplicity was higher in patients with the GG+GA genotype than in those with the AA genotype (chi-square test: P = 0.008; odds ratio  =  4.79; 95% confidence interval, 1.44-15.92; Yates' correction for chi-square test: P = 0.013. In conclusion, our results provide evidence supporting the genetic effects of CASR on the pathogenesis of calcium nephrolithiasis.

  17. Trimetazidine protects cardiomyocytes against hypoxia-induced injury through ameliorates calcium homeostasis.

    Wei, Jinhong; Xu, Hao; Shi, Liang; Tong, Jie; Zhang, Jianbao


    Intracellular calcium (Ca(2+)i) overload induced by chronic hypoxia alters Ca(2+)i homeostasis, which plays an important role on mediating myocardial injury. We tested the hypothesis that treatment with trimetazidine (TMZ) would improve Ca(2+)i handling in hypoxic myocardial injury. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to chronic hypoxia (1% O2, 5% CO2, 37 °C). Intracellular calcium concentration ([Ca(2+)]i) was measured with Fura-2/AM. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). For TMZ-treated cardiomyocytes exposured in hypoxia, we observed a decrease in mRNA expression of proapoptotic Bax, caspase-3 activation and enhanced expression of anti-apoptotic Bcl-2. The cardiomyocyte hypertrophy were also alleviated in hypoxic cardiomyocyte treated with TMZ. Moreover, we found that TMZ treatment cardiomyocytes enhanced "metabolic shift" from lipid oxidation to glucose oxidation. Compared with hypoxic cardiomyocyte, the diastolic [Ca(2+)]i was decreased, the amplitude of Ca(2+)i oscillations and sarcoplasmic reticulum Ca(2+) load were recovered, the activities of ryanodine receptor 2 (RyR2), NCX and SERCA2a were increased in cardiomyocytes treated with TMZ. TMZ attenuated abnormal changes of RyR2 and SERCA2a genes in hypoxic cardiomyocytes. In addition, cholinergic signaling are involved in hypoxic stress and the cardioprotective effects of TMZ. These results suggest that TMZ ameliorates Ca(2+)i homeostasis through switch of lipid to glucose metabolism, thereby producing the cardioprotective effect and reduction in hypoxic cardiomyocytes damage. PMID:25937560

  18. Alpha hemolysin induces an increase of erythrocytes calcium: a FLIM 2-photon phasor analysis approach.

    Susana Sanchez

    Full Text Available α-Hemolysin (HlyA from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin, a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM. Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells.

  19. 5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

    张小玲; 阎宏涛; 闫炀


    With a new fluorescence probe of Ca2+, STDIn-AM, 5-hydroxytryptamino (5-HT)-induced spontaneous calcium release events (calcium sparks) in cultured rat stomach fundus smooth muscle cells (SFSMC) are investigated by laser scanning confocal microscope. The mechanisms of initiation of Ca2+ sparks, propagating Ca2+ waves and their relation to E-C coupling are discussed. After the extracellular [Ca2+] is increased to 10 mmol/L, addition of 5-HT causes hot spots throughout the cytoplasm, which is brighter near the plasmalemma. The amplitude of the event is at least two times greater than the standard deviation of fluorescence intensity fluctuations measured in the neighboring region and the duration of the Ca2+ signal is over 100 ms. The results suggest that 5-HT acts by the way of 5-HT2 receptors on SFSMC, then through 5-HT2 receptors couples IP3/Ca2+ and DG/PKC double signal transduction pathways to cause Ca2+ release from intracellular Ca2+ stores and followed Ca2+ influx possibly through calcium release-activated calcium influx. The acceptor of activated 5-HT2 can also cause membrane depolarization, which then stimulates the L-type Ca2+ channels leading to Ca2+ influx. Thenthe local Ca2+ entry mentioned above activates ryanodine-sensitive Ca2+ releasechannels (RyR) on sarcoplasmic reticulum (SR) to cause local Ca2+ release events (Ca2+ sparks) through calcium-induced calcium release (CICR).

  20. Calcium-Activated Potassium (BK) Channels Are Encoded by Duplicate slo1 Genes in Teleost Fishes

    Rohmann, Kevin N.; Deitcher, David L.; Bass, Andrew H.


    Calcium-activated, large conductance potassium (BK) channels in tetrapods are encoded by a single slo1 gene, which undergoes extensive alternative splicing. Alternative splicing generates a high level of functional diversity in BK channels that contributes to the wide range of frequencies electrically tuned by the inner ear hair cells of many tetrapods. To date, the role of BK channels in hearing among teleost fishes has not been investigated at the molecular level, although teleosts account ...

  1. Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

    Yasuaki Kobayashi

    Full Text Available Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

  2. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David


    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  3. Differential expression of calcium-related genes in gastric cancer cells transfected with cellular prion protein.

    Liang, Jie; Luo, Guanhong; Ning, Xiaoxuan; Shi, Yongquan; Zhai, Huihong; Sun, Shiren; Jin, Haifeng; Liu, Zhenxiong; Zhang, Faming; Lu, Yuanyuan; Zhao, Yunping; Chen, Xiong; Zhang, Hongbo; Guo, Xuegang; Wu, Kaichun; Fan, Daiming


    The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies, which causes prion disorders partially due to Ca2+ dysregulation. In our previous work, we found that overexpressed PrPC in gastric cancer was involved in apoptosis, cell proliferation, and metastasis of gastric cancer. To better understand how PrPC acts in gastric cancer, a human microarray was performed to select differentially regulated genes that correlate with the biological function of PrPC. The microarray data were analyzed and revealed 3798 genes whose expression increased at least 2-fold in gastric cancer cells transfected with PrPC. These genes encode proteins involved in several aspects of cell biology, among which, we specially detected molecules related to calcium, especially the S100 calcium-binding proteins, and found that PrPC upregulates S100A1, S100A6, S100B, and S100P but downregulates CacyBP in gastric cancer cells. We also found that intracellular Ca2+ levels in cells transfected with PrPC increased, whereas these levels decreased in knockdowns of these cells. Taken together, PrPC might increase intracellular Ca2+, partially through calcium-binding proteins, or PrPC might upregulate the expression of S100 proteins, partially through stimulating the intracellular calcium level in gastric cancer. Though the underlying mechanisms need further exploration, this study provides a new insight into the role of PrPC in gastric cancer and enriches our knowledge of prion protein. PMID:17612632

  4. Effect of calcium on strawberry fruit flavonoid pathway gene expression and anthocyanin accumulation.

    Xu, Wenping; Peng, Hui; Yang, Tianbao; Whitaker, Bruce; Huang, Luhong; Sun, Jianghao; Chen, Pei


    Two diploid woodland strawberry (Fragaria vesca) inbred lines, Ruegen F7-4 (red fruit-bearing) and YW5AF7 (yellow fruit-bearing) were used to study the regulation of anthocyanin biosynthesis in fruit. Ruegen F7-4 fruit had similar total phenolics and anthocyanin contents to commercial octoploid (F. × ananassa) cultivar Seascape, while YW5AF7 exhibited relatively low total phenolics content and no anthocyanin accumulation. Foliar spray of CaCl2 boosted fruit total phenolics content, especially anthocyanins, by more than 20% in both Seascape and RF7-4. Expression levels of almost all the flavonoid pathway genes were comparable in Ruegen F7-4 and YW5AF7 green-stage fruit. However, at the turning and ripe stages, key anthocyanin structural genes, including flavanone 3-hydroxylase (F3H1), dihydroflavonol 4-reductase (DFR2), anthocyanidin synthase (ANS1), and UDP-glucosyltransferase (UGT1), were highly expressed in Ruegen F7-4 compared with YW5AF7 fruit. Calcium treatment further stimulated the expression of those genes in Ruegen F7-4 fruit. Anthocyanins isolated from petioles of YW5AF7 and Ruegen F-7 had the same HPLC-DAD profile, which differed from that of Ruegen F-7 fruit anthocyanins. All the anthocyanin structural genes except FvUGT1 were detected in petioles of YW5AF7 and Ruegen F-7. Taken together, these results indicate that the "yellow" gene in YW5AF7 is a fruit specific regulatory gene(s) for anthocyanin biosynthesis. Calcium can enhance accumulation of anthocyanins and total phenolics in fruit possibly via upregulation of anthocyanin structural genes. Our results also suggest that the anthocyanin biosynthesis machinery in petioles is different from that in fruit. PMID:25036468

  5. Transcription Factor Oscillations Induce Differential Gene Expressions

    Wee, Keng Boon; Yio, Wee Kheng; Surana, Uttam; Chiam, Keng Hwee


    Intracellular protein levels of diverse transcription factors (TFs) vary periodically with time. However, the effects of TF oscillations on gene expression, the primary role of TFs, are poorly understood. In this study, we determined these effects by comparing gene expression levels induced in the presence and in the absence of TF oscillations under same mean intracellular protein level of TF. For all the nonlinear TF transcription kinetics studied, an oscillatory TF is predicted to induce ge...

  6. Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

    A. Pedro Gonçalves


    Full Text Available Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS. Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.

  7. Transcription factor oscillations induce differential gene expressions.

    Wee, Keng Boon; Yio, Wee Kheng; Surana, Uttam; Chiam, Keng Hwee


    Intracellular protein levels of diverse transcription factors (TFs) vary periodically with time. However, the effects of TF oscillations on gene expression, the primary role of TFs, are poorly understood. In this study, we determined these effects by comparing gene expression levels induced in the presence and in the absence of TF oscillations under same mean intracellular protein level of TF. For all the nonlinear TF transcription kinetics studied, an oscillatory TF is predicted to induce gene expression levels that are distinct from a nonoscillatory TF. The conditions dictating whether TF oscillations induce either higher or lower average gene expression levels were elucidated. Subsequently, the predicted effects from an oscillatory TF, which follows sigmoid transcription kinetics, were applied to demonstrate how oscillatory dynamics provide a mechanism for differential target gene transactivation. Generally, the mean TF concentration at which oscillations occur relative to the promoter binding affinity of a target gene determines whether the gene is up- or downregulated whereas the oscillation amplitude amplifies the magnitude of the differential regulation. Notably, the predicted trends of differential gene expressions induced by oscillatory NF-κB and glucocorticoid receptor match the reported experimental observations. Furthermore, the biological function of p53 oscillations is predicted to prime the cell for death upon DNA damage via differential upregulation of apoptotic genes. Lastly, given N target genes, an oscillatory TF can generate between (N-1) and (2N-1) distinct patterns of differential transactivation. This study provides insights into the mechanism for TF oscillations to induce differential gene expressions, and underscores the importance of TF oscillations in biological regulations. PMID:22713556

  8. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello


    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  9. Formation of ring calcium oxalate patterns induced by domains in DPPC Langmuir-Blodgett films

    Yi Ming Liu; Sui Ping Deng; Hui Zheng; Jian Ming Ouyang


    The ring patterns of calcium oxalate crystals were induced by domains in Langmuir-Blodgett (LB) films of dipalmitoylpho-sphatidylcholine (DPPC). The result was explained by the defects at the ring boundaries of liquid condensed (LC) and liquid expanded (LE) phases of LB film. These boundaries could provide less free energy and much more nucleating sites for COM crystals.

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

    Salomonsson, Max; Arendshorst, William J


    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 ratiom...

  11. BMP-2 and ALP gene expression induced by a BMP-2 gene-fibronectin-apatite composite layer

    Wang Xiupeng; Sogo, Yu; Li Xia; Ito, Atsuo [Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Oyane, Ayako [Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Tsurushima, Hideo, E-mail:, E-mail: [Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575 (Japan)


    The bone morphogenetic protein 2 (BMP-2) gene delivery system with a gene-fibronectin (Fn)-apatite composite layer was fabricated on the surface of a hydroxyapatite ceramic scaffold. The BMP-2 gene-Fn-apatite composite layer was coated on the scaffold using a supersaturated calcium phosphate solution supplemented with BMP-2 DNA and Fn. The scaffolds were ectopically implanted into the dorsal subcutaneous tissue of rats. Four weeks after the implantation, the hydroxyapatite scaffold coated with the BMP-2 gene-Fn-apatite composite layer showed improved gene expressions of BMP-2 and alkaline phosphatase as compared with the scaffold coated with the apatite layer. Although these results suggest the possibility of ectopic bone formation induced by the present gene delivery system, further study is necessary to prove this.

  12. Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

    Highlights: ► Infra-red laser beam generates microscopic heat pulses. ► Heat pulses induce contraction of cardiomyocytes. ► Ca2+ transients during the contraction were not detected. ► Skinned cardiomyocytes in free Ca2+ solution also contracted. ► Heat pulses regulated the contractions without Ca2+ dynamics. -- Abstract: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heat pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, ΔT, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, ΔT at physiological temperature was lower than that at room temperature. Ca2+ transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca2+ solution. This heat pulse-induced Ca2+-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.

  13. Fas mRNA expression and calcium influx change in H2O2-induced apoptotic hepatocytes in vitro

    Qi-Ping Lu; Lei Tian


    AIM: To investigate the relationship between Fas gene expression and calcium influx change in peroxide-induced apoptotic hepatocytes and the possible molecular mechanism of Rxa in protecting hepatocytes.METHODS: Single-cell Fas mRNA expression in H2O2-exposed L02 hepatocytes with or without treatment of Rxa,an extract from an anti-peroxidant, Radix Salviae Miltiorrhizae,was determined by all-cell patch clamp and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Transient calcium influx change ([Ca2+]i) in the cells was evaluated with all-cell patch clamp micro-fluorescence single-cytosolic free Ca2+ concentration technique. Fas protein expression, early apoptotic index (annexin-V+) and cell membrane change inthe cells were evaluated by immunohistochemistry, flow cytometry (FCM) and scan electron microscopy respectively.RESULTS: In cells exposed to H2O2 for 2 h, the specific lane for Fas mRNA was vivid on electrophoresis, with increased Fas protein expression, [Ca2+]i (from 143.66±34.21 to 1115.28±227.16), annexin-V+ index (from 4.00±0.79 to 16.18±0.72) and membrane vesicle formation. However, in cells exposed to H2O2 but pre-treated with Rxa, there was no increase in Fas mRNA or protein expression and [Ca2+]i (103.56±28.92). Annexin-V+ index (8.92±1.44) was lower than the controls (P<0.01), and the cell membrane was intact.CONCLUSION: H2O2 induces apoptosis of L02 cells by increasing cytosolic [Ca2+]i, and inducing Fas mRNA and protein expression. Rxa protects the L02 cells from apoptosis through anti-peroxidation, inhibition of calcium overloading and prevention of the activation of cytosolic Fas signal pathway.

  14. Heat induces gene amplification in cancer cells

    Highlights: ► This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. ► Hyperthermia induces DNA double strand breaks. ► DNA double strand breaks are considered to be required for the initiation of gene amplification. ► The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 °C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) γH2AX immunostaining to detect γH2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 °C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 °C for 30 min induces DNA double strand breaks in HCT116 cells as shown by γH2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

  15. Heat induces gene amplification in cancer cells

    Yan, Bin, E-mail: [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)


    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  16. Serca1 Truncated Proteins Unable to Pump Calcium Reduce the Endoplasmic Reticulum Calcium Concentration and Induce Apoptosis

    Chami, Mounia; Gozuacik, Devrim; Lagorce, David; Brini, Marisa; Falson, Pierre; Peaucellier, Gérard; Pinton, Paolo; Lecoeur, Hervé; Gougeon, Marie-Lyse; le Maire, Marc; Rizzuto, Rosario; Bréchot, Christian; Paterlini-Bréchot, Patrizia


    By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human...

  17. Effects of Arecoline on Calcium Channel Currents and Caffeine-induced Calcium Release in Isolated Single Ventricular Myocyte of Guinea Pig

    林先明; 李真; 胡本容; 夏国瑾; 姚伟星; 向继洲


    Summary: The effects of Arecoline (Are) on calcium mobilization were investigated. In isolatedsingle ventricular myocyte of guinea pig, patch clamp whole cell recording techniques were used torecord the current of L-type calcium channel and cytosolic Ca2+ level ([Ca2+]i) labeled with fluo-rescence probe Fluo-3/AM was measured under a laser scanning confocal microscope. Results re-vealed that Are (3-100 μmol/L) could inhibit L-type calcium current in a concentration-depen-dent manner and the value of IC50 was 33. 73μmol/L (n= 5). In the absence of extracellular calci-um, the resting levels of [Ca2+]i was not affected by Are (n=6, P>0. 05), but pretreatmentwith Are (30 μmol/L) could significantly inhibit the [Ca2+]i elevation induced by caffeine (10mmol/L, n = 6, P < 0. 01). It was concluded that Are could inhibit not only calcium influxthrough L-type calcium channel but also calcium release from sarcoplasmic reticulum.

  18. A Single Nucleotide Polymorphism (rs4236480 in TRPV5 Calcium Channel Gene Is Associated with Stone Multiplicity in Calcium Nephrolithiasis Patients

    Anas Khaleel


    Full Text Available Nephrolithiasis is characterized by calcification of stones in the kidneys from an unknown cause. Animal models demonstrated the functional roles of the transient receptor potential vanilloid member 5 (TRPV5 gene in calcium renal reabsorption and hypercalciuria. Therefore, TRPV5 was suggested to be involved in calcium homeostasis. However, whether genetic polymorphisms of TRPV5 are associated with kidney stone multiplicity or recurrence is unclear. In this study, 365 Taiwanese kidney-stone patients were recruited. Both biochemical data and DNA samples were collected. Genotyping was performed by a TaqMan allelic discrimination assay. We found that a TRPV5 polymorphism (rs4236480 was observed to be associated with stone multiplicity of calcium nephrolithiasis, as the risk of stone multiplicity was higher in patients with the TT+CT genotype than in patients with the CC genotype (p=0.0271. In summary, despite the complexity of nephrolithiasis and the potential association of numerous calcium homeostatic absorption/reabsorption factors, TRPV5 plays an important role in the pathogenesis of calcium nephrolithiasis.

  19. Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus

    Yin Weilun


    Full Text Available Abstract Background CBL1 is a calcium sensor that regulates drought, cold and salt signals in Arabidopsis. Overexpression of CBL1 gene in Arabidopsis and in Ammopiptanthus mongolicus showed different tolerant activities. We are interested in understanding the molecular mechanism of the upstream region of the CBL1 gene of A. mongolicus (AmCBL1. We investigated and characterized the promoter of the AmCBL1 gene, for promoters play a very important role in regulating gene expression in eukaryotes. Results A 1683-bp 5' flanking region was isolated from A. mongolicus. The sequence was identified as AmCBL1 promoter. Analysis of the promoter sequence indicated a 690-bp intron and some basic cis-acting elements were related to various environmental stresses and plant hormones. To identify the functional region of the AmCBL1 promoter, five plant expression vectors fused with the GUS (β-glucuronidase gene, driven by series deleted fragments of AmCBL1 promoter at different lengths from -1659, -1414, -1048, -296 to -167 bp relative to the transcriptional start site were constructed and transformed into Nicotiana tabacum L. cv. 89. Functional properties of each promoter segment were examined by GUS staining and fluorescence quantitative analyses using at least three single-copy PCR-positive plants of transgenic tobacco, treated with various environmental stresses and plant hormones for different times. We demonstrated that the AmCBL1 promoter was a vascular-specific and multiple-stress-inducible promoter. Our results further imply that the promoter fragment B1S3 possessed sufficient essential cis-acting elements, accounting for vascular-specific and stress-induced expression patterns. It may also indicate that for response to some stresses certain cis-elements are required in tissues outside the region of the B1S3 construct. Conclusions To help resolve uncertainties about the upstream regulatory mechanism of the CBL1 gene in desert plants, we suggest that

  20. Inhibitory effect of rutin and curcumin on experimentally-induced calcium oxalate urolithiasis in rats

    Jaydip Ghodasara


    Full Text Available Background: Renal epithelial cell injury by reactive oxygen species is pre-requisite step in the pathogenesis of urolithiasis. Rutin and curcumin are polyphenolic compounds known to have antioxidant and anti-inflammatory activities, but their effect on urolithiasis is yet to be elucidated. In the present study, we have investigated the inhibitory effect of rutin and curcumin on calcium oxalate urolithiasis in Wistar albino rats. Methods: Calcium oxalate urolithiasis was induced experimentally by administration of 0.75% v/v ethylene glycol with 1% w/v ammonium chloride in drinking water for three days followed by only 0.75% v/v ethylene glycol for 25 days. Rutin (20 mg/kg body weight and curcumin (60 mg/kg body weight were given once daily for 28 days by oral route. After treatment period, calcium and oxalate levels in urine and kidney tissue homogenate were measured. Kidney was also used for histopathological examination. Results: Stone-induction with ethylene glycol and ammonium chloride resulted in elevated levels of calcium and oxalate in the urine and kidney sample, whereas supplementation of rutin and curcumin restored it near to normal. Histopathological study revealed minimum tissue damage and less number of calcium oxalate deposits in kidney of animal treated with rutin and curcumin as compared to calculi-induced animal. Conclusion: The data suggest that the rutin and curcumin inhibits calcium oxalate urolithiasis. This effect is mediated possibly through a lowering of urinary concentration of stone forming constituents, anti-inflammatory and antioxidant effects.

  1. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng


    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α’, α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

  2. Plasma Calcium, Inorganic Phosphate and Magnesium During Hypocalcaemia Induced by a Standardized EDTA Infusion in Cows

    Enemark JMD


    Full Text Available The intravenous Na2EDTA infusion technique allows effective specific chelation of circulating Ca2+ leading to a progressive hypocalcaemia. Methods previously used were not described in detail and results obtained by monitoring total and free ionic calcium were not comparable due to differences in sampling and analysis. This paper describes a standardized EDTA infusion technique that allowed comparison of the response of calcium, phosphorus and magnesium between 2 groups of experimental cows. The concentration of the Na2EDTA solution was 0.134 mol/l and the flow rate was standardized at 1.2 ml/kg per hour. Involuntary recumbency occurred when ionised calcium dropped to 0.39 – 0.52 mmol/l due to chelation. An initial fast drop of ionized calcium was observed during the first 20 min of infusion followed by a fluctuation leading to a further drop until recumbency. Pre-infusion [Ca2+] between tests does not correlate with the amount of EDTA required to induce involuntary recumbence. Total calcium concentration measured by atomic absorption remained almost constant during the first 100 min of infusion but declined gradually when the infusion was prolonged. The concentration of inorganic phosphate declined gradually in a fluctuating manner until recumbency. Magnesium concentration remained constant during infusion. Such electrolyte responses during infusion were comparable to those in spontaneous milk fever. The standardized infusion technique might be useful in future experimental studies.

  3. RNA-induced silencing attenuates G protein-mediated calcium signals.

    Philip, Finly; Sahu, Shriya; Golebiewska, Urszula; Scarlata, Suzanne


    Phospholipase Cβ (PLCβ) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCβ is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCβ raises the possibility that RNA silencing activity can affect the ability of PLCβ to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCβ stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCβ-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCβ-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals. PMID:26862135

  4. Transition in complex calcium bursting induced by IP3 degradation

    Complex intracellular Ca2+ oscillations are systematically investigated in a mathematical model based on the mechanism of Ca2+-induced Ca2+ release (CICR), taking account of the Ca2+-stimulated degradation of inositol 1,4,5-trisphosphate (IP3) by a 3-kinase. Periodic, quasi-periodic and chaotic bursting oscillations exist in a wide range of parameter values and occur alternatively as the parameters change slightly. The transition among them can be observed by the evidence in their interspike interval and the Lyapunov exponent. These results reveal the role of agonist-stimulated of IP3 degradation as a possible source for complex patterns in Ca2+ signaling.

  5. Role of Calcium Ion in Apoptosis of MD Cancer Cells Induced by Arsenic Trioxide

    ZHANG Jiuli; WANG Jintao; XU Shiwen


    In order to observe the role of calcium ion in apoptosis of MD cancer cells induced by arsenic trioxide, inhibition percentage was detected by MTT assay;morphology changes were examined by fluorescence microscope;apoptosis was examined by DNA Ladder;[Ca2+]i was investigated by spectrofluorimeter in vitro on MDCC-MSB1 cells. The results showed that As2O3 inhibited the proliferation of MDCC-MSB1 cells in concentration dependent manner (P<0.05 or P<0.01);typical apoptosis character was observed by fluorescence microscope;DNA Ladder was observed;the [Ca2+]i was elevated significantly after the treatment of As203 (P<0.05 or P<0.01) and showed a dose-dependent manner. It is concluded that the calcium may play an important role in apoptosis of MD cancer cells induced by arsenic trioxide.

  6. Calcium hydroxide induced apical barrier in fractured nonvital immature permanent incisors

    Vellore K


    Full Text Available Management of trauma to an immature permanent incisor is a challenge, especially when the pulp is necrotic. The aim of this study is to discuss the management of fractured maxillary right and left central incisors with immature root apex. Radiographic evaluation revealed that the tooth 11 had convergent apical walls, whereas tooth 21 had parallel apical walls; both the incisors were treated with pure calcium hydroxide paste to induce apical development. Follow-up clinical and radiographic examinations confirmed apical barrier at 3 months. Radiograph of tooth 11 with convergent roots showed continued apical development, whereas tooth 21 showed apical bridging, following which root canal was completed in both the teeth. Therefore, in view of the simplicity of this method, we believe that calcium hydroxide can be reemphasized as one of the treatment alternatives for fractured nonvital immature permanent incisors to induce apical barrier.

  7. Calcium channel blocker prevents stress-induced activation of renin and aldosterone in conscious pig

    A considerable amount of data suggest the involvement of calcium-mediated processes in the activation of the renin-angiotensin-aldosterone (RAA) cascade. To investigate the effect of calcium-channel inhibition on the RAA system, the authors studied 21 conscious pigs. Blood renin and aldosterone levels increased by subjecting animals to 24 hours of immobilization stress. Renin and aldosterone levels were repeatedly measured by radioimmunoassay in blood samples taken periodically over 24 hours from a chronically implanted arterial cannula. Pretreatment of the animals (N = 11) with nisoldipine, 2 x 20 mg p.o. daily for 2 days before and on the day of immobilization, transiently attenuated the stress-induced increase of plasma renin activity and completely prevented the rise of aldosterone, as compared to nontreated controls (N = 10). The finding that nisoldipine suppresses RAA activation induced by a nonpharmacologic stimulus in the conscious intact animal may have clinical implications

  8. Circular patterns of calcium oxalate monohydrate induced by defective Langmuir-Blodgett film on quartz substrates

    The defective Langmuir-Blodgett (LB) film of dipalmitoylphosphatidylcholine (DPPC) on quartz injured by potassium oxalate (K2C2O4) was used as a model system to induce growth of calcium oxalate crystals. Atomic force microscopy (AFM) indicated that circular defective domains with a diameter of 1-200 μm existed in the LB film. Scanning electron microscopy (SEM) showed circular patterns of aggregated calcium oxalate monohydrate (COM) crystallites were induced by these defective domains. It was ascribed to that the interaction between the negatively-charged oxalate ions and the phosphatidyl groups in DPPC headgroups makes the phospholipid molecules rearranged and exist in an out-of-order state in the LB film, especially at the boundaries of liquid-condensed (LC)/liquid-expanded (LE) phases, which provide much more nucleating sites for COM crystals

  9. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism

    Millay, Douglas P.; Goonasekera, Sanjeewa A.; Sargent, Michelle A.; Maillet, Marjorie; Aronow, Bruce J; Molkentin, Jeffery D.


    Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx ...

  10. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis.

    Jiang, Shanshan; Zhang, Dan; Wang, Li; Pan, Jiaowen; Liu, Yang; Kong, Xiangpei; Zhou, Yan; Li, Dequan


    Calcium-dependent protein kinases (CDPKs) play essential roles in calcium-mediated signal transductions in plant response to abiotic stress. Several members have been identified to be regulators for plants response to abscisic acid (ABA) signaling. Here, we isolated a subgroup I CDPK gene, ZmCPK4, from maize. Quantitative real time PCR (qRT-PCR) analysis revealed that the ZmCPK4 transcripts were induced by various stresses and signal molecules. Transient and stable expression of the ZmCPK4-GFP fusion proteins revealed ZmCPK4 localized to the membrane. Moreover, overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth and stomatal movement. The transgenic plants also enhanced drought stress tolerance. Taken together, the results suggest that ZmCPK4 might be involved in ABA-mediated regulation of stomatal closure in response to drought stress. PMID:23911729

  11. Sodium induces simultaneous changes in cytosolic calcium and pH in salt-tolerant quince protoplasts.

    D'Onofrio, Cladio; Lindberg, Sylvia


    Previous experiments with salt-resistant quince BA29 (Cydonia oblonga cv. Mill.) have shown that this cultivar takes up sodium transiently into the cytosol of shoot protoplasts only in the absence of calcium chloride, or at or =100mM to single protoplasts from in vitro-cultivated quince in the presence of 1.0mM calcium induced instant changes in the cytosolic concentrations of calcium and protons. These changes were investigated by use of tetra [acetoxymethyl] esters of the fluorescent stilbene chromophores Fura 2 and bis-carboxyethyl-carboxyfluorescein (BCECF), respectively. The cytosolic Ca(2+) dynamics in the protoplasts were dependent on the concentration of NaCl added. The changes in calcium differed in amplitude and final concentration and were correlated in time mainly with changes in pH. Addition of 100-400mM NaCl to the protoplasts caused an oscillating increase in the cytosolic level of calcium, and then a decrease. Addition of mannitol, of equiosmolar concentration to NaCl, did not increase the cytosolic calcium concentration. Moreover, there was no increase in cytosolic calcium when NaCl was added in the presence of calcium binding ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA), or lantan or verapamil, two inhibitors of plasma membrane calcium channels. Therefore, we conclude that, in salt-resistant quince, sodium induces an influx of calcium into the cytosol by plasma membrane calcium channels, and a simultaneous increase in cytosolic pH. Because these changes were obtained in the presence of 1mM calcium in the medium, they were not due to sodium uptake into the cytosol. PMID:19556023

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

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


    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  13. Osteogenesis-inducing calcium phosphate nanoparticle precursors applied to titanium surfaces

    This study investigated the effects of the morphology and physicochemical properties of calcium phosphate (CaP) nanoparticles on osteogenesis. Two types of CaP nanoparticles were compared, namely amorphous calcium phosphate (ACP) nano-spheres (diameter: 9–13 nm) and poorly crystalline apatite (PCA) nano-needles (30–50 nm × 2–4 nm) that closely resemble bone apatite. CaP particles were spin-coated onto titanium discs and implants; they were evaluated in cultured mouse calvarial osteoblasts, as well as after implantation in rabbit femurs. A significant dependence of CaP coatings was observed in osteoblast-related gene expression (Runx2, Col1a1 and Spp1). Specifically, the PCA group presented an up-regulation of the osteospecific genes, while the ACP group suppressed the Runx2 and Col1a1 expression when compared to blank titanium substrates. Both the ACP and PCA groups presented a more than three-fold increase of calcium deposition, as suggested by Alizarin red staining. The removal torque results implied a slight tendency in favour of the PCA group. Different forms of CaP nanostructures presented different biologic differences; the obtained information can be used to optimize surface coatings on biomaterials. (paper)

  14. Calmodulin-dependent protein kinases mediate calcium-induced slow motility of mammalian outer hair cells.

    Puschner, B; Schacht, J


    Cochlear outer hair cells in vitro respond to elevation of intracellular calcium with slow shape changes over seconds to minutes ('slow motility'). This process is blocked by general calmodulin antagonists suggesting the participation of calcium/calmodulin-dependent enzymatic reactions. The present study proposes a mechanism for these reactions. Length changes of outer hair cells isolated from the guinea pig cochlea were induced by exposure to the calcium ionophore ionomycin. ATP levels remained unaffected by this treatment ruling out depletion of ATP (by activation of calcium-dependent ATPases) as a cause of the observed shape changes. Involvement of protein kinases was suggested by the inhibition of shape changes by K252a, a broad-spectrum inhibitor of protein kinase activity. Furthermore, the inhibitors ML-7 and ML-9 blocked the shape changes at concentrations compatible with inhibition of myosin light chain kinase (MLCK). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), also attenuated the length changes. Inhibitors with selectivity for cyclic nucleotide-dependent protein kinases (H-89, staurosporine) were tested to assess potential additional contributions by such enzymes. The dose dependence of their action supported the notion that the most likely mechanism of slow motility involves phosphorylation reactions catalyzed by MLCK or CaMKII or both. PMID:9282907

  15. The role of prenucleation clusters in surface-induced calcium phosphate crystallization

    Dey, Archan; Bomans, Paul H. H.; Müller, Frank A.; Will, Julia; Frederik, Peter M.; de With, Gijsbertus; Sommerdijk, Nico A. J. M.


    Unravelling the processes of calcium phosphate formation is important in our understanding of both bone and tooth formation, and also of pathological mineralization, for example in cardiovascular disease. Serum is a metastable solution from which calcium phosphate precipitates in the presence of calcifiable templates such as collagen, elastin and cell debris. A pathological deficiency of inhibitors leads to the uncontrolled deposition of calcium phosphate. In bone and teeth the formation of apatite crystals is preceded by an amorphous calcium phosphate (ACP) precursor phase. ACP formation is thought to proceed through prenucleation clusters-stable clusters that are present in solution already before nucleation-as was recently demonstrated for CaCO3 (refs 15,16). However, the role of such nanometre-sized clusters as building blocks for ACP has been debated for many years. Here we demonstrate that the surface-induced formation of apatite from simulated body fluid starts with the aggregation of prenucleation clusters leading to the nucleation of ACP before the development of oriented apatite crystals.

  16. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.

    Dan Zhang

    Full Text Available BACKGROUND: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. METHODOLOGY/PRINCIPAL FINDINGS: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR or Catestatin (CAT induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate, a store operated channels (SOCs blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. CONCLUSIONS/SIGNIFICANCE: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated i

  17. Comparison of calcium phosphate coatings formed on femtosecond laser-induced and sand-blasted titanium

    Liang, C. Y.; Yang, X. J.; Wei, Q.; Cui, Z. D.


    High energy femtosecond laser process was employed to create regular surface patterning on titanium while sand blasting treatment made a coarse surface. Both laser-induced titanium and blasted titanium could promote the formation of calcium phosphate compounds after the acid and alkali treatment, but little crystallized hydroxyapatite was grown on the laser-induced titanium in 1.5SBF only for 6 h, whereas Ca 4P 6O 19 was formed on the sand-blasted titanium. The femtosecond laser process together with common acid and alkali treatment might provide potential choice to enhance the biocompatibility of titanium and its alloys.

  18. P12 - PTHC1: A Continuing Cell Line Expressing PTH and Genes Involved in Calcium Homeostasis

    Fabbri, S.; Mazzotta, C.; Ciuffi, S.; Mavilia, C.; Galli, G.; Zonefrati, R; Strigoli, D.; Cavalli, L.; Cavalli, T.; Brandi, M.L.


    The main organs regulating serum levels of ionised calcium (Ca2+) are the parathyroids, which are composed of two different cell types: chief cells and oxyphil cells. Chief cells, through the calcium sensing receptor (CaSR), are affected by changes in calcium concentration, modifying PTH secretion in proportion to calcium levels. Current understanding of calcium regulation mechanisms connected to PTH and of the signalling pathways involved derive from in vitro studies carried out on primary c...

  19. Dissecting the calcium-induced differentiation of human primary keratinocytes stem cells by integrative and structural network analyses.

    Kiana Toufighi


    Full Text Available The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55% are composed of non-dynamic and dynamic gene products ('di-chromatic', 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation.

  20. Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

    Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina


    The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651


    马欣; 厉英倩; 白宇飞; 刘明


    Objective To explore the toxic effects of mercuric chloride (HgCl2) on vascular smooth muscle as well as its relationship to calcium antagonist. Methods By using isolated vascular tension methods, we studied the effect of HgCl2 on isolated rabbit aortic rings. Results HgCl2 (1-100μmol*L-1) caused a concentration-dependent contraction of rabbit aortic rings, which did not change with phentolamin or without endothelium. In KH solution with Ca2+ , the maximum contraction amplitude reduced by(61.2±3.3)%. Nifedipine produced a concentration-dependent decrease of the maximum contraction amplitude. Conclusion Calcium antagonist has protective effects on vascular smooth muscle against damage induced by HgCl2.

  2. Mechanism for Alternating Electric Fields Induced-Effects on Cytosolic Calcium

    LUO Ming-Yan; SONG Kun; ZHANG Xu; LEE Imshik


    In this work, from the point of calcium ions in the cytosol, we extend a Vm-[Ca2+]cyt model to explain the changes of action potential Vm of the plasma membrane and the calcium concentration in the cytosol [Ca2+]cyt under an alternating electric field in cells. An alternating external electric field may exert an oscillating force to each of the free electrolytes, existing on both sides of the plasma membrane. The mechanism for the alternating electric field induced-effects on Vm and [Ca2+]cyt is elucidated. The simulation results show a correlation between the changes of [Ca2+]cyt and the alternating electric field. When the numerical ratio between the intensity Eo(mV/m) and the frequency ν, (Hz) of the field was about 1-2, the [Ca2+]cyt signal is changed dramatically. The bioactive changes of [Ca2+]cyt appear at low frequency, in the range of 0-100 Hz.

  3. Spontaneous calcium signals induced by gap junctions in a network model of astrocytes

    Kazantsev, V. B.


    The dynamics of a network model of astrocytes coupled by gap junctions is investigated. Calcium dynamics of the single cell is described by the biophysical model comprising the set of three nonlinear differential equations. Intercellular dynamics is provided by the diffusion of inositol 1,4,5-trisphosphate (IP3) through gap junctions between neighboring astrocytes. It is found that the diffusion induces the appearance of spontaneous activity patterns in the network. Stability of the network steady state is analyzed. It is proved that the increase of the diffusion coefficient above a certain critical value yields the generation of low-amplitude subthreshold oscillatory signals in a certain frequency range. It is shown that such spontaneous oscillations can facilitate calcium pulse generation and provide a certain time scale in astrocyte signaling.

  4. Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus.

    Alberto Muñoz

    Full Text Available Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

  5. Specific gene mutations induced by heavy ions

    This report summarizes our heavy-ion research rationale, progress, and plans for the near future. The major project involves selecting a group of maize Adh1 mutants induced by heavy ions and correlating their altered behavior with altered DNA nucleotide sequences and sequence arrangements. This research requires merging the techniques of classical genetics and recombinant DNA technology. Our secondary projects involve (1) the use of the Adh gene in the fruit fly, Drosophila melanogaster, as a second system with which to quantify the sort of specific gene mutants induced by heavy ions as compared to x rays, and (2) the development of a maize Adh1 pollen in situ monitor for environmental mutagens

  6. Tetracycline inducible gene manipulation in serotonergic neurons.

    Tillmann Weber

    Full Text Available The serotonergic (5-HT neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA mouse line (TPH2-tTA that allows temporal and spatial control of tetracycline (Ptet controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ. In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox. Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20 were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We

  7. HIFU-induced gene activation in vitro

    Liu, Yunbo; Zhong, Pei; Kon, Takashi; Li, Chuanyuan


    This work investigated the inducible gene activation in cancer cells that were sublethally injured during HIFU treatment. HeLa cells were transfected by an adenovirus vector that encodes GFP under the control of hsp70B promoter, leading to about 65% transfection efficiency. A volume of 10 μL transfected HeLa cells in suspension (5×107 cells/ml) were placed at the bottom of a PCR tube so that the cell suspension could be heated to a peak temperature of 50°C, 60°C, and 70°C for 120, 10, and 1 s, respectively, by a focused 1.1-MHz HIFU transducer operated at a peak negative pressure of -2.7 MPa at different duty cycles. One day after HIFU treatment, cell viability was determined to be 63%, 35%, and 18%, respectively, based on Trypan Blue exclusion test. Importantly, in all test groups, inducible GFP expression was detected in about 40%-50% of the surviving cells with GFP intensity increased by 25-fold based on flow cytometry analysis. These results demonstrate that even under the short exposure duration of HIFU treatment, inducible gene expression could be produced in sublethally injured cell population in vitro. Further studies are underway to explore the optimal HIFU condition for gene activation in vivo.

  8. Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

    Rogers, E J; Lovett, P S


    The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.

  9. Dysfunction and changes of gene expression in sarcoplasmic reticulum calcium pump in endotoxin-induced diaphragm%内毒素血症大鼠膈肌功能障碍和肌浆网钙泵基因表达的变化

    方迎艳; 郭晓磊; 马丽; 高琴; 叶红伟; 关宿东


    Objective To investigate endotoxemia inducing the changes of diaphragm dysfunction and gene expression in sarcoplasmic reticulum calcium pump in rats. Methods Rats were given saline (0. 5 ml ip, saline control group) and endotoxin (12 mg/kg ip, en-dotoxin group) respectively. Animals were killed at 24, 48 and 96 hours after injections. Assessments were made of the diaphragm contractility, such as peak twitch tension (Pt) , maximum tetanic tension (Po) , time to peak contraction (CT) , half relaxation time ( 1/2RT) and diaphragm force-frequency relationships, and the change of the ultrastructures and the content of sarcoplasmic reticulum calcium pump (SERCA mRNA) was analyzed by reverse transcriptase polymerize chain reactive. Results Compared with control group, Pt and Po of endotoxin group were lower (P <0. 01) , CT and 1/2RT of endotoxin group were significantly longer (P <0. 01) ; Tetanic force under the stimulus frequency of 10, 20, 40, 60, 100 Hz in endotoxin group were decreased significantly (P<0. 01). Transmission electron microscopic morphometry of diaphragm in endotoxin group revealed diaphragm myoneme confused and broken, sarcoplasmic reticulum was distended, the quantity of mitochondria was decreased, mitochondria edema and expanded, its cristae broken and vague, a great quantity of mitochondria vacuolization or vesiculation. The expression of SERCA mRNA in diaphragm was lower in endotoxin group than that of control group (P < 0.01). Conclusions Endotoxin destroys the diaphragmatic ultrastructure and induces diaphragmatic dysfunction, it could also decrease SERCA mRNA contents.%目的 探讨内毒素血症大鼠膈肌功能和膈肌肌浆网钙泵基因(SERCA)表达的变化.方法 直接采用腹腔注射内毒素12 mg/kg建立大鼠内毒素血症模型,32只成年雄性SD大鼠随机分成四组:生理盐水对照组和内毒素24 h组,48 h组,96 h组,即分别在注射内毒素24、48、96 h后处死大鼠,应用体外灌流大鼠膈肌条的方

  10. A model of calcium signaling and degranulation dynamics induced by laser irradiation in mast cells

    SHI XiaoMin; ZHENG YuFan; LIU ZengRong; YANG WenZhong


    Recent experiments show that calcium signaling and degranulation dynamics induced by low power laser irradiation in mast cells must rely on extracellular Ca2+ influx. An analytical expression of Ca2+ flux through TRPV4 cation channel in response to interaction of laser photon energy and extracellular Ca2+ is deduced, and a model characterizing dynamics of calcium signaling and degranulation activated by laser irradiation in mast cells is established. The model indicates that the characteristics of calcium signaling and degranulation dynamics are determined by interaction between laser photon energy and Ca2+ influx. Extracellular Ca2+ concentration is so high that even small photon energy can activate mast cells, thus avoiding the possible injury caused by laser irradiation with shorter wavelengths. The model predicts that there exists a narrow parameter domain of photon energy and extracellular Ca2+ concentration of which results in cytosolic Ca2+ limit cycle oscillations, and shows that PKC activity is in direct proportion to the frequency of Ca2+ oscillations. With the model it is found that sustained and stable maximum plateau of cytosolic Ca2+ concentration can get optimal degranulation rate. Furthermore, the idea of introducing the realistic physical energy into model is applicable to modeling other physical signal transduction systems.

  11. Role of calcium in neutrophil activation by Japanese encephalitis virus - induced macrophage derived factor

    The role of cytosolic Ca2+ concentration in neutrophils stimulated with macrophage derived neutrophil chemotactic factor (MDF) produced following Japanese encephalitis virus infection in mice was correlated with cell functions. MDF-induced Ca2+ influx from extracellular milieu and release from intracellular store resulted in rise of cytosolic Ca2+ in a dose-dependent manner and was independent of protein kinase C. Macrophages and B cells did not show cytosolic Ca2+ changes wile T lymphocytes showed slight rise when stimulated with MDF. Neutrophil chemotaxis in the absence of Ca2+ was slightly different from that in presence of Ca2+. Pretreatment of neutrophils with 3,4,5-trimethoxy-benzoic acid-8-(dimethylamino)-octylester (TMB-8) inhibited the chemotaxis. It was observed that superoxide production and de-granulation by neutrophils after stimulation with MDF was not depend on the presence of extracellular calcium, but stripping of intracellular calcium resulted in abrogation of neutrophil activation. Thus, mobilization of intracellular calcium seems to be necessary for neutrophil activation by MDF. (author)

  12. Changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats

    陈立华; 于嘉; 刘丽旭; 曹美鸿


    To explore changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats, and to investigate the relationship between cytosolic free calcium concentration ( [ Ca2 + ] i ) in the synaptosome and Ca2 + -ATPase activities of mitochondria. Methods: The level of [ Ca2+ ]i in the synaptosome and Ca2+ -ATPase activities of mitochondria in the acute brain damage induced by injection of pertussis bacilli (PB)in rat was determined and nimodipine was administrated to show its effects on [ Ca2+ ]i in the synaptosome and on alteration of Ca2+ -ATPase activity in the mitochondria.Seventy-three rats were randomly divided into four groups,ie, normal control group (Group A ), sham-operation control group (Group B), PB group (Group C) and nimodipine treatment group (Group D). Results: The level of [ Ca2+ ]i was significantly increased in the PB-injected cerebral hemisphere in the Group C as compared with that in the Group A and the Group B at 30 minutes after injection of PB. The level of [ Ca2+ ]i was kept higher in the 4 hours and 24 hours subgroups after the injection in the Group C ( P < 0.05).In contrast, the Ca2+ -ATPase activities were decreased remarkably among all of the subgroups in the Group C.Nimodipine, which was administered after injection of PB,could significantly decrease the [ Ca2+ ]i and increase the activity of Ca2 + -ATPase ( P < 0.05 ). Conclusions: The neuronal calcium channel is opened after injection of PB. There is a negative correlation between activities of Ca2 +-ATPase and [ Ca2 + ]i.Nimodipine can reduce brain damage through stimulating the activities of Ca2+ -ATPase in the mitochondria, and decrease the level of [ Ca2+ ]i in the synaptosome.Treatment with nimodipine dramatically reduces the effects of brain damage induced by injection of PB.

  13. Calcium citrate improves the epithelial-to-mesenchymal transition induced by acidosis in proximal tubular cells

    Maria José Rodriguez Cabalgante; Liliana Gadola; Leonella Luzardo; María Márquez; José Boggia; Mirian Aparecida Boim


    INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is a key event in renal fibrosis. The aims of the study were to evaluate acidosis induced EMT, transforming-growth-factor (TGF) β1 role and citrate effect on it. METHODS: HK2 cells (ATCC 2290) were cultured in DMEM/HAM F12 medium, pH 7.4. At 80% confluence, after 24 hr under serum free conditions, cells were distributed in three groups (24 hours): A) Control: pH 7.4, B) Acidosis: pH 7.0 and C) Calcium citrate (0.2 mmol/L) + pH 7.0. Chan...

  14. Role of calcium signaling in down-regulation of aggrecan induced by cyclic tensile strain in annulus fibrosus cells

    GUO Zhi-liang; ZHOU Yue; LI Hua-zhuang; CAO Guo-yong; TENG Hai-jun


    Objective:To study the role of intracellular calcium signal pathway in the down-regulation of aggrecan induced by cyclic tensile strain in the annulus fibrosus cells. Methods :The expression of aggrecan mRNA and core protein were respectively detected with RT-PCR and western blot after the channels transmitting calcium ions were blocked with EGTA, gadolinium and verapamil. Results:EGTA, gadolinium and verapamil partially prevented the effects of cyclic tensile strain on the expression of aggrecan in annulus fibrosus cells. Conclusion:The calcium signaling is involved in the down-regulation of proteoglycan resulting from cyclic tensile strain in the annulus fibrosus cells.

  15. Stimulation of Adrenal Chromaffin Cell Proliferation by Hypercalcemia Induced by Intravenous Infusion of Calcium Gluconate in Rats

    Isobe, Kaori; Ito, Tsuneo; Komatsu, Shun-ichiro; Asanuma, Kentaro; Fujii, Etsuko; Kato, Chie; Adachi, Kenji; Kato, Atsuhiko; Sugimoto, Tetsuro; Suzuki, Masami


    Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by ...

  16. Enhanced permeability transition explains the reduced calcium uptake in cardiac mitochondria from streptozotocin-induced diabetic rats

    Oliveira, Paulo J; Seiça, Raquel; Coxito, Pedro M.; Rolo, Anabela P.; Palmeira, Carlos M.; Santos, Maria S.; Moreno, António J. M.


    Cardiac dysfunction is associated with diabetes. It was previously shown that heart mitochondria from diabetic rats have a reduced calcium accumulation capacity. The objective of this work was to determine whether the reduction in calcium accumulation by cardiac mitochondria from diabetic rats is related to an enhanced susceptibility to induction of the mitochondrial permeability transition. Streptozotocin-induced diabetic rats were used as a model to study the alterations caused by diabetes ...

  17. Multidrug resistance 1 gene polymorphism in amlodipine-induced gingival enlargement

    Naik, Kumaraswamy Naik Lambani Rama; Jhajharia, Kapil; Chaudhary, Roopam; Tatikonda, Aravind; Dhaliwal, Aprinderpal Singh; Kaur, Rose Kanwaljeet


    Gingival enlargement comprises any clinical condition in which an increase in the size of the gingiva is observed. It is a side effect associated with some distinct classes of drugs, such as anticonvulsants, immunosuppressant, and calcium channel blockers. Among calcium channel blockers, nifedipine causes gingival enlargement in about 10% of patients, whereas the incidence of amlodipine, a third-generation calcium channel blocker, induced gingival enlargement is very limited. Because the calcium antagonists, albeit to a variable degree, act as inhibitors of P-glycoprotein (P-gp), the gene product of multidrug resistance 1 (MDR1), and inflammation may modify P-gp expression. We hereby, report a case of amlodipine-induced gingival enlargement with MDR1 3435C/T polymorphism, associated with inflammatory changes due to plaque accumulation, in a 50-year-old hypertensive male patient. The genotype obtained for the polymorphism was a heteromutant genotype, thus supporting the contention that the MDR1 polymorphism may alter the inflammatory response to the drug. PMID:26015682

  18. Mechanism of histamine release from rat mast cells induced by the ionophore A23187: effects of calcium and temperature

    Johansen, Torben


    1 The mechanism of histamine release from a pure population of rat mast cells induced by the lipid soluble antibiotic, A23187, has been studied and compared with data for anaphylactic histamine release reported in the literature. 2 Histamine release induced by A23187 in the presence of calcium 10...

  19. Histamine release induced from rat mast cells by the ionophore A23187 in the absence of extracellular calcium

    Johansen, Torben


    Isolated rat mast cells were used to study whether ionophore A23187 could induce histamine release by mobilizing cellular calcium. The histamine release was a slow process which was completed after about 20 min incubation with A23187. The A23187-induced histamine release was inhibited after...

  20. Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists

    Rui ZHANG; Pang-ke YAN; Cai-hong ZHOU; Jia-yu LIAO; Ming-wei WANG


    Aim: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts, cAMP measurements were camed out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 recep-tor agonists following HTS of 16 000 samples. Results: EC50 values of the positive control compounds (β-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z' factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA1 receptor showing ≥20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screen- ing confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conclusion: A series of validation studies demonstrated that the homogeneous calcium mobili-zation assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation.

  1. Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells.

    Yutaka Maruyama

    Full Text Available 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 Ca(2+ concentration ([Ca(2+](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 Ca(2+. 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.

  2. Preserved frontal lobe oxygenation following calcium chloride for treatment of anesthesia-induced hypotension

    Carl-Christian eKitchen


    Full Text Available Vasopressor agents may affect cerebral oxygenation (rScO2 as determined by near-infrared spectroscopy on the forehead. This case series evaluated the effect of calcium chloride vs. α and β-adrenergic receptor agonists on rScO2 in patients (n = 47 undergoing surgery during i.v. anesthesia. Mean arterial pressure (MAP and cardiac output (CO were assessed by Model-flow® and ephedrine (55 ± 3 vs. 74 ± 9 mmHg; 10 mg, n = 9, phenylephrine (51 ± 5 vs. 78 ± 9 mmHg, 0.1 mg, n = 11, adrenaline (53 ± 3 vs. 72 ± 11 mmHg; 1 - 2 µg, n = 6, noradrenaline (53 ± 5 vs. 72 ± 12 mmHg; 2-4 µg, n = 11, and calcium chloride (49 ± 7 vs. 57 ± 16 mmHg; 5 mmol, n = 10 increased MAP (all P < 0.05. CO increased with ephedrine (4.3 ± 0.9 vs. 5.3 ± 1.2, P < 0.05 and adrenaline (4.7 ± 1.2 vs. 5.9 ± 1.1 l/min; P = 0.07 but was not significantly affected by phenylephrine (3.9 ± 0.7 vs. 3.6 ± 1.0 l/min, noradrenaline (3.8 ± 1.2 vs. 3.7 ± 0.7 l/min, or calcium chloride (4.0 ± 1.4 vs. 4.1 ± 1.5 l/min. Following administration of β-adrenergic agents and calcium chloride rScO2 was preserved while after administration of α-adrenergic drugs rScO2 was reduced by app. 2% (P < 0.05. Following α-adrenergic drugs to treat anesthesia-induced hypotension tissue oxygenation is reduced while the use of β-adrenergic agonists and calcium chloride preserve tissue oxygenation.

  3. Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process

    Gower, Laurie B.; Odom, Damian J.


    A polypeptide additive has been used to transform the solution crystallization of calcium carbonate to a solidification process of a liquid-phase mineral precursor. In situ observations reveal that polyaspartate induces liquid-liquid phase separation of droplets of a mineral precursor. The droplets deposit on the substrate and coalesce to form a coating, which then solidifies into calcitic tablets and films. Transition bars form during the amorphous to crystalline transition, leading to sectorization of calcite tablets, and the defect textures and crystal morphologies are atypical of solution grown crystals. The formation of nonequilibrium crystal morphologies using an acidic polypeptide may have implications in the field of biomineralization, and the environmentally friendly aspects of this polymer-induced liquid-precursor (PILP) process may offer new techniques for aqueous-based processing of ceramic films, coatings, and particulates.

  4. Genome-wide analysis of the calcium-dependent protein kinase gene family inGossypium raimondii

    LI Li-bei; YU Ding-wei; ZHAO Feng-li; PANG Chao-you; SONG Mei-zhen; WEI Heng-ling; FAN Shu-li; YU Shu-xun


    Plant calcium-dependent protein kinases (CDPKs) play important roles in diverse physiological processes by regulating the downstream components of calcium signaling. To date, only a few species of the plant CDPK gene family have been functionaly identiifed. In addition, there has been no systematic analysis of the CDPK family in cotton. Here, 41 putative cotton CDPK (GrCDPK) genes were identiifedvia bioinformatics analysis of the entire genome ofGossypium raimondi and were classiifed into four groups based on evolutionary relatedness. Gene structure analysis indicated that most of these GrCDPK genes share a similar intron-exon structure (7 or 8 exons), strongly supporting their close evolutionary relationships. Chromosomal distributions and phylogenetics analysis showed that 13 pairs of GrCDPK genes arosevia segmental duplication events. Furthermore, using microarray data of upland cotton (G. hirsutumL.), comparative proifles analysis of these GhCDPKs indicated that some of the encoding genes might be involved in the responses to multiple abiotic stresses and play important regulatory roles during cotton ifber development. This study is the ifrst genome-wide analysis of the CDPK family in cotton, and it wil provide valuable information for the further functional characterization of cotton CDPK genes.

  5. Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases

    The present studies establish that there are specific, significant decreases in the neuronal calcium-binding protein (28-kDa calbindin-D) gene expression in aging and in neurodegenerative diseases. The specificity of the changes observed in calbindin mRNA levels was tested by reprobing blots with calmodulin, cyclophilin, and B-actin cDNAs. Gross brain regions of the aging rat exhibited specific, significant decreases in calbindin·mRNA and protein levels in the cerebellum, corpus striatum, and brain-stem region but not in the cerebral cortex or hippocampus. Discrete areas of the aging human brain exhibited significant decreases in calbindin protein and mRNA in the cerebellum, corpus striatum, and nucleus basalis but not in the neocortex, hippocampus, amygdala, locus ceruleus, or nucleus raphe dorsalis. Comparison of diseased human brain tissue with age- and sex-matched controls yielded significant decreases calbindin protein and mRNA in the substantia nigra (Parkinson disease), in the corpus striatum (Huntington disease), in the nucleus basalis (Alzheimer disease), and in the hippocampus and nucleus raphe dorsalis (Parkinson, Huntington, and Alzheimer diseases) but not in the cerebellum, neocortex, amygdala, or locus ceruleus. These findings suggest that decreased calbindin gene expression may lead to a failure of calcium buffering or intraneuronal calcium homeostasis, which contributes to calcium-mediated cytotoxic events during aging and in the pathogenesis of neurodegenerative diseases

  6. Caffeine-induced myocardial injury in calcium-free perfused rat hearts.

    Vander Heide, R. S.; Ganote, C. E.


    Hearts depleted of extracellular calcium become susceptible to injury caused by repletion of extracellular calcium (calcium paradox). It has been suggested that calcium-free perfusion causes weakening of intercalated disks and that the physical stress of contracture may cause sarcolemmal membrane rupture and creatine kinase (CK) release. To further investigate this hypothesis, the effects of caffeine on contracture, cellular morphology, and CK release were studied in control and calcium-free ...

  7. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    S.C.F. Olinto


    Full Text Available The amino acid arginine (Arg is a recognized secretagogue of growth hormone (GH, and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO, which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g were removed, divided into two halves, pooled (three hemi-pituitaries and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM, the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM and a cyclic guanosine monophosphate (cGMP analogue (8-Br-cGMP, 1 mM increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS inhibitor, 55 mM abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

  8. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression

  9. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    Olinto, S.C.F. [Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Ituiutaba, MG (Brazil); Adrião, M.G. [Departamento de Morfologia e Fisiologia, Universidade Federal Rural de Pernambuco, Recife, PE (Brazil); Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)


    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

  10. Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes

    Meng YANG; Wen-xie XU; Xing-lan LI; Hui-ying XU; Jia-bin SUN; Bin MEI; Hai-feng ZHENG; Lian-hua PIAO; De-gang XING; Zhai-liu LI


    Aim: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (IKCa) in gastric myocytes. Methods: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. Results: Hyposmotic membrane stretch and AA increased both IK(Ca) and spontaneous transient outward currents significantly.Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in IK(Ca). The hyposmotic membrane stretch-induced increase in IK(Ca) was significantly suppressed by dimethyleicosadienoic acid (100 μmol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in IK(Ca). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in IK(Ca) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in IK(Ca); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in IK(Ca). Conclusion:Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca2+ influx, which triggers Ca2+-induced Ca2+ release and elicits activation of IK(Ca) in gastric antral circular myocytes of the guinea pig.

  11. Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

    Guauque-Olarte, Sandra; Messika-Zeitoun, David; Droit, Arnaud; Lamontagne, Maxime; Tremblay-Marchand, Joël; Lavoie-Charland, Emilie; Gaudreault, Nathalie; Arsenault, Benoit J.; Dubé, Marie-Pierre; Tardif, Jean-Claude; Body, Simon C.; Seidman, Jonathan G.; Boileau, Catherine; Mathieu, Patrick; Pibarot, Philippe; Bossé, Yohan


    Background Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had Pmeta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway. PMID:26553695

  12. A novel mutation in the calcium-sensing receptor gene in an Irish pedigree showing familial hypocalciuric hypercalcemia: a case report.

    Elamin, Wael F


    Familial hypocalciuric hypercalcemia is a rare autosomal dominant disorder characterized by asymptomatic and non-progressive hypercalcemia due to mutations of the calcium-sensing receptor gene. Disorders of calcium metabolism are very common in the elderly, and they can coexist with familial hypocalciuric hypercalcemia in affected families.

  13. Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

    High blood levels of ammonium/ammonia (NH4+/NH3) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca2+]i) in astrocytoma (EC50 = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca2+]i was due to an intracellular effect of NH4+/NH3 and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by Gq protein-coupled receptors (H1 and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH3/NH4+ from neuronal cells. Cellular uptake of NH4+/NH3 was mainly by passive diffusion of NH3. Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia

  14. Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.

    Guang Qin Zhang

    Full Text Available INTRODUCTION: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs-derived cardiomyocytes (hiPSC-CMs may display unique structural and functional properties than mature adult cardiomyocytes. METHODS AND RESULTS: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM and full duration at half maximum (FDHM of spontaneous Ca2+ sparks. CONCLUSIONS: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

  15. Polymorphisms in the calcium-sensing receptor gene are associated with clinical outcome of neuroblastoma.

    Laia Masvidal

    Full Text Available BACKGROUND: Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726 to assess if genetic variants producing a less active receptor are associated with more aggressive disease course. METHODS: Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays. RESULTS: Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002 and the histological subgroup of undifferentiated neuroblastomas (P = 0.046. Patients harboring this polymorphism had significantly lower overall (P = 0.022 and event-free survival (P = 0.01 rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14-5.29]; P = 0.022 and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20-6.25]; P = 0.016. CONCLUSIONS: The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636

  16. Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

    The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity

  17. Inhibition of Store-Operated Calcium Entry Protects Endothelial Progenitor Cells from H2O2-Induced Apoptosis.

    Wang, Yan-Wei; Zhang, Ji-Hang; Yu, Yang; Yu, Jie; Huang, Lan


    Store-operated calcium entry (SOCE), a major mode of extracellular calcium entry, plays roles in a variety of cell activities. Accumulating evidence indicates that the intracellular calcium ion concentration and calcium signaling are critical for the responses induced by oxidative stress. The present study was designed to investigate the potential effect of SOCE inhibition on H2O2-induced apoptosis in endothelial progenitor cells (EPCs), which are the predominant cells involved in endothelial repair. The results showed that H2O2-induced EPC apoptosis was reversed by SOCE inhibition induced either using the SOCE antagonist ML-9 or via silencing of stromal interaction molecule 1 (STIM1), a component of SOCE. Furthermore, SOCE inhibition repressed the increases in intracellular reactive oxygen species (ROS) levels and endoplasmic reticulum (ER) stress and ameliorated the mitochondrial dysfunction caused by H2O2. Our findings provide evidence that SOCE inhibition exerts a protective effect on EPCs in response to oxidative stress induced by H2O2 and may serve as a potential therapeutic strategy against vascular endothelial injury. PMID:27169819

  18. Calcium-dependent trichosanthin-induced generation of reactive oxygen species involved in apoptosis of human choriocarcinoma cells

    Zhang, Chunyang; Ma, Hui; Chen, Die Yan


    The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced production of reactive oxygen species (ROS) in JAR cells by using fluorescent probe 2',7'-dichlorofluorescin diacetate with confocal laser scanning microscopy. TCS-induced ROS showed dependence on the increase in intracellular calcium and on the presence of extracellular calcium. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-indued increase in intracellular calcium monitored using fluo 3-AM, suggesting that TCS-induced ROS might mediate by the increase in intracellular Ca2PLU concentration. Simultaneous observation of the nuclear morphological changes and production of ROS in JAR cells with two-photon laser scanning microscopy and confocal laser scanning microscopy revealed that ROS involved in the apoptosis of JAR cells, which was confirmed by that antioxidant (alpha) -tocopherol prevented TCS-induced ROS formation and cell death. The finding that calcium-dependent TCS-induced ROS involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.

  19. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long


    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

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

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


    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: 4.503, year: 2014

  1. Rapid insight into heating-induced phase transformations in the solid state of the calcium salt of atorvastatin using multivariate data analysis

    Christensen, Niels Peter Aae; Van Eerdenbrugh, Bernard; Kwok, Kaho; Taylor, Lynne S.; Bond, Andrew; Rades, Thomas; Rantanen, Jukka; Cornett, Claus


    To investigate the heating-induced dehydration and melting behavior of the trihydrate phase of the calcium salt of atorvastatin.......To investigate the heating-induced dehydration and melting behavior of the trihydrate phase of the calcium salt of atorvastatin....

  2. Monitoring of hardening and hygroscopic induced strains in a calcium phosphate bone cement using FBG sensor.

    Bimis, A; Karalekas, D; Bouropoulos, N; Mouzakis, D; Zaoutsos, S


    This study initially deals with the investigation of the induced strains during hardening stage of a self-setting calcium phosphate bone cement using fiber-Bragg grating (FBG) optical sensors. A complementary Scanning Electron Microscopy (SEM) investigation was also conducted at different time intervals of the hardening period and its findings were related to the FBG recordings. From the obtained results, it is demonstrated that the FBG response is affected by the microstructural changes taking place when the bone cement is immersed into the hardening liquid media. Subsequently, the FBG sensor was used to monitor the absorption process and hygroscopic response of the hardened and dried biocement when exposed to a liquid/humid environment. From the FBG-based calculated hygric strains as a function of moisture concentration, the coefficient of moisture expansion (CME) of the examined bone cement was obtained, exhibiting two distinct linear regions. PMID:26807773

  3. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara


    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. PMID:26652403

  4. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

    Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent

  5. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

    Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H.


    Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using /sup 125/I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

  6. Blockade of L-type calcium channel in myocardium and calcium-induced contractions of vascular smooth muscle by by CPU 86017

    De-zai DAI; Hui-juan HU; Jing ZHAO; Xue-mei HAO; Dong-mei YANG; Pei-ai ZHOU; Cai-hong WU


    AIM: To assess the blockade by CPU 86017 on the L-type calcium channels in the myocardium and on the Ca2+related contractions of vascular smooth muscle. METHODS: The whole-cell patch-clamp was applied to investigate the blocking effect of CPU 86017 on the L-type calcium current in isolated guinea pig myocytes and contractions by KC1 or phenylephrine (Phe) of the isolated rat tail arteries were measured. RESULTS: Suppression of the L-type current of the isolated myocytes by CPU 86017 was moderate, in time- and concentration-dependent manner and with no influence on the activation and inactivation curves. The IC50 was 11.5 μmol/L. Suppressive effect of CPU 86017 on vaso-contractions induced by KC1 100 mmol/L, phenylephrine I μmol/Lin KH solution (phase 1),Ca2+ free KH solution ( phase 2), and by addition of CaCI2 into Ca2+-free KH solution (phase 3) were observed. The IC50 to suppress vaso-contractions by calcium entry via the receptor operated channel (ROC) and Voltage-dependent channel (VDC) was 0.324 μmol/L and 16.3 μmol/L, respectively. The relative potency of CPU 86017 to suppress vascular tone by Ca2+ entry through ROC and VDC is 1/187 of prazosin and 1/37 of verapamil, respectively.CONCLUSION: The blocking effects of CPU 86017 on the L-type calcium channel of myocardium and vessel are moderate and non-selective. CPU 86017 is approximately 50 times more potent in inhibiting ROC than VDC.

  7. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah


    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  8. Bupivacaine-induced Vasodilation Is Mediated by Decreased Calcium Sensitization in Isolated Endothelium-denuded Rat Aortas Precontracted with Phenylephrine

    Ok, Seong Ho; Bae, Sung Il; Kwon, Seong Chun; Park, Jung Chul; Kim, Woo Chan; Park, Kyeong Eon; Shin, Il Woo; Lee, Heon Keun; Chung, Young Kyun; Choi, Mun Jeoung


    Background A toxic dose of bupivacaine produces vasodilation in isolated aortas. The goal of this in vitro study was to investigate the cellular mechanism associated with bupivacaine-induced vasodilation in isolated endotheliumdenuded rat aortas precontracted with phenylephrine. Methods Isolated endothelium-denuded rat aortas were suspended for isometric tension recordings. The effects of nifedipine, verapamil, iberiotoxin, 4-aminopyridine, barium chloride, and glibenclamide on bupivacaine concentration-response curves were assessed in endothelium-denuded aortas precontracted with phenylephrine. The effect of phenylephrine and KCl used for precontraction on bupivacaine-induced concentration-response curves was assessed. The effects of verapamil on phenylephrine concentration-response curves were assessed. The effects of bupivacaine on the intracellular calcium concentration ([Ca2+]i) and tension in aortas precontracted with phenylephrine were measured simultaneously with the acetoxymethyl ester of a fura-2-loaded aortic strip. Results Pretreatment with potassium channel inhibitors had no effect on bupivacaine-induced relaxation in the endothelium-denuded aortas precontracted with phenylephrine, whereas verapamil or nifedipine attenuated bupivacaine-induced relaxation. The magnitude of the bupivacaine-induced relaxation was enhanced in the 100 mM KCl-induced precontracted aortas compared with the phenylephrine-induced precontracted aortas. Verapamil attenuated the phenylephrine-induced contraction. The magnitude of the bupivacaine-induced relaxation was higher than that of the bupivacaine-induced [Ca2+]i decrease in the aortas precontracted with phenylephrine. Conclusions Taken together, these results suggest that toxic-dose bupivacaine-induced vasodilation appears to be mediated by decreased calcium sensitization in endothelium-denuded aortas precontracted with phenylephrine. In addition, potassium channel inhibitors had no effect on bupivacaine-induced relaxation

  9. Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability.

    Wang, Hsiu-Jung; Hsiao, Yu-Yuan; Chen, Yu-Pei; Ma, Tien-Yang; Tseng, Ching-Ping


    Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca(2+)-free and Ca(2+)-bounded conformations of the calcium site, we found that Ca(2+) induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca(2+)), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca(2+) added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca(2+) in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature. PMID:26729722

  10. Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

    Yang, Xinhui; Jiang, Jiangtao; Yang, Xinyan; Han, Jichun; Zheng, Qiusheng


    Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain 2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be

  11. Sources of calcium in agonist-induced contraction of rat distal colon smooth muscle in vitro

    Hua Zhou; De-Hu Kong; QunWan Pan; HaiHua Wang


    AIM:To study the origin of calcium necessary foragonist-induced contraction of the distal colon in rats.METHODS:The change in intracellular calcium concentration ([Ca2+]i)evoked by elevating external Ca2+was detected by fura 2/AM fluorescence.Contractile activity was measured with a force displacement transducer.Tension was continuously monitored and recorded using a Powerlab 4/25T data acquisition system with an ML110 bridge bioelectric physiographic amplifier.RESULTS:Store depletion induced Ca2+ influx had an effect on [Ca2+]i.In nominally Ca2+-free medium,the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1 μmol/L) increased [Ca2+]i from 68 to 241 nmol/L,and to 458 (P<0.01) and 1006 nmol/L (P<0.01),respectively,when 1.5 mmol/L and 3.0 mmol/L extracellular Ca2+ was reintroduced.Furthermore,the change in [Ca2+]1.was observed with verapamil (5 μmol/L),La3+(1 mmol/L) or KCI (40 mmol/L) in the bathing solution.These channels were sensitive to La3+(P<0.01),insensitive to verapamil,and voltage independent.In isolated distal colons we found that in normal Krebs solution,contraction induced by acetylcholine (ACh) was partially inhibited by verapamil,and the inhibitory rate was 41% (P<0.05).On the other hand,in Ca2+-free Krebs solution,ACh induced transient contraction due to Ca2+ release from the inLracellular stores.The transient contraction lasted until the Ca2+ store was depleted.Restoration of extracellular Ca2+ in the presence of atropine produced contraction,mainly due to Ca2+ influx.Such contraction was not inhibited by verapamil,but was decreased by La3+ (50 μmol/L) from 0.96 to 0.72 g (P<0.01).CONCLUSION:The predominant source of activator Ca2+ for the contractile response to agonist is extracellular Ca2+,and intracellular Caz+ has little role to play in mediating excitation-contraction coupling by agonists in rat distal colon smooth muscle in vitro.The influx of extracellular Ca2+ is mainly mediated through voltage-,receptor- and

  12. Calcium channel activity of purified human synexin and structure of the human synexin gene

    Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. The authors have isolated and sequenced a set of overlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. These include endonexin II, lipocortin I, calpactin I heavy chain (p36), protein II, and calelectrin 67K. The Mr 51,000 synexin molecule can be divided into a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Analysis of the entire structure reveals possible insights into such diverse properties as voltage-sensitive calcium channel activity, ion selectivity, affinity for phospholipids, and membrane fusion

  13. Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion.

    Wegner, J A; Martinez-Zaguilan, R; Gillies, R J; Hoyer, P B


    A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in

  14. Controlled superficial assembly of DNA-amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery.

    Oyane, Ayako; Araki, Hiroko; Nakamura, Maki; Shimizu, Yoshiki; Shubhra, Quazi T H; Ito, Atsuo; Tsurushima, Hideo


    Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres. PMID:26896659

  15. Depolarization-induced contractile activity of smooth muscle in calcium-free solution.

    Mangel, A W; Nelson, D O; Rabovsky, J L; Prosser, C L; Connor, J A


    In calcium-free solution, strips of cat intestinal muscle developed slow, rhythmic electrical potential changes that triggered contractions. Some strips failed to develop spontaneous electrical activity in calcium-free solution but responded with contractions to depolarization by direct electrical stimulation or by treatment with barium chloride, potassium chloride, or acetylcholine. Similar results were obtained with segments of cat stomach, colon, esophagus, bladder, uterus, and vena cava, as well as with rabbit vena cava. In calcium-free saline, rat small intestinal muscle showed fast electrical activity with accompanying development of a tetanuslike contraction. After 60 min in calcium-free solution, cat small intestinal muscle retained 17.7% of its original concentration of calcium. It is concluded that in some smooth muscles, depolarization-triggered release of intracellular calcium does not require an associated influx of calcium. PMID:7058877

  16. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

    Padma P Srinivasan

    Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

  17. Mercury-induced apoptosis and necrosis in murine macrophages: role of calcium-induced reactive oxygen species and p38 mitogen-activated protein kinase signaling

    The current study characterizes the mechanism by which mercury, a toxic metal, induces death in murine macrophages. The cytotoxic EC50 of mercury ranged from 62.7 to 81.1 μM by various assays in J774A.1 cultures; accordingly, we employed 70 μM of mercuric chloride in most experiments. Mercury-induced intracellular calcium modulated reactive oxygen species (ROS) production, which resulted in both cell apoptosis and necrosis indicated by annexin V binding and caspase-3 activity, and propidium-iodide binding. Calcium antagonists abolished ROS production. Mercury stimulated p38 mitogen-activated protein kinase (MAPK) and additively stimulated lipopolysaccharide-activated p38. Mercury-activated p38 was decreased by pretreatment of cells with antioxidants, N-acetylcysteine (NAC) and silymarin, indicating that mercury-induced ROS were involved in p38 activation. Mercury increased the expression of tumor necrosis factor α (TNFα); antioxidants and a specific p38 inhibitor decreased this effect. Pretreatment with antioxidants, p38 inhibitor, and anti-TNFα antibody decreased mercury-induced necrosis; however, anti-TNFα antibody did not decrease mercury-induced apoptosis. Results suggest that mercury-induced macrophage death is a mix of apoptosis and necrosis employing different pathways. P38-mediated caspase activation regulates mercury-induced apoptosis and p38-mediated TNFα regulates necrosis in these cells. Calcium regulates ROS production and mercury-induced ROS modulate downstream p38 that regulates both apoptosis and necrosis

  18. Comparison of adenosine-induced myocardial ischemia and atherosclerosis measured by coronary calcium tomography

    Coronary artery calcium shows a anatomic information and coronary atherosclerotic burde, but myocardial perfusion SPECT shows a physiologic significance of coronary stenosis and stress induced ischemia. Both are valuable in the noninvasive assessment of patients with suspected coronary artery disease. There has been little evaluation regarding the relationship between CAC and adenosine-induced ischemia and how to integrate CAC with myocardial perfusion SPECT. We assessed the relationship between adenosine-induced myocardial ischemia on myocardial perfusion single-photon emission computed tomography (MPS) and magnitude of coronary calcification (CAC) by MDCT in patients undergoing both tests. A total of 111 patients underwent adenosine-induced MPS and CAC within 2days. Coronary angiography was done in 55 patients. The frequency of ischemia by MPS was compared to the magnitude of CAC. Among 56 patients with ischemic MPS, the CAC scores were >0 in 87.5%, >100 in 76.8%, and > 400 in 50.0%. Of 25 normal MPS, the CAC scores were >0 in 70.9%. >100 in 34.5%, and > 400 in 14.5%, respectively. Of 38 patient with coronary artery stenosis proved by coronary angiography, the CAC scores were >0 in 92.1%, >100 in 78.9%, and > 400 in 50.0 %, respectively. Of 12 patient without coronary artery stenosis, the CAC scores were >100 in 66.7%, and > 400 in 41.7%. Ischemic MPS is associated with a high likelihood of subclinical atherosclerosis by CAC, but it can be also seen for CAC scores <100. The patient without significant coronary artery stenosis, however, may have extensive atherosclerosis by CAC criteria. Although, low CAC scores appear to obviate the need for subsequent testing, but MPS is still needed to diagnosis the myocardial ischemia

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

    Yoneda, Akihiro, E-mail: [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan); Division of Molecular Therapeutics, Center for Food & Medical Innovation, Hokkaido University (Japan); Watanabe, Tomomasa [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan)


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

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

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca2+) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca2+ oscillatory pattern, whether PLCζ-induced Ca2+ oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca2+ oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca2+ oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca2+ oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca2+ oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca2+ oscillations continued after pronuclear formation. • The Ca2+ oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts

  1. Maitotoxin-induced myocardial cell injury: Calcium accumulation followed by ATP depletion precedes cell death

    Maitotoxin, the most potent marine toxin, is known to increase the uptake and the accumulation of Ca2+ into cells, and was used in the present study to investigate the mechanisms of myocardial cell damage induced by Ca2+ overload. In cultured cardiomyocytes, isolated from 2-day-old rats, maitotoxin affected cell viability, as indicated by the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) and of radiolabeled adenine nucleotides into the extracellular medium. Maitotoxin-induced leakage of LDH steadily increased between 30 min and 24 hr, and was preceded by a marked depletion of intracellular ATP. Addition of maitotoxin resulted in a rapid influx of extracellular Ca2+, as detected by preincubating the cells in the presence of 45Ca; this effect evolved in a few minutes, thus preceding the signs of cell death. Cytosolic levels of free Ca2+ ([Ca2+]i) were monitored by loading freshly isolated, suspended cardiomyocytes with the intracellular fluorescent probe fura-2; in these cells, maitotoxin induced a dose-dependent increase in [Ca2+]i, with a lag phase of less than a minute. All these effects of maitotoxin were inhibited by reducing Ca2+ concentration in the culture medium or by incubating the cells with the calcium-channel blocking drug verapamil. It is thus demonstrated that maitotoxin-induced cardiotoxicity is secondary to an inordinate influx of Ca2+ into the cells. It is also suggested that, in those conditions that lead to an inordinate accumulation of Ca2+ into myocardial cells, the unmatched demands of energy and the depletion of ATP play a primary role in the irreversible stage of cell damage

  2. Impaired Compensation for Salt-Induced Urinary Calcium Loss in a Space Flight Model

    Navidi, Meena; Harper, J. S.; Evans, J.; Fung, P.; Wolinsky, I.; Arnaud, S. B.; Wade, Charles E. (Technical Monitor)


    The loss of urinary calcium (UCa) induced by high sodium (HiNa) diets is compensated for by an increase in net intestinal Ca absorption (abs.). To determine the capacity of the intestine to absorb Ca in a space flight model in which the formation of 1,25-dihydroxyvitamin D (1,25-D) is suppressed, we induced Ca loss with HiNa diets (8%) and restricted dietary Ca (0.2%). In 200 g rats with hind limbs unloaded by tail suspension (S), we examined intestinal Ca abs. by direct measurement in the duodenum (everted gut sac or S/M), vitamin D receptors (VDR) and Ca balance. We also measured serum ionized calcium (ICa), pH, parathyroid hormone (PTH) and 1,25D. PTH was related to ICa (r = -0.44, p is less than 0.02), pH (r = -0.47, p is less than 0.02) and %Ca abs. (r = -0.40, p is less than 0.05). 1,25-D was related to %Ca abs. (r = 0.60, p is less than 0.001) but not VDR or S/M. Effects of the model were lower serum 1,25-D (110 +/- 59 vs. 199 +/- 80 pg/ml, p is less than 0.005), %Ca abs. (83 +/- 6.9 vs. 93 +/- 3.2, p is less than 0.03) and Ca balance (27 +/- 0.2 vs. 30 +/- 0.3 mg/d, p is less than 0.001) in S than controls (C). The HiNa diet increased UCa excretion from 2 to 13% of dietary Ca. Responses to HiNa diets, compared to normal Na, revealed no differences in 1,25-D, Ca abs. or VDR. Ca balances were lower in HiNa (27 +/- 0.3 vs. 30 +/- 0.4 mg/d, p is less than 0.001) in spite of higher Ca intakes. The failure of S rats fed HiNa diets to increase Ca abs. in response to Na-induced Ca loss appears to be related to suppressed 1,25-D in the space flight model, the cause of which remains obscure.

  3. Chronic calcium pyrophosphate crystal inflammatory arthritis induced by extreme hypomagnesemia in short bowel syndrome

    Hahn Markus


    Full Text Available Abstract Background Short bowel syndrome (SBS may induce a plethora of clinical symptoms ranging from underweight to nutrient-, vitamin- and electrolyte deficiencies. The objective of this case report is to illustrate how demanding the management of a 60 year old patient with SBS and recurrent joint attacks was for different medical disciplines. Case presentation The patient with SBS presented with a body mass index of 16.5 kg/m2 after partial jejunoileal resection of the small intestine with a six year long history of recurrent pain attacks in multiple peripheral joints, chronic diarrhoea and food intolerances. Pain attacks occurred 4–5 times a week with a median consumption of 15 mg prednisone per day. The interdisciplinary workup after several gastroenterologic, rheumatologic, radiologic, psychiatric and orthopedic consultations is shown including successful treatment steps. Clinical diagnosis revealed no systemic inflammatory disease, but confirmed extreme hypomagnesemia (0.2 mmol/l after reproducible pathological magnesium resorption tests as causative for chronic calcium pyrophosphate crystal inflammatory arthritis (pseudogout, chondrocalcinosis. Multidisciplinary treatment included application of colchicines, parenteral nutrition and magnesium substitution, antiperistaltic agents and avoidance of intolerant foods. Normalization of magnesium levels and a marked remission of joint attacks were achieved after six months with significant reduction of prednisone to 1.5 mg/day. Conclusion Despite the rarity of this condition, it is important to know that hypomagnesaemia may be associated with calcium pyrophosphate crystal inflammatory arthritis (chondrocalcinosis and that SBS patients may be prone to develop extreme hypomagnesaemia causing recurrent joint attacks without systemic inflammation.

  4. Hypoxia-Inducible Factor-1α Causes Renal Cyst Expansion through Calcium-Activated Chloride Secretion

    Buchholz, Bjoern; Schley, Gunnar; Faria, Diana; Kroening, Sven; Willam, Carsten; Schreiber, Rainer; Klanke, Bernd; Burzlaff, Nicolai; Jantsch, Jonathan; Kunzelmann, Karl; Eckardt, Kai-Uwe


    Polycystic kidney diseases are characterized by numerous bilateral renal cysts that continuously enlarge and, through compression of intact nephrons, lead to a decline in kidney function over time. We previously showed that cyst enlargement is accompanied by regional hypoxia, which results in the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) in the cyst epithelium. Here we demonstrate a correlation between cyst size and the expression of the HIF-1α–target gene, glucose t...

  5. Calcium hydroxide-induced resorption of deciduous teeth: A possible explanation

    G R Ravi


    Full Text Available Introduction: Calcium hydroxide (CaH is customarily used for permanent teeth but not for deciduous dentition because it is known to cause internal resorption in the latter. Though this has been attributed to chronic inflammation and odontoclasts, the exact mechanism has not been elucidated. The Hypothesis: The authors propose an explanation that CaH-induced odontoclastogenesis could be multifactorial. Odontoclasts may result from fusion of cells of monocyte/macrophage series either due to inflammatory mediators or through stimulation by stromal odontoblasts /fibroblasts. Pre-existing progenitor cells of primary tooth pulp because of their inherent propensity may transform into odontoclasts. Evaluation of the Hypothesis: The hypothesis discusses the role of various inflammatory cytokines that may be responsible for CaH-induced transformation of pre-odontoclasts to odontoclasts. Alternatively, pre-existing progenitor cells with proclivity to change into odontoclasts may cause internal resorption. The loss of protective layer of predentin over mineralized dentin may also make the primary tooth more susceptible to resorption.

  6. Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser

    Zhao, Yuan; Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zeng, Shaoqun; Luo, Qingming


    Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

  7. Gene activation by induced DNA rearrangements

    A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10(-7), whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10(6) and 67 per 10(6) cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33 degrees C) for varying periods of time (1-5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5 degrees C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9-52 per 10(6) cells over 1-5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome

  8. Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family

    Kaufman, I.; Luchinsky, D. G.; Tindjong, R.; McClintock, P. V. E.; Eisenberg, R. S.


    We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Qf at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Qf=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Qf for the sodium-calcium channels family. An increase of Qf leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Qf(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca2+/Na+ valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls.

  9. Inducible gene expression system by 3-hydroxypropionic acid

    Zhou, Shengfang; Ainala, Satish Kumar; Seol, Eunhee; Nguyen, Trinh Thi; Park, Sunghoon


    Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion. Results Here we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP ...

  10. Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway.

    Lee, Eunkyung; Choi, So-Young; Yang, Jae-Ho; Lee, Youn Ju


    Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway. PMID:27382356

  11. Lysophosphatidic acid induced nuclear translocation of nuclear factor-κB in Panc-1 cells by mobilizing cytosolic free calcium

    Yoshiyuki Arita; Tetsuhide Ito; Takamasa Pond; Ken Kawabe; Terumasa Hisano; Ryoichi Takayanagi


    AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer.METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout the study. The expression of LPA receptors was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). Cytosolic free calcium was measured by fluorescent calcium indicator fura-2, and the localization of NF-κB was visualized by immunofluorescent method with or without various agents, which effect cell signaling.RESULTS: Panc-1 expressed LPA receptors, LPAA1,LPA2 and LPA3. LPA caused the elevation of cytosolic free calcium dose-dependently. LPA also caused the nuclear translocation of NF-κB. Cytosolic free calcium was attenuated by pertussis toxin (PTX) and U73122,an inhibitor of phospholipase C. The translocation of NF-κB was similarly attenuated by PTX and U73122,but phorbol ester, an activator of protein kinase C,alone did not translocate NF-κB. Furthermore, the transtocation of NF-κB was completely blocked by Ca2+ chelator BAPTA-AM. Thapsigargin, an endoplasmic-reticulum Ca2+-ATPase pump inhibitor, also promoted the translocation of NF-κB. Staurosporine, a protein kinase C inhibitor, attenuated translocation of NF-κB induced by LPA.CONCLUSlON: These findings suggest that protein kinase C is activated endogenously in Panc-1, and protein kinase C is essential for activating NF-κB with cytosolic calcium and that LPA induces the nuclear translocation of NF-κB in Panc-1 by mobilizing cytosolic free calcium.

  12. Effects of calcium phosphate/chitosan composite on bone healing in rats: calcium phosphate induces osteon formation.

    Fernández, Tulio; Olave, Gilberto; Valencia, Carlos H; Arce, Sandra; Quinn, Julian M W; Thouas, George A; Chen, Qi-Zhi


    Vascularization of an artificial graft represents one of the most significant challenges facing the field of bone tissue engineering. Over the past decade, strategies to vascularize artificial scaffolds have been intensively evaluated using osteoinductive calcium phosphate (CaP) biomaterials in animal models. In this work, we observed that CaP-based biomaterials implanted into rat calvarial defects showed remarkably accelerated formation and mineralization of new woven bone in defects in the initial stages, at a rate of ∼60 μm/day (0.8 mg/day), which was considerably higher than normal bone growth rates (several μm/day, 0.1 mg/day) in implant-free controls of the same age. Surprisingly, we also observed histological evidence of primary osteon formation, indicated by blood vessels in early-region fibrous tissue, which was encapsulated by lamellar osteocyte structures. These were later fully replaced by compact bone, indicating complete regeneration of calvarial bone. Thus, the CaP biomaterial used here is not only osteoinductive, but vasculogenic, and it may have contributed to the bone regeneration, despite an absence of osteons in normal rat calvaria. Further investigation will involve how this strategy can regulate formation of vascularized cortical bone such as by control of degradation rate, and use of models of long, dense bones, to more closely approximate repair of human cortical bone. PMID:24460696

  13. Gene Expression of Atrial Calcium-Handling Proteins in Patients with Rheumatic Heart Disease and Atrial Fibrillation

    伍伟锋; 黄从新; 刘唐威; 朱树雄


    Objectives To investigate the gene expression of calcium-handling proteins inpatients with rheumatic heart disease (RHD) and atrialfibrillation (AF) . Methods A total of 50 patientswith rheumatic mitral valve disease were included.According to cardiac rhythm and duration of episode ofAF, patients were divided into four groups: sinusrhythm group, paroxysmal AF group, persistent AF forless than 6 months group and persistent AF for morethan 6 months group. Atrial tissue was obtained fromthe right atrial appendage, the right atrial free wall andthe left atrial appendage respectively during open heartsurgery. Total RNA was isolated and reversly tran-scribed into cDNA. In a semi -quantitative polymerasechain reaction the cDNA of interest and of glyceralde-hyde3 -phosphate dehydrogenase (GAPDH) were am-plified and separated by ethidium bromide - stained gelelectrophoresis. Multiple liner regress was used forcorrelation between the mRNA amount and age, sex,right atrial diameter (RAd) and left atrial diameter(LAd) Results The mRNA of L- type calciumchannelα1c subunit, of Ca2 + - ATPase and of ryanodinereceptor in patients with persistent AF for more than 6months were significantly decreased ( P all < 0. 01 ). But no alterations of the mRNA levels for SR phos-pholamban and calsequestrin were observed in patientswith persistent AF for more than 6 months comparedwith patients with sinus rhythm, paroxysmal AF andpersistent AF for less than 6 months( P all > 0.05) .There was no difference of the gene expression amongthe three atrial tissue sampling sites(P all > 0.05). Age, gender, RAd and LAd had no significant effectson the gene expression of calcium- handling proteins( P all> 0. 05). Conclusions The mRNA expressionof calcium -handling proteins is down -regulated onlyin patients with RHD and long- term persistent AF.Such abnormalities may be related to the initiationand/or perpetuation of AF in the patients with RHD.

  14. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance

    Manmathan, Harish; Shaner, Dale; Snelling, Jacob; Tisserat, Ned; Lapitan, Nora


    In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways i...

  15. Mefloquine-Induced Disruption of Calcium Homeostasis in Mammalian Cells Is Similar to That Induced by Ionomycin▿

    Caridha, D.; Yourick, D.; Cabezas, M.; De Wolf, L.; Hudson, T. H.; Dow, G. S.


    In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Sec...

  16. Elevating calcium in Th2 cells activates multiple pathways to induce IL-4 transcription and mRNA stabilization

    Guo, Liying; Joseph F. Urban; Zhu, Jinfang; Paul, William E.


    PMA and ionomycin cause T cell cytokine production. We report that ionomycin alone induces IL-4 and IFNγ, but not IL-2, from in vivo and in vitro generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires nuclear factor of activated T cells (NFAT), p38; and calmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporine A-inhibitable pathway. “Knocking-down” apoptosis signal-regulating kinase 1 (ASK1) inhibits io...

  17. Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9.

    Long, Shaojun; Wang, Qiuling; Sibley, L David


    Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii. PMID:26755159

  18. An efficient calcium phosphate nanoparticle-based nonviral vector for gene delivery

    Liu YC


    Full Text Available Yachun Liu1,2,*, Tao Wang1,*, Fangli He1,*, Qian Liu1,*, Dexi Zhang2, Shuanglin Xiang1, Shengpei Su2, Jian Zhang11Key Laboratory of Protein Chemistry and Developmental Biology, Ministry of Education of China, College of Life Sciences; 2Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research and Key Laboratory of Sustainable Resources Processing and Advanced Materials of Hunan Province, Ministry of Education of China, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, China*These authors contributed equally to this workBackground: Smaller nanoparticles facilitate the delivery of DNA into cells through endocytosis and improve transfection efficiency. The aim of this study was to determine whether protamine sulfate-coated calcium phosphate (PS-CaP could stabilize particle size and enhance transfection efficiency.Methods: pEGFP-C1 green fluorescence protein was employed as an indicator of transfection efficiency. Atomic force microscopy was used to evaluate the morphology and the size of the particles, and an MTT assay was introduced to detect cell viability and inhibition. The classical calcium phosphate method was used as the control.Results: Atomic force microscopy images showed that the PS-CaP were much smaller than classical calcium phosphate particles. In 293 FT, HEK 293, and NIH 3T3 cells, the transfection efficiency of PS-CaP was higher than for the classical calcium phosphate particles. The difference in efficiencies implies that the smaller nanoparticles may promote the delivery of DNA into cells through endocytosis and could improve transfection efficiency. In addition, PS-CaP could be used to transfect HEK 293 cells after one week of storage at 4°C with a lesser extent of efficiency loss compared with classical calcium phosphate, indicating that protamine sulfate may increase the stability of calcium phosphate nanoparticles. The cell viability inhibition assay indicated that

  19. Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

    Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung


    For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa. PMID:26408812

  20. Inhibitory effect of MgSO4 on calcium overload after radiation-induced brain injuries

    Objective: To explore the neuroprotective effect of magnesium sulfate (MgSO4 ) on radiation-induced acute brain injuries. Methods: A total of 60 mature Sprague-Dawley rats were randomly divided into 3 groups: blank control group, experimental control group and experimental therapy group. The whole brain of SD rats of experimental control group and experimental therapy group was irradiated to a dose of 20 Gy using 6 MeV electrons. Magnesium sulfate was injected intraperitoneally into the rats of experimental therapy group before and after irradiation for five times. At different time points (24 h, 7 days, 14 days, 30 days after irradiation), the brain tissue was taken. Plasma direct reading spectrography was used to measure the contents of Ca2+, Mg2+ in brain tissue, and the percentage of brain water content was calculated with the wet-dry weight formula. Results: Compared with the blank control group, the percentage of brain water and content of Ca2+ in brain of the experimental control group increased markedly (P2+ decreased significantly (P2+ in brain of the experimental therapy group were significantly lower than those of the experimental control group (P<0.05). Conclusion: Magnesium sulfate used in the early stage after irradiation can inhibit the calcium overload in rat brain , and attenuate brain edema and injuries. (authors)

  1. Somatic ATP release from guinea pig sympathetic neurons does not require calcium-induced calcium release from internal stores

    Merriam, Laura A.; Locknar, Sarah A.; Girard, Beatrice M.; Parsons, Rodney L.


    Prior studies indicated that a Ca2+-dependent release of ATP can be initiated from the soma of sympathetic neurons dissociated from guinea pig stellate ganglia. Previous studies also indicated that Ca2+-induced Ca2+ release (CICR) can modulate membrane excitability in these same neurons. As Ca2+ release from internal stores is thought to support somatodendritic transmitter release in other neurons, the present study investigated whether CICR is essential for somatic ATP release from dissociat...

  2. Effect of dietary calcium and magnesium on experimental renal tubular deposition of calcium oxalate crystal induced by ethylene glycol administration and its prevention with phytin and citrate.

    Ebisuno, S; Morimoto, S; Yoshida, T; Fukatani, T; Yasukawa, S; Ohkawa, T


    Oral administration of ethylene glycol to rats, and the resultant intratubular depositions of microcrystals of calcium oxalate were studied investigating the influences of dietary calcium or magnesium and assessing the protective efficacies against the crystallizations by treatment with phytin and sodium citrate. With increase of calcium intake and consequent increase of urinary calcium excretion there was a marked increase in the amount of tubular deposit of calcium oxalate crystal and in the calcium content of renal tissue. Although magnesium deficiency accelerated renal tubular calcium oxalate deposition, the protection against the crystal formation was not observed with excessive dietary magnesium. When rats were fed a high-calcium diet supplemented with phytin, a significant inhibition of the intratubular crystallization was observed. It appeared obvious that a hypocalciuric action of phytin was attributed to the effect of the prevention. There was vigorous protection of crystal formation by treatment with sodium citrate, which correlated with the level of citrate concentration in the drinking water. PMID:3433579

  3. Influence factors and gene expression patterns during MeJa-induced gummosis in peach.

    Li, Minji; Liu, Meiyan; Peng, Futian; Fang, Long


    Jasmonates (JAs) play important roles in gummosis in peach. Mechanical damage, methyl jasmonate (MeJa), and ethylene can induce gummosis on peach shoots in the field. In this study, we used MeJa (2%, w/w) to induce gummosis on current-year shoots in peach on high temperature (35°C). Based on the experimental model, we studied the influence of factors on the development of peach gummosis. Our experimental results showed that high temperature could promote gummosis development induced by MeJa. Exogenous CaCl2 treatment reduced the degree of gummosis by increasing the calcium content in shoots, which is conducive to the synthesis and maintenance of the cell wall. Using digital gene expression (DGE), 3831 differentially expressed genes were identified in the MeJa treatment versus the control. By analyzing changes in gene expression associated with cell wall degradation, genes encoding pectin methylesterase (PME) and endo-polygalacturonase (PG) were found to be significantly induced, suggesting that they are key enzymes in cell wall degradation that occurs during MeJa-induced gummosis. Genes for glycosyltransferase (GT) and cellulose synthase (CS) were also significantly upregulated by MeJa. This result suggests that MeJa treatment not only promotes the degradation of polysaccharides to destroy the cell wall, but also promotes the synthesis of new polysaccharides. We also analyzed changes in gene expression associated with sugar metabolism, senescence, and defense. MeJa treatment affected the expression of genes related to sugar metabolism and promoted plant senescence. Among the defense genes, the expression pattern of phenylalanine ammonium lyase (PAL) suggested that PAL may play an important role in protecting against the effects of MeJa treatment. Our experimental results showed that MeJa treatment can promote the biosynthesis and signal transduction of ethylene in peach shoots; they can induce gummosis on peach shoots respectively, and there are overlaps between

  4. TRPV1 Activation Exacerbates Hypoxia/Reoxygenation-Induced Apoptosis in H9C2 Cells via Calcium Overload and Mitochondrial Dysfunction

    Zewei Sun


    Full Text Available Transient potential receptor vanilloid 1 (TRPV1 channels, which are expressed on sensory neurons, elicit cardioprotective effects during ischemia reperfusion injury by stimulating the release of neuropeptides, namely calcitonin gene-related peptide (CGRP and substance P (SP. Recent studies show that TRPV1 channels are also expressed on cardiomyocytes and can exacerbate air pollutant-induced apoptosis. However, whether these channels present on cardiomyocytes directly modulate cell death and survival pathways during hypoxia/reoxygenation (H/R injury remains unclear. In the present study, we investigated the role of TRPV1 in H/R induced apoptosis of H9C2 cardiomyocytes. We demonstrated that TRPV1 was indeed expressed in H9C2 cells, and activated by H/R injury. Although neuropeptide release caused by TRPV1 activation on sensory neurons elicits a cardioprotective effect, we found that capsaicin (CAP; a TRPV1 agonist treatment of H9C2 cells paradoxically enhanced the level of apoptosis by increasing intracellular calcium and mitochondrial superoxide levels, attenuating mitochondrial membrane potential, and inhibiting mitochondrial biogenesis (measured by the expression of ATP synthase β. In contrast, treatment of cells with capsazepine (CPZ; a TRPV1 antagonist or TRPV1 siRNA attenuated H/R induced-apoptosis. Furthermore, CAP and CPZ treatment revealed a similar effect on cell viability and mitochondrial superoxide production in primary cardiomyocytes. Finally, using both CGRP8–37 (a CGRP receptor antagonist and RP67580 (a SP receptor antagonist to exclude the confounding effects of neuropeptides, we confirmed aforementioned detrimental effects as TRPV1−/− mouse hearts exhibited improved cardiac function during ischemia/reperfusion. In summary, direct activation of TRPV1 in myocytes exacerbates H/R-induced apoptosis, likely through calcium overload and associated mitochondrial dysfunction. Our study provides a novel understanding of the role of


    Janir Alves Soares; Mário Roberto Leonardo; Léa Assed Bezerra da Silva; Mário Tanomaru Filho; Izabel Yoko Ito


    The aim of this study was to evaluate the antiseptic efficacy of rotary instrumentation associated with calcium hydroxide-based pastes prepared with different vehicles and antiseptics. Chronic periapical lesions were experimentally induced in 72 premolar root canals of four dogs. Under controlled asepsis, after initial microbiological sampling (A1), the root canals were instrumented using the ProFile system in conjunction with 5.25% sodium hypochlorite and the intracanal medication was placed...

  6. Differential modulation of ATP-induced calcium signalling by A1 and A2 adenosine receptors in cultured cortical astrocytes

    Alloisio, Susanna; Cugnoli, Carlo; Ferroni, Stefano; Nobile, Mario


    Despite the accumulating evidence that under various pathological conditions the extracellular elevation of adenine-based nucleotides and nucleosides plays a key role in the control of astroglial reactivity, how these signalling molecules interact in the regulation of astrocyte function is still largely elusive.The action of the nucleoside adenosine in the modulation of the intracellular calcium signalling ([Ca2+]i) elicited by adenosine 5′-triphosphate (ATP)-induced activation of P2 purinoce...

  7. In Vitro Spectrophotometry of Tooth Discoloration Induced by Tooth-Colored Mineral Trioxide Aggregate and Calcium-Enriched Mixture Cement

    Arman, Marjan; Khalilak, Zohreh; Rajabi, Moones; Esnaashari, Ehsan; Saati, Keyvan


    Introduction: There are numerous factors that can lead to tooth discoloration after endodontic treatment, such as penetration of endodontic materials into the dentinal tubules during root canal treatment. The aim of this in vitro study was to compare discoloration induced by tooth colored mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) cement in extracted human teeth. Methods and Materials: Thirty two dentin-enamel cuboid blocks (7×7×2 mm) were prepared from extracted maxi...

  8. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    Ostrow, Lyle W., E-mail: [Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States); Suchyna, Thomas M.; Sachs, Frederick [Department of Physiology and Biophysical Sciences, State University of New York at Buffalo, Buffalo, NY 14214 (United States)


    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  9. Parathyroid Hormone Induces Bone Cell Motility and Loss of Mature Osteocyte Phenotype through L-Calcium Channel Dependent and Independent Mechanisms.

    Matthew Prideaux

    Full Text Available Parathyroid Hormone (PTH can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R, which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1 promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA, exchange proteins activated by cAMP (Epac, protein kinase C (PKC or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these

  10. Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family

    Kaufman, I; Tindjong, R; McClintock, P V E; Eisenberg, R S


    We use Brownian dynamics simulations to study the permeation properties of a generic electrostatic model of a biological ion channel as a function of the fixed charge Q_f at its selectivity filter. We reconcile the recently-discovered discrete calcium conduction bands M0 (Q_f=1e), M1 (3e), M2 (5e) with the set of sodium conduction bands L0 (0.5-0.7e), L1 (1.5-2e) thereby obtaining a completed pattern of conduction and selectivity bands v Q_f for the sodium-calcium channels family. An increase of Q_f leads to an increase of calcium selectivity: L0 (sodium selective, non-blocking channel) -> M0 (non-selective channel) -> L1 (sodium selective channel with divalent block) -> M1 (calcium selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L1 band is identified with the eukaryotic (DEKA) sodium channel, and L0 (speculatively) with the bacterial NaChBac channel. The scheme created is able to account for the experimentally observed mutation-induced ...

  11. Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

    Hudecová, S.; Vadászová, Adriana; Soukup, Tomáš; Križanová, O.


    Roč. 75, č. 8 (2004), s. 923-931. ISSN 0024-3205 R&D Projects: GA ČR GA309/03/0752 Grant ostatní: VEGA(SK) 2/3008; NATO(XX) 979876; SAV(SK) APVT-51-013802 Institutional research plan: CEZ:AV0Z5011922 Keywords : thyroid hormones * calcium transport systems Subject RIV: ED - Physiology Impact factor: 2.158, year: 2004

  12. Analysis of calcium-induced effects on the conformation of fengycin

    Nasir, Mehmet Nail; Laurent, Pascal; Flore, Christelle; Lins, Laurence; Ongena, Marc; Deleu, Magali


    Fengycin is a natural lipopeptide with antifungal and eliciting properties and able to inhibit the activity of phospholipase A2. A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate its conformation in a membrane-mimicking environment and to investigate the effect of calcium ions on it. We mainly observed that fengycin adopts a turn conformation. Our results showed that calcium ions are bound by the two charged glutamates. The calcium binding has an influence on the fengycin conformation and more particularly, on the environment of the tyrosine residues. The modulation of the fengycin conformation by the environmental conditions may influence its biological properties.

  13. Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth*

    Takemura, Miyohiko; Mishima, Toshiaki; Wang, Yan; Kasahara, Jiro; Fukunaga, Kohji; Ohashi, Kazumasa; Mizuno, Kensaku


    Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppres...

  14. Cardioprotective activity of alcoholic extract of Tinospora cordifolia (Willd.) Miers in calcium chloride-induced cardiac arrhythmia in rats

    Sharma, Ashish Kumar; Kishore, Kunal; Sharma, Divya; B P Srinivasan; Agarwal, Shyam Sunder; Sharma, Ashok; Singh, Santosh Kumar; Gaur, Samir; Jatav, Vijay Singh


    The present study investigated the antiarrhythmic activity of alcoholic extract of Tinospora cordifolia (T. cordifolia) in CaCl2 induced arrhythmia. CaCl2 (25 mg/kg) was administered by intravenous infusion (iv) to produce arrhythmia in rats. The animals were then treated with T. cordifolia extract (150, 250, and 450 mg/kg) and verapamil (5 mg/kg,iv). Lead II electrocardiogram was monitored. Plasma calcium, sodium and potassium levels were measured. In CaCl2 induced arrhythmia, heart rate was...

  15. IL-10 induces gene expression in macrophages: partial overlap with IL-5 but not with IL-4 induced genes.

    Stumpo, Rita; Kauer, Manfred; Martin, Stephan; Kolb, Hubert


    The hypothesis that IL-10, in addition to down-regulating pro-inflammatory activities of macrophages, induces an alternative state of macrophage reactivity was tested. We therefore conducted a systematic search for genes induced by IL-10 using the method of suppression subtractive hybridisation. Of an initial 1,300 candidate clones obtained, several screening rounds led to the identification of 51 clones which were reproducibly at least twofold up-regulated in mouse J774 macrophages in response to treatment with IL-10. Of these, 41 genes were homologous to known genes involved in cell metabolism or immunoregulation, five contained novel sequences and another five were homologous to ESTs without known function. One major finding was that about 25% of the IL-10 genes were also found expressed in response to IFNgamma, and several of these also reappeared in IL-4 or IL-5 induced mRNA species. Hence, Th1 and Th2 type cytokines may elicit a common basal activation response in macrophages. The second major finding was that 57% of IL-10 induced genes reappeared in IL-5 induced mRNA but no more than 18% were also found in IL-4 induced mRNA of J774 cells. We conclude that the gene expression response to IL-10 in macrophages is partially different from the response to IL-5 and is substantially different from the response to IL-4, which suggests an unexpected diversity of biological phenotypes induced by different Th2 type cytokines. PMID:14561490

  16. Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille;


    Electroporation of cells with short, high-voltage pulses causes a transient permeabilization of cell membranes that permits passage of otherwise nonpermeating ions and molecules. In this study, we illustrate how electroporation with isotonic calcium can achieve highly effective cancer cell kill in......, 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....... Electroporation equipment is already used clinically to enhance the delivery of chemotherapy to superficial tumors, with trials on internal tumors in progress, enabling the introduction of calcium electroporation to clinical use. Moreover, the safety profile, availability, and low cost of calcium facilitate...

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

    Francesco Chemello


    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 ( (GSE60931.

  18. Genome-wide survey and expression analysis of the calcium-dependent protein kinase gene family in cassava.

    Hu, Wei; Hou, Xiaowan; Xia, Zhiqiang; Yan, Yan; Wei, Yunxie; Wang, Lianzhe; Zou, Meiling; Lu, Cheng; Wang, Wenquan; Peng, Ming


    Calcium-dependent protein kinases (CPKs) play important roles in regulating plant tolerance to abiotic stress and signal transduction; however, no data are currently available regarding the CPK family in cassava. Herein, we identified 27 CPK genes from cassava based on our previous genome sequencing data. Phylogenetic analysis showed that cassava CPKs could be clustered into three groups, which was further supported by gene structure and conserved protein motif analyses. Global expression analysis suggested that MeCPK genes showed distinct expression patterns in different tissues between wild subspecies and cultivated varieties, indicating their involvement in the functional diversity of different varieties. Transcriptomics, interaction networks, and co-expression assays revealed a broad transcriptional response of cassava CPKs and CPK-mediated networks to drought stress and their differential expression profiles in different varieties, implying their contribution to drought stress tolerance in cassava. Expression analysis of eight MeCPK genes suggested a comprehensive response to osmotic stress, salt, cold, abscisic acid, and H2O2, which indicated that cassava CPKs might be convergence points for different signaling pathways. This study provides a basis for crop improvements and understanding of abiotic stress responses and signal transduction mediated by CPKs in cassava. PMID:26272723

  19. Vascularization of plastic calcium phosphate cement in vivo induced by in-situ-generated hollow channels.

    Yu, Tao; Dong, Chao; Shen, Zhonghua; Chen, Yan; Yu, Bo; Shi, Haishan; Zhou, Changren; Ye, Jiandong


    Despite calcium phosphate cement (CPC) is promising for bone repair therapy, slow biodegradation and insufficient vascularization in constructs negatively impacts its clinical application. A self-setting CPC composited with gelatin fiber is investigated to test the utility of this tissue engineering strategy to support rapid and extensive vascularization process. The interconnected hollow channels in CPC are formed after dissolution of gelatin fibers in vivo. The CPC-gelatin samples exhibit relatively decent/enhanced mechanical property, compared to the control. When implanted in vivo, the pre-established vascular networks in material anastomose with host vessels and accelerate vascular infiltration throughout the whole tissue construct. Different channel sizes induce different vascularization behaviors in vivo. Results indicate that the channel with the size of 250μm increases the expression of the representative angiogenic factors HIF1α, PLGF and migration factor CXCR4, which benefit the formation of small vessels. On the other hand, the channel with the size of 500μm enhances VEGF-A expression, which benefit the development of large vessels. Notably, the intersection area of channels has high invasive, sprouting and vasculogenesis potential under hypoxic condition, because more HIF1α-positive cells are observed there. Observation of the CD31-positive lumen in the border of scaffold indicates the ingrowth of blood vessels from its host into material through channel, benefited from gradually increased HIF1α expression. This kind of material was suggested to promote the effective application of bone regeneration through the combination of in situ self-setting, plasticity, angiogenesis, and osteoconductivity. PMID:27524007

  20. Surface expressed nucleolin is constantly induced in tumor cells to mediate calcium-dependent ligand internalization.

    Ara G Hovanessian

    Full Text Available BACKGROUND: Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in tumorigenesis and angiogenesis. Emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS: By monitoring the expression of nucleolin mRNA, and by measuring the level of nucleolin protein recovered from the surface and nucleus of cells, here we show that the presence of nucleolin at the cell surface is dependent on the constant induction of nucleolin mRNA. Indeed, inhibitors of RNA transcription or translation block expression of surface nucleolin while no apparent effect is observed on the level of nucleolin in the nucleus. The estimated half-life of surface nucleolin is less than one hour, whereas that of nuclear nucleolin is more than 8 hours. Nucleolin mRNA induction is reduced markedly in normal fibroblasts that reach confluence, while it occurs continuously even in post-confluent epithelial tumor cells consistent with their capacity to proliferate without contact inhibition. Interestingly, cold and heat shock induce nucleolin mRNA concomitantly to enhanced mRNA expression of the heat shock protein 70, thus suggesting that surface nucleolin induction also occurs in response to an environmental insult. At the cell surface, one of the main functions of nucleolin is to shuttle specific extracellular ligands by an active transport mechanism, which we show here to be calcium dependent. CONCLUSION/SIGNIFICANCE: Our results demonstrate that the expression of surface nucleolin is an early metabolic event coupled with tumor cell proliferation and stress response. The fact that surface nucleolin is constantly and abundantly expressed on the surface of tumor cells, makes them a preferential target for the inhibitory action of anticancer agents that target

  1. Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cell Seeding on Biofunctionalized Calcium Phosphate Cements

    WahWah TheinHan; Jun Liu; Minghui Tang; Wenchuan Chen; Linzhao Cheng; Hockin H. K. Xu


    Induced pluripotent stem cells (iPSCs) have great potential due to their proliferation and differentiation capability. The objectives of this study were to generate iPSC-derived mesenchymal stem cells (iPSC-MSCs), and investigate iPSC-MSC proliferation and osteogenic differentiation on calcium phosphate cement (CPC) containing biofunctional agents for the first time. Human iPSCs were derived from marrow CD34+ cells which were reprogrammed by a single episomal vector. iPSCs were cultured to form embryoid bodies (EBs), and MSCs migrated out of EBs. Five biofunctional agents were incorporated into CPC:RGD (Arg-Gly-Asp) peptides, fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and platelet concentrate. iPSC-MSCs were seeded on five biofunctionalized CPCs:CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. iPSC-MSCs on biofunctional CPCs had enhanced proliferation, actin fiber expression, osteogenic differentiation and mineralization, compared to control. Cell proliferation was greatly increased on biofunctional CPCs. iPSC-MSCs underwent osteogenic differentiation with increased alkaline phosphatase, Runx2 and collagen-I expressions. Mineral synthesis by iPSC-MSCs on CPC-Platelets was 3-fold that of CPC control. In conclusion, iPSCs showed high potential for bone engineering. iPSC-MSCs on biofunctionalized CPCs had cell proliferation and bone mineralization that were much better than traditional CPC. iPSC-MSC-CPC constructs are promising to promote bone regeneration in craniofacial/orthopedic repairs.

  2. PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina

    Nancy Scardua Binda


    Full Text Available The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-d-Aspartate-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a, a spider toxin that blocks N-P/Q calcium channels.

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

    Yutaka Maruyama; Reiko Yasuda; Motonaka Kuroda; Yuzuru Eto


    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 su...

  4. Oxidant-Induced Inhibition of Myocardial Calcium-Independent Phospholipase A2

    McHowat, Jane; Swift, Luther M.; Sarvazyan, Narine


    We discovered the acute inhibition of myocardial phospholipase A2 activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A2 activity was assessed in cell subcellular fractions using (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A2 activity was inhibi...

  5. Different effects of R 56865 and calcium entry blockers on K+- and noradrenaline-induced contractions and45Ca uptake in rat aorta

    Koch, P.; Wilhelm, D.; Wermelskirchen, D.; Nebel, U.; Wilffert, B.; Peters, Thies


    The effects of R 56865, nifedipine, verapamil, diltiazem and flunarizine on K+- and NA-induced contractions and K+-induced45Ca uptake were compared in the isolated rat aorta. The calcium entry blockers concentration dependently inhibited the K+-induced contraction and45Ca uptake over the same dose-r

  6. Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D3 are suppressed by calcium channel blockers

    Previous investigations have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] stimulates muscle Ca uptake through a nuclear mechanism. The possibility that 1,25-(OH)2D3 would induce rapid changes in muscle Ca fluxes independent of de novo protein synthesis was investigated in the present work. In vitro preparations of soleus muscles obtained from vitamin D-deficient chicks were used. A significant increase in 45Ca labeling of the tissue was already observed after 3-min treatment with 2.4 X 10(-10) M 1,25-(OH)2D3. This early stimulation in muscle Ca uptake became maximal at 10-15 min. Cycloheximide (50 microM) did not block the effect of the metabolite at 15 and 30 min. However, the antibiotic effectively blocked the increase in Ca uptake induced by 1,25-(OH)2D3 after 1-h treatment. The rapid 1,25-(OH)2D3-dependent stimulation of 45Ca labeling of soleus muscle was not associated to changes in lipid synthesis as assessed by measurements of 3H-glycerol incorporation into the tissue lipids. However, the calcium antagonists verapamil and nifedipine (50 microM) abolished the stimulation in Ca uptake produced by 1,25-(OH)2D3 in 5 min. These results suggest that 1,25-(OH)2D3 can act directly at the muscle membrane level affecting Ca fluxes through Ca channels

  7. Biophysical characterization and functional studies on calbindin-D28K: A vitamin D-induced calcium-binding protein

    Vitamin D dependent calcium binding protein, or calbindin-D, is the principal protein induced in the intestine in response to the steroid hormone 1,25(OH)2-vitamin D3. A definitive role for calbindin-D in vitamin D3 mediated biological responses remains unclear. Biophysical and functional studies on chick intestinal calbindin-D28K (CaBP) were initiated so that some insight might be gained into its relevance to the process of intestinal calcium transport. Calbindin-D belongs to a class of high affinity calcium binding proteins which includes calmodulin, parvalbumin and troponin C. The Ca 2+ binding stoichiometry and binding constants for calbindin-D28K were quantitated by Quin 2 titration analysis. The protein was found to bind 5-6 Ca 2+ ions with a KD on the order of 10-8, in agreement with the 6 domains identified from the amino acid sequence. A slow Ca 2+ exchange rate (80 s-1) as assessed by 43Ca NMR and extensive calcium dependent conformational changes in 1H NMR spectra were also observed. Functional studies on chick intestinal CaBP were carried out by two different methods. Interactions between CaBP and intestinal cellular components were assessed via photoaffinity labeling techniques. Specific calcium dependent complexes for CaBP were identified with bovine intestinal alkaline phosphatase and brush border membrane proteins of 60 and 150 kD. CaBP was also found to co-migrate with the alkaline phosphatase activity of chick intestinal brush border membranes as evaluated by gel filtration chromatography. The second procedure for evaluating CaBP functionality has involved the quantitation of CaBP association with vesicular transport components as assessed by ELISA. CaBP, immunoreactivity was observed in purified lysosomes, microsomes and microtubules

  8. Screening of hypoxia-inducible genes in sporadic ALS.

    Cronin, Simon


    Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

  9. Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

    Goichi Beck; Koei Shinzawa; Hideki Hayakawa; Kousuke Baba; Toru Yasuda; Hisae Sumi-Akamaru; Yoshihide Tsujimoto; Hideki Mochizuki


    Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA2β), which is encoded by the PLA2G6 gene. Perl's staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron hom...

  10. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  11. Mechanisms of radiation-induced gene responses

    In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5' region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3' region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts; however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process

  12. Localization of large conductance calcium-activated potassium channels and their effect on calcitonin gene-related peptide release in the rat trigemino-neuronal pathway

    Wulf-Johansson, H.; Amrutkar, D.V.; Hay-Schmidt, Anders;


    Large conductance calcium-activated potassium (BK(Ca)) channels are membrane proteins contributing to electrical propagation through neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide found in the trigeminovascular system (TGVS). Both BK(Ca) channels and CGRP are involved in migraine...

  13. Inducible gene expression and environmentally regulated genes in lactic acid bacteria

    Kok, Jan


    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transc

  14. Calcium induced regulation of skeletal troponin--computational insights from molecular dynamics simulations.

    Georgi Z Genchev

    Full Text Available The interaction between calcium and the regulatory site(s of striated muscle regulatory protein troponin switches on and off muscle contraction. In skeletal troponin binding of calcium to sites I and II of the TnC subunit results in a set of structural changes in the troponin complex, displaces tropomyosin along the actin filament and allows myosin-actin interaction to produce mechanical force. In this study, we used molecular dynamics simulations to characterize the calcium dependent dynamics of the fast skeletal troponin molecule and its TnC subunit in the calcium saturated and depleted states. We focused on the N-lobe and on describing the atomic level events that take place subsequent to removal of the calcium ion from the regulatory sites I and II. A main structural event - a closure of the A/B helix hydrophobic pocket results from the integrated effect of the following conformational changes: the breakage of H-bond interactions between the backbone nitrogen atoms of the residues at positions 2, 9 and sidechain oxygen atoms of the residue at position 12 (N(2-OE(12/N(9-OE(12 in sites I and II; expansion of sites I and II and increased site II N-terminal end-segment flexibility; strengthening of the β-sheet scaffold; and the subsequent re-packing of the N-lobe hydrophobic residues. Additionally, the calcium release allows the N-lobe to rotate relative to the rest of the Tn molecule. Based on the findings presented herein we propose a novel model of skeletal thin filament regulation.

  15. Low doses of neutrons induce changes in gene expression

    Studies were designed to identify genes induced following low-dose neutron but not following γ-ray exposure in fibroblasts. Our past work had shown differences in the expression of β-protein kinase C and c-fos genes, both being induced following γ-ray but not neutron exposure. We have identified two genes that are induced following neutron, but not γ-ray, exposure: Rp-8 (a gene induced by apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency (HIV). Rp-8 mRNA induction was demonstrated in Syrian hamster embryo fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.5 cGy/min) and at high dose rate (12 cGy/min). The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measures of CAT activity and CAT transcripts following irradiation demonstrated an unresponsiveness to γ rays over a broad range of doses. Twofold induction of the HIV-LTR was detected following neutron exposure (48 cGy) administered at low (0.5 cGy/min) but not high (12 cGy/min) dose rates. Ultraviolet-mediated HIV-LTR induction was inhibited by low-dose-rate neutron exposure

  16. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

    Rami Shinnawi


    Full Text Available The advent of the human-induced pluripotent stem cell (hiPSC technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs. To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight and calcium (GCaMP5G fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.

  17. Epac2 contributes to PACAP-induced astrocytic differentiation through calcium ion influx in neural precursor cells.

    Seo, Hyunhyo; Lee, Kyungmin


    Astrocytes play a critical role in normal brain functions and maintaining the brain microenvironment, and defects in astrocytogenesis during neurodevelopment could give rise to severe mental illness and psychiatric disorders. During neuro-embryogenesis, astrocytogenesis involves astrocytic differentiation of neural precursor cells (NPCs) induced by signals from ciliary neurotrophic factor (CNTF) or pituitary adenylate cyclase-activating peptide (PACAP). However, in contrast to the CNTF signaling pathway, the exact mechanism underlying astrocytic differentiation induced by PACAP is unknown. In the present study, we aimed to verify a signaling pathway specific to PACAP-induced astrocytogenesis, using exchange protein directly activated by cAMP2 (Epac2)-knockout mice. We found that PACAP could trigger astrocytic differentiation of NPCs via Epac2 activation and an increase in the intracellular calcium concentration via a calcium ion influx. Taken together, we concluded that astrocytogenesis stimulated by PACAP occurs through a novel signaling pathway independent from CNTF-JAK/STAT signaling, that is the well-known pathway of astrocytogenesis. [BMB Reports 2016; 49(2): 128-133]. PMID:26645637

  18. Calcium ionophore (A-23187) induced peritoneal eicosanoid biosynthesis: a rapid method to evaluate inhibitors of arachidonic acid metabolism in vivo

    Rao, T S; Currie, J. L.; A. F. Shaffer; Isakson, P C


    The present investigation characterizes calcium ionophore (A-23187) induced peritoneal eicosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat) stimulated marked biosynthesis of 6-keto-PGF1α (6-KPA), TxB2, LTC4 and LTB4, with no detectable changes on levels of PGE2. Levels of all eicosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased r...

  19. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z


    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart. PMID:20720179

  20. Comparison of the Species-Sensitive Effects of Different Dosages of Calcium and Verapamil on Gentamicin-Induced Nephrotoxicity in Rats and Rabbits

    Amol N Patil; Arora, Tarun; Desai, Amrita; Tripathi, Chakra Dhar


    Aim: To compare the effects of different dosages of calcium and verapamil on gentamicin-induced nephrotoxicity in rats and rabbits. Materials and Methods: Rabbits and rats of either sex in weight range of 1.5–2.5 kg and 175–225 g, respectively were used in study. Gentamicin 80 mg/kg i.m., calcium carbonate 0.5 g/kg/day oral, calcium carbonate 1.0 g/kg/day oral, and verapamil 7 mg/kg/day i.m. were administered for 6 days in either species containing 7 groups. Blood urea nitrogen (BUN), serum c...

  1. Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons

    Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated Ca2+ calcium chann...

  2. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  3. Induced marker gene mutations in soybean

    Full text: Non-fluorescent root mutants in soybean are useful as markers in genetic studies. 13 such mutants were detected among more than 150 000 seedlings derived from soybean lines treated with 6 mutagens. One of them, derived from variety 'Williams' treated with 20 kR gamma rays, did not correspond to the already known spontaneous non-fluorescent mutants. It was assigned the identification no. T285 and the gene symbol fr5. The other mutants corresponded with known loci fr1, fr2 or fr4. (author)

  4. The effects of calcium channel blockade on agouti-induced obesity

    Kim, Jung Han; Moustaid, N.; Zemel, M.B. [Univ. of Tennessee, Knoxville, TN (United States)] [and others


    We have previously observed that obese viable yellow (A{sup vy}/a) mice exhibit increased intracellular Ca{sup 2+} ([Ca{sup 2+}]i) and fatty acid synthase (FAS) gene expression; further, recombinant agouti protein increases in cultured adipocytes and these effects are inhibited by Ca{sup 2+} channel blockade. Accordingly, we determined the effect of Ca{sup 2+} channel blockade (nifedipine for 4 wk) on FAS and obesity in transgenic mice expressing the agouti gene in a ubiquitous manner. The transgenic mice initially were significantly heavier (30.5 {+-} 0.6 vs. 27.3 {+-} 0.3 g; P<0.001) and exhibited a 0.81{degrees}C lower initial core temperature (P<0.0005), an approximately twofold increase in fat pad weights (P=0.002), a sevenfold increase in adipose FAS activity (P=0.009), and a twofold increase in plasma insulin level (P<0.05) compared to control mice. Nifedipine treatment resulted in an 18% decrease in fat pad weights (P<0.007) and a 74% decrease in adipose FAS activity (P=0.03), normalized circulating insulin levels and insulin sensitivity (P,0.05), and transiently elevated core temperature in the transgenic mice, but was without effect in the control mice. These data suggest that agouti regulates FAS, fat storage, and possibly thermogenesis, at least partially, via a [Ca{sup 2+}]{sub i}-dependent mechanism, and that Ca{sup 2+} channel blockade may partially attenuate agouti-induced obesity. 42 refs., 4 figs., 1 tab.

  5. Different sensitivities for four calcium entry blockers of serotonin-, but not K+-induced contractions of isolated jugular vein and aorta of the rat

    Gouw, M.A.M.; Wilffert, B.; Van Zwieten, P.A.


    We investigated the inhibitory effect of the calcium entry blockers (CEBs) nifedipine, diltiazem, flunarizine and gallopamil on K+- and serotonin (5-HT)-induced contractions of the rat jugular vein and aorta in vitro. In both tissues all four CEBs inhibited K+-induced contractions concentration-depe

  6. CNS Voltage-gated Calcium Channel Gene Variation And Prolonged Recovery Following Sport-related Concussion

    McDevitt, Jane


    Objectives: To examine the association between concussion duration and two calcium channel, voltage-dependent, R type, alpha 1E subunit (CACNA1E) single nucleotide polymorphisms (i.e., rs35737760 and rs704326). A secondary purpose was to examine the association between CACNA1E single nucleotide polymorphisms (SNPs) and three acute concussion severity scores (i.e., vestibule-ocular reflex test, balance error scoring scale, and Immediate Post-Concussion Assessment and Cognitive Testing). Methods: Forty athletes with a diagnosed concussion from a hospital concussion program completed a standardized initial evaluation. Concussion injury characteristics, acute signs and symptoms followed by an objective screening (i.e., vestibular ocular assessments, balance error scoring system test, and Immediate Post-Concussion Assessment and Cognitive Testing exam) were assessed. Enrolled participants provided salivary samples for isolation of DNA. Two exon SNPs rs35737760 and rs704326 within CACNA1E were genotyped. Results: There was a significant difference found between acute balance deficits and prolonged recovery group (X2 = 5.66, p = 0.017). There was an association found between the dominant model GG genotype (X2 = 5.41, p = 0.027) within the rs704326 SNP and prolonged recovery group. Significant differences were identified for the rs704326 SNP within the dominant model GG genotype (p = 0.030) for VOR scores by recovery. A significant difference was found between the rs704326 SNP codominant model AA (p = 0.042) and visual memory. There was an association between acute balance deficits and prolonged recovery (X2 = 5.66, p = 0.017) for the rs35737760 SNP. No significant associations between concussion severity and genotype for rs35737760 SNP. Conclusion: Athletes carrying the CACNA1E rs704326 homozygous genotype GG are at a greater risk of a prolonged recovery. Athletes that reported balance deficits at the time of injury were more likely to have prolonged recovery. These

  7. Types of voltage—dependent calcium channels involved in high potassium depolarization—induced amylase secretion in the exocrine pancreatic tumour cell line AR4—2J



    In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium,Attached cells when stimulated with high potassium secreted large amount of amylase.High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation.High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel anatagonists with an order of potency as follows:nifedipine>ω-agatoxin IVA>ω-conotoxin GVIA.In contrast,the L-type calcium channel anatagonist nifedipine almost completely inhibited potassium-induced amylase secretion,whereas the N-type channel antagonist ω-conotoxin GVIA was without effect.The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect,but this inhibition was not significant at the level of amylase secretion.In conclusion,the AR4-2J cell line posesses different voltage-dependent calcium channels(L,P,N)with the L-type predominantly involved in depolarization induced amylase secretion.

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

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


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

  9. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation.

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang


    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  10. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang


    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.