Extra Ca2 Research Articles

Basket anode filled with CaCO3 particles: A membrane-free electrochemical system for boosting phosphate recovery and product purity

Phosphorus (P) is often regarded as the primary stimulant for eutrophication, while its importance as a crucial life element is also well acknowledged. Given its future scarcity, P recycling from waste streams is suggested and practiced. Electrochemically mediated precipitation (EMP) is a robust and chemical-free process for P removal and recovery, yet it requires further developments. The first generation of the CaCO3-packed electrochemical precipitation column successfully solved the problem of H+-OH− recombination, achieving enhanced P removal efficiency with less energy consumption but suffering from low Ca-phosphate purity in recovered products. Herein, a new concept of a basket-anode electrochemical system is proposed and validated to prevent direct H+-OH− recombination and enhance product purity. The CaCO3 pellets packed basket anode alleviates the OH− depletion by CaCO3-H+ interaction and provides extra Ca2+ for enhanced P removal. The novel structure of the basket anode, by its derived acidic anode region and alkaline cathode region, completely avoids the precipitation of Ca-phosphate on the packed CaCO3 and greatly facilitates the collection of high-quality Ca-phosphate product. Our results suggest that almost 100% of the removed P was in high-purity, highly crystalline Ca-phosphate on the cathode. The recovered products contained significantly more P (13.5 wt%) than in the previous study (0.1 wt%) at similar energy consumptions (29.8 kWh/kg P). The applied current density, pellets size, and influent P concentration were critical for P removal performance, product purity, and power consumption. We further demonstrated the long-term stability of this novel system and its technical and economic feasibility in treating real stored urine. Our study provides new cell architectural designs to enhance the performance of EMP systems and may inspire innovations and developments in other electrochemical water treatment processes.

Germination Kinetics and Chlorophyll Fluorescence Imaging Allow for Early Detection of Alkalinity Stress in Rhododendron Species

Rhododendron species are typical calcifuges that do not grow well in calcareous soils characterized by alkaline pH and high concentrations of Ca2+. In this study, we investigated the effects of three pH levels and a Ca2+ treatment on the in vitro germination and seedling growth of three Rhododendron species: R. chihsinianum, R. fortunei, and R. vernicosum. Alkaline pH had no significant effect on germination parameters (gMAX, mean germination time and germination uniformity) but significantly increased abnormal leaf development (AL) and mortality in all three species. Adding extra Ca2+ reduced the mean germination time for R. vernicosum. The negative influence of alkaline pH on seedlings was already visible on the second day of treatment as measured by chlorophyll fluorescence imaging parameters (Fv/Fm and ΦPSII) on R. fortunei. Extra Ca2+ alleviated the negative effect of alkaline pH and increased Fv/Fm 41 days after seed germination in R. fortunei and R. chihsinianum and reduced mortality for all three species. In conclusion, alkaline pH mainly influenced seedling development and growth but not the germination process itself. Chlorophyll fluorescence imaging can be an efficient way to perform high-throughput in vitro screening of Rhododendron seedlings for alkalinity tolerance.

NMDA Receptor-mediated Ca2+ Influx in the Absence of Mg2+ Block Disrupts Rest: Activity Rhythms in Drosophila.

The correlated activation of pre- and postsynaptic neurons is essential for the NMDA receptor-mediated Ca2+ influx by removing Mg2+ from block site and NMDA receptors have been implicated in phase resetting of circadian clocks. So we assessed rest:activity rhythms in Mg2+ block defective animals. Using Drosophila locomotor monitoring system, we checked circadian rest:activity rhythms of different mutants under constant darkness (DD) and light:dark (LD) conditions. We recorded NMDA receptor-mediated currents or Ca2+ increase in neurons using patch-clamp and Ca2+ imaging techniques. We found that Mg2+ block defective mutant flies were completely arrhythmic under DD. To further understand the role of Mg2+ block in daily circadian rest:activity, we observed the mutant files under LD cycles, and we found severely reduced morning anticipation and advanced evening peak compared to control flies. We also used tissue-specific expression of Mg2+ block defective NMDA receptors and demonstrated pigment-dispersing factor receptor (PDFR)-expressing circadian neurons were implicated in mediating the circadian rest:activity deficits. Endogenous functional NMDA receptors are expressed in most Drosophila neurons, including in a subgroup of dorsal neurons (DN1s). Subsequently, we determined that the uncorrelated extra Ca2+ influx may act in part through Ca2+/Calmodulin (CaM)-stimulated PDE1c pathway leading to morning behavior phenotypes. These results demonstrate that Mg2+ block of NMDA receptors at resting potential is essential for the daily circadian rest:activity rhythms and we propose that Mg2+ block functions to suppress CaM-stimulated PDE1c activation at resting potential, thus regulating Ca2+ and cyclic AMP oscillations in circadian and sleep circuits.

A comparative transcriptome and proteomics analysis reveals the positive effect of supplementary Ca2 + on soybean sprout yield and nutritional qualities

Effects of Ca2+ on yield and nutritional qualities of soybean sprout were investigated. Ca-treated sprouts had higher yield than water-treated ones. Metabolism of selected storage materials and bioactive substances in soybean sprouts was strengthened by Ca2+. The phytic acid and saponin content of Ca-treated soybean sprouts were lower than those of control. Supplemental Ca2+ increased content of gamma-aminobutyric acid, isoflavones, phenolics, and vitamins, respectively. These findings indicate that supplemental Ca2+ can increase soybean sprout yield and improve its nutritional qualities. The comparative transcriptome and proteomics between water-treated and Ca-treated soybean sprouts were studied. As consequence 1912 genes and 460 proteins were up- or down-regulated after 4days of Ca2+ treatment. The functional classification of these differentially expressed genes and proteins indicated their connection with primary/secondary metabolic pathways, ion transport, signal transduction, and transcriptional regulation. The results obtained here will enable to understand how changes in yield and nutritional quality are regulated by extra Ca2+ in soybean sprouts. Biological significanceIn this study, a total of 1912 genes and 460 proteins involved in the growth, storage material decomposition, and bioactive substance synthesis in soybean sprouts after treated with Ca2+ were identified. This is the first report of a comprehensive transcriptomic and proteomic analysis of soybean sprout in response to supplemental Ca2+.

Cytoskeleton and Chromosome Damage Leading to Abnormal Mitosis Were Involved in Multinucleated Cells Induced by Silicon Nanoparticles

Recently, amorphous silicon nanoparticles (SNPs) are widely used in a variety of fields, especially in biological and medical science. Thus, the adverse effect of these nanoparticles should be carefully investigated. The multinucleation effect of SNPs was firstly reported in our previous studies, while the relative mechanism is still unclear. Therefore, the purpose of this study is to investigate the mechanisms with regard to the formation of multinucleated cells. Two sizes of amorphous SNPs (Nano‐Si64 and Nano‐Si46) are carefully characterized. Cytotoxicity and rate of multinucleated cells are firstly determined after human hepatic L‐02 cells are treated with two SNPs for 24 h. Then cell fusion and abnormal mitosis, two ways could form multinucleated cells, are investigated, respectively. Results indicated that SNPs produce a dose‐dependent and size‐related multinucleation effect in L‐02 cells. Abnormal mitosis instead of cell fusion is the main reason for the formation of multinucleated cells caused by SNPs. Both two SNPs could affect the quantity and distribution of cytoskeleton through extra ROS and Ca2+ leading to abnormal mitosis and cytokinesis. Additionally, chromosome damage resulting in corresponding G2/M cell cycle arrest should be another aspect, which finally leads to the formation of multinucleated cells in L‐02 cell line.

Adipokinetic hormone exerts its anti-oxidative effects using a conserved signal-transduction mechanism involving both PKC and cAMP by mobilizing extra- and intracellular Ca2 + stores

The involvement of members of the adipokinetic hormone (AKH) family in regulation of response to oxidative stress (OS) has been reported recently. However, despite these neuropeptides being the best studied family of insect hormones, their precise signaling pathways in their OS responsive role remain to be elucidated. In this study, we have used an in vitro assay to determine the importance of extra and intra-cellular Ca2+ stores as well as the involvement of protein kinase C (PKC) and cyclic adenosine 3′,5′-monophosphate (cAMP) pathways by which AKH exerts its anti-oxidative effects. Lipid peroxidation product (4-HNE) was significantly enhanced and membrane fluidity reduced in microsomal fractions of isolated brains (CNS) of Pyrrhocoris apterus when treated with hydrogen peroxide (H2O2), whereas these biomarkers of OS were reduced to control levels when H2O2 was co-treated with Pyrap-AKH. The effects of mitigation of OS in isolated CNS by AKH were negated when these treatments were conducted in the presence of Ca2+ channel inhibitors (CdCl2 and thapsigargin). Presence of either bisindolylmaliemide or chelyrythrine chloride (inhibitors of PKC) in the incubating medium also compromised the anti-oxidative function of AKH. However, supplementing the medium with either phorbol myristate acetate (PMA, an activator of PKC) or forskolin (an activator of cAMP) restored the protective effects of exogenous AKH treatment by reducing 4-HNE levels and increasing membrane fluidity to control levels. Taken together, our results strongly implicate the importance of both PKC and cAMP pathways in AKHs' anti-oxidative action by mobilizing both extra and intra-cellular stores of Ca2+.

PH-Dependent improvement of pyrophosphate removal on amorphous ferric hydroxide by incorporating Ca2+

The interaction of calcium and pyrophosphate (PP) on the surface of amorphous ferric hydroxide (AFH) was investigated at pH 3.5–11.0. Zeta potential analysis of solid samples after PP removal indicates that the removal of PP by Ca2+ on the AFH was relative to the interaction of Ca2+ with different species of PP. The reduction of PP removal amount with the pH increasing was observed in all cases at the initial PP concentration ranging from 5 to 50mg/L. The lowest PP removal amount at the initial PP concentration of 25mg/L was 5.0, 11.1 and 21.7mg/g with the initial [Ca2+] of 0, 25 and 50mg/L, respectively. Moreover, this improvement of the PP removal in the presence of Ca2+ was pH-dependent. At pH<7.5, the formation of surface complex FeOCa+ was supposed, which is expected to absorb and remove PP. This resulted in a Ca/P ratio ranging from 2.5 to 25.6 in the solid sample. In contrast, the precipitation of Ca2P2O7 was responsible for the increasing of PP removal amount in basic solution, which resulted in the reduction of Ca/P in the solid sample to 2.0 at pH⩾7.5. The Ca/P molar ratio higher than 1.0 in Ca2P2O7 indicates extra Ca2+ adsorbed in the sample during the precipitation. Therefore, our results suggest an improved treatment of PP wastewater through the adsorption on AFH with a small Ca addition at low pH.

Mitochondrial handling of excess Ca2+ is substrate‐dependent with implications on ROS generation

Mitochondrial complex III is a major source of ROS during ischemia and reperfusion (I/R). Several mechanisms modulate ROS production such as mitochondrial Ca2+ uptake. Here we sought out the mechanisms of ROS generation from complex III due to excess extra matrix Ca2+. Guinea pig heart mitochondria were suspended in respiration media containing different concentrations of Ca2+, and either amplex red/horseradish peroxidase, rhodamine 123, or calcium green to measure H2O2 generation, membrane potential (ΔΨ), or [Ca2+], respectively. Either pyruvate or succinate (10 mM) was added next, followed by antimycin A (AA; 5 μM) to inhibit complex III. Mitochondria established a fully polarized ΔΨ under excess extra matrix Ca2+ with pyruvate, whereas they remained partially depolarized with succinate as the substrate. AA caused Δ Ψ depolarization for both substrates, and matrix Ca2+ extrusion which was more with succinate than pyruvate and coincided with a larger H2O2 generation that was significantly decreased with cyclosporine A, an inhibitor of permeability transition pore (PTP) opening. Succinate accumulates during I/R and becomes the major substrate utilized by mitochondria. Mitochondrial inability to maintain a polarized Δ Ψ under excess extra matrix Ca2+ when succinate, but not pyruvate, is the substrate may lead to PTP opening and enhancing ROS formation from complex III during I/R.

A Two Phase Decrease in Force and Intracellular Ca2+ during the Relaxation of Mouse Lumbricle Muscle

Intracellular Ca2+ and force were measured simultaneously in intact mouse lumbricle muscle (toe muscle). All motor units were stimulated simultaneously for a fixed period of time at varying frequencies. During the fixed period of stimulation average force and intracellular Ca2+ were computed. Results showed that there was a sigmoidal increase in both average force and intracellular Ca2+ as a function of log10 of the increasing frequency of the muscle stimulation. At the end of the stimulation period the isometric contracting muscle relaxed in two phases. Immediately following the end of stimulation there was an initial slow phase of relaxation associated with a very rapid fall in intracellular Ca2+ (Phase 1). Following the initial phase of slow relaxation (Phase 1) there was a very rapid relaxation associated with a much slower rate of decrease in intracellular Ca2+ (Phase 2). Analysis of Ca2+ records during the Phase 2 relaxation showed that the rapid decrease in force was associated with extra Ca2+ being released into the myoplasm. The initial slow phase of relaxation (Phase 1) was found to be dependent upon the initial isometric force (active state) at the time of cessation of the muscle stimulus. The relationship between phase 1 relaxation rate and active state was linear showing that a 50% decrease in maximum active state would result in approximately a 200% increase in phase 1 relaxation rate. The results suggest that the initial slow rate of cross-bridge dissociation (Phase 1) is dependent upon the activation state of the myofilaments and that the rapid relaxation during Phase 2 is associated with Ca2+ being released from the myofilaments. Supported by AHA 10GRNT4450042

Cross-Talk among Intracellular Signaling Pathways Mediates the Diphenyl Ditelluride Actions on the Hippocampal Cytoskeleton of Young Rats

In the present report, we showed that diphenyl ditelluride (PhTe)(2) induced in vitro hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) subunits in hippocampus of 21 day-old rats. Hyperphosphorylation was dependent on L-voltage dependent Ca(2+) channels (L-VDCC), N-methyl-d-aspartate (NMDA) and metabotropic glutamate receptors, as demonstrated by the specific inhibitors verapamil, DL-AP5 and MCPG, respectively. Also, dantrolene, a ryanodine channel blocker, EGTA and Bapta-AM, extra and intracellular Ca(2+) chelators respectively, totally prevented this effect. Activation of metabotropic glutamate receptors by (PhTe)(2) upregulates phospholipase C (PLC), producing inositol 1, 4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). Therefore, high Ca(2+) levels and DAG directly activate Ca(2+)/calmodulin-dependent protein kinase (PKCaMII) and protein kinase C (PCK), resulting in the hyperphosphorylation of Ser-57 in the carboxyl-terminal tail domain of the low molecular weight NF subunit (NF-L). Also, the activation of Erk1/2, and p38MAPK resulted in hyperphosphorylation of KSP repeats of the medium molecular weight NF subunit (NF-M). It is noteworthy that PKCaMII and PKC inhibitors prevented (PhTe)(2)-induced Erk1/2MAPK and p38MAPK activation as well as hyperphosphorylation of KSP repeats on NF-M, suggesting that PKCaMII and PKC could be upstream of this activation. Taken together, our results highlight the role of Ca(2+) as a mediator of the (PhTe)(2)-elicited signaling targeting specific phosphorylation sites on IF proteins of neural cells of rat hippocampus. Interestingly, this action shows a significant cross-talk among signaling pathways elicited by (PhTe)(2), connecting glutamate metabotropic cascade with activation of Ca(2+) channels. The extensively phosphorylated amino- and carboxyl- terminal sites could explain, at least in part, the neural dysfunction associated with (PhTe)(2) exposure.

Effects of Na+, K+, and Ca2+ on the Structures of Anionic Lipid Bilayers and Biological Implication

Ion-membrane interactions are essential to the regulation of cell functions. While numerous molecular dynamics (MD) simulations have been carried out to study the effects of ions on neutral lipid bilayers, few have been conducted on anionic lipid bilayers. Moreover, there is a lack of long-time simulations. Here, submicrosecond MD simulations were performed to investigate the effects of pure cations (K(+), Na(+), and Ca(2+), respectively) on the anionic palmitoyloleoylphosphatidylglycerol (POPG) bilayer first. The results reveal how K(+), Na(+), and Ca(2+) ions influence the structure of anionic lipid bilayers. In general, cations tighten the anionic lipid bilayer and increase the ordering of the lipids. Subsequently, two MD simulations were carried out to elucidate the effects of extra cations added to the bilayers in addition to counterions. It is found that the extra Ca(2+) ions result in stronger effects on the structures of the lipid bilayer, whereas extra Na(+) ions do not. Finally, simulations of ion mixture effects on the structure of the POPG bilayer were conducted, and it is observed that Ca(2+), over K(+) and Na(+), plays a dominant role in affecting the bilayer structures. These results may cast new insights on the distinct functions of Ca(2+) in the biological systems. In addition, our simulations indicate that long-time simulations are necessary to address the effects of ions on lipid bilayer structures.

Complexin: Does It Deserve Its Name?

Knockout and other perturbations of complexins have provided important insights and elicited controversies about their role in neurotransmitter release. New work by Yang etal. in this issue of Neuron adds important detail and complexity to existing concepts-particularly on the nature of a Ca(2+)-dependent complexin-synaptotagmin switch for the triggering of exocytosis. But it also provokes thoughts about alternative interpretations, which might result in a simpler model of complexin function.

Magnetic fields promote a pro-survival non-capacitative Ca2+ entry via phospholipase C signaling

The ability of magnetic fields (MFs) to promote/increase Ca2+ influx into cells is widely recognized, but the underlying mechanisms remain obscure. Here we analyze how static MFs of 6mT modulates thapsigargin-induced Ca2+ movements in non-excitable U937 monocytes, and how this relates to the anti-apoptotic effect of MFs. Magnetic fields do not affect thapsigargin-induced Ca2+ mobilization from endoplasmic reticulum, but significantly increase the resulting Ca2+ influx; this increase requires intracellular signal transduction actors including G protein, phospholipase C, diacylglycerol lipase and nitric oxide synthase, and behaves as a non-capacitative Ca2+ entry (NCCE), a type of influx with an inherent signaling function, rather than a capacitative Ca2+ entry (CCE). All treatments abrogating the extra Ca2+ influx also abrogate the anti-apoptotic effect of MFs, demonstrating that MF-induced NCCE elicits an anti-apoptotic survival pathway.

Changes in intracellular Na+in heart failure following SERCA knockout - more of a solution or more of a problem?

One of the difficulties in understanding and treating heart failure (HF) is that it involves many structural, molecular and physiological defects (Bers, 2006). Diastolic and systolic dysfunction arise from distinct defects that may or may not contribute to disease development in an individual patient. For example, downregulation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) has been reported in HF and the level of SERCA expression was related to both type and severity of HF, so that variability in expression would be expected among a mixed patient population (Hasenfuss et al. 1999). Reduced SERCA means that less Ca2+ is taken up into the SR at a slower rate so less Ca2+ is available for contraction (systolic dysfunction) and relaxation slows (diastolic dysfunction). Intracellular Na+ concentration ([Na+]i) is increased in both experimental and clinical HF, possibly as a result of an increase in the late component of rapid Na+ current, reduced Na+, K+-ATPase (NKA) expression/activity and increased Na+–Ca2+ exchange (NCX) expression/activity. This increase in diastolic [Na+]i might maintain contractility by offsetting the negative inotropic consequences of reduced SERCA function. However, there is a cost to this mechanism of inotropic compensation in the form of increased [Ca2+]i which contributes to slower relaxation and possibly triggered arrhythmias. In a recent issue of The Journal of Physiology, Louch et al. (2010) investigated the mechanisms by which excitation–contraction coupling compensates for a conditional knock-out of SERCA in a mouse model of HF. As SERCA levels decline, the myocyte must compensate for the loss of its normal means of removing Ca2+ from the cytoplasm. The initial response was an increase in NCX expression along with increases in the L-type Ca2+ channel protein and current, providing the means by which trans-sarcolemmal Ca2+ entry is increased (activating contraction and maintaining cardiac output) and by which this extra Ca2+ is removed from the cell in the face of a non-functional SR. However, things become more complicated in the prolonged absence of SERCA as HF develops and additional compensatory changes occur as both systolic and diastolic dysfunction develop. These involve reduced expression of both α1 and α2 isoforms of NKA, leading to reduced Na+ removal and a resultant increase in resting [Na+]i. The authors demonstrate quite convincingly that this Na+ accumulation in itself contributes to slower relaxation (via reduced Ca2+ efflux through the NCX) and therefore to diastolic dysfunction. This is an interesting and potentially important observation about how specific molecular changes affect the integrative physiology of cardiac function and contribute to the development of a specific disease state (HF). Curiously, the authors found no associated change in diastolic [Ca2+] although no data were presented addressing this point nor was there any discussion of how this could be. Questions remain about why these particular forms of compensation occur and, of course, whether or not changes in mouse also occur in human HF. It is not clear why expression of NKA overall and/or specific isoforms are reduced in response to SERCA deletion. NKA expression is also decreased in human HF but there are likely to be marked differences in both NKA expression levels and the balance of isoform expression in humans where NKA expression is comprised mostly of α2 and α3 with little α1 expression (Wasserstrom & Aistrup, 2005). Moreover, the fact that late INa is increased in most HF models as well as in humans (Sossalla et al. 2008) means that [Na+]i regulation is likely to play a critical role in HF onset and severity, depending on when changes occur in Na+ current kinetics, NKA and NCX expression and a multitude of other defects in contractile filaments, mitochondrial function, reactive oxygen species generation and so forth. It appears this interesting compensatory mechanism develops in response to a specific molecular defect and is initially able to deal effectively with altered physiological function but then becomes less a part of the solution and becomes more a part of the problem.

Calcium Can Prevent Toxic Effects of Na+ on Tomato Leaf Photosynthesis but Does Not Restore Growth

The availability of good-quality irrigation water is decreasing worldwide, and salinity is an increasingly important agricultural problem. To determine whether detrimental effects of NaCl on plant growth and leaf physiology can be minimized by additional Ca2+ supply, tomato (Solanum lycopersicum L.) ‘Supersweet 100’ was grown hydroponically. The basic nutrient solution contained 11.1 mm NO3 − and 2.8 mm Ca2+. Three levels of NaCl (14.1, 44.4, and 70.4 mm) were added to the basic solution to determine Na+ effects on leaf physiology and growth. To determine if Ca2+ could alleviate the toxic effects of Na+, treatments with 10 or 20 mm Ca2+ combined with 44.4 or 70 mm NaCl were included as well. To distinguish between osmotic and ion-specific effects, there were three treatments in which all nutrient concentrations were increased (without NaCl) to obtain electrical conductivity (EC) levels similar to those of the NaCl treatments. Nutrient solutions with 70.4 mm NaCl reduced leaf photosynthesis, chlorophyll content, gas-phase conductance, carboxylation efficiency, and dark-adapted quantum yield. Inclusion of 20 mm Ca2+ prevented these effects of NaCl. NaCl also decreased leaf length and elongation rate. This could not be prevented by adding extra Ca2+ to the solution; reductions in leaf elongation were due to osmotic effects rather than to Na+ specifically. Likewise, plant dry weight was negatively correlated with solution EC, suggesting an osmotic effect. Leaf area development apparently was more important for dry matter accumulation than leaf photosynthesis. Adding 20 mm Ca2+ to the 70 mm NaCl solution reduced the Na+ concentration in the leaf from 79 to 24 mg·g−1.

NaCl Stress in Hydroponic Tomatoes Can Be Alleviated by Calcium

The availability of good quality irrigation water is decreasing worldwide, and salinity is an increasingly important agricultural problem. To determine whether detrimental effects of NaCl can be minimized by additional Ca2+, tomato (Lycopersicon esculentum Mill.) `Super-sweet 100' was grown hydroponically. The basic nutrient solution contained 11.9 mM NO -3 and 3.2 mM Ca 2+. We added 14.1, 44.4 and 70.4 mM of NaCl to this solution to determine the effect of NaCl and there were treatments with 70 mM NaCl and 10 or 20 mM Ca2+ to look at Ca2+ effects. We also included three treatments in which all nutrient concentrations were increased (without NaCl) to distinguish between osmotic and ion-specific effects. 70.4 mM NaCl reduced leaf photosynthesis, chlorophyll content, gas phase conductance for CO2 diffusion, carboxylation efficiency, and dark-adapted quantum yield of photosystem II. The inclusion of 20 mM Ca2+ prevented these effects of NaCl. NaCl also decreased leaf size and elongation rate, but this could not be prevented by adding extra Ca2+ to the nutrient solution; these were caused by osmotic effects, rather than Na+ specifically. Likewise, plant dry mass was negatively correlated with solution EC, suggesting an osmotic effect. Our results indicate that leaf area development, which was inhibited by high EC, is more important for dry matter accumulation than leaf photosynthesis, which was inhibited by high Na+. Adding 20 mM Ca2+ to the 70 mM NaCl solution reduced the Na+ concentration in the leaf from 79 to 24 mg·g-1, which may explain why Ca2+ alleviates Na+ toxicity.

Human Prothrombin Fragment 1 Conformational Changes Following Binding to Phospholipid Vesicles: A Computational Study.

Our long term goal is to create a model of the prothrombinase complex. We have recently proposed a complete model in solution of human coagulation factor Va (pdb entry 1y61). In the present study we create the framework needed to accomplish our final goal. We first created a lipid bilayer that mimics the cell membrane surface. The lipid bilayer mimetic was set up as a random mixture of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) using a 4:1 ratio. Human prothrombin fragment one is a highly homologous region among species, except zebrafish and hagfish ( Jagadeeswaran et al., Blood Cells Mol. Dis., 2000). In the absence of a crystal structure of the entire human prothrombin molecule, we created a homology model of human prothrombin fragment 1 (amino acid residues 1–155). In the homology modeling procedure we used three X-ray structures available for the bovine prothrombin fragment 1 molecule (Soriano-Garcia et al., Biochemistry, 1992, Seshadri et al., J. Mol. Biol., 1991, and Huang et al., Nat. Struct. Biol., 2003). One extra Ca2+ was added as proposed in an earlier human prothrombin fragment 1 homology model (Leping et al., Protein Sci., 1995). The modeling was performed using the Modeller software package. Prothrombin fragment 1 was then inserted into the lipid bilayer so the region composed of amino acid residues 1–10 were positioned in the hydrophobic core, whereas the γ-carboxyglutamic acid rich region was on the surface of the lipid bilayer as proposed (Falls et al., J. Biol. Chem., 2001). Molecular dynamics simulations on the 10 ns time scale of the prothrombin fragment 1 bound to the POPC/POPS lipid bilayer showed a conformational change in prothrombin fragment 1. This conformational change can be described as a rearrangement of three regions: region A (composed of amino acid residues 1–30), region B (composed of amino acid residues 31–69), and region C (composed of amino acid residues 70–155). Root mean square deviation (RMSD) of region A compared to the starting model was found to be 1.61 Å whereas for region C was found to be 1.91 Å. The RMSD of region B was found to be 1.99 Å. Because region B contains two α- helix motifs the deviation is due to the amino acids found at both ends (i.e., the ones that connect region A and region C with region B). During the simulations the initial Ca2+ ions positions were found to be preserved in the vicinity of the γ-carboxyglutamic amino acid residues. The secondary structure elements were preserved during simulations. Following a 10 ns simulation three γ-carboxyglutamic residues (7, 14 and 25) of prothrombin fragment 1 were found to form polar contacts with POPS molecules. Due to the conformational change, Asp55 was also found to interact with one POPS molecule. This amino acid residue was previously shown to be located near the active site of meizothrombin. Overall, our studies present for the first time a POPC/POPS lipid bilayer model, and show that in the presence of Ca2+ ions a conformational change occurs in prothrombin fragment 1 following its interaction with the lipid bilayer.

Phospholemman

See related article, pages 558–565 The control of intracellular Na+ levels has long been known to be a crucial part of the regulation of cardiac contractility and the treatment of heart failure. Cardiac glycosides have been used to improve the symptoms of heart failure since William Withering published trials of a foxglove extract obtained from a gypsy woman in the late 1700’s. The active ingredients, digitalis and digoxin, were found to target sodium pump activity in the 1950’s, and the sodium pump was identified as Na,K-ATPase by Nobel Laureate Jens Skou in the 1970’s. Recent studies have established that digitalis does not improve survival in heart failure, but American College of Cardiology/American Heart Association guidelines recommend its use in combination with ACE inhibitors and β-adrenergic blockade in patients with symptomatic left ventricular systolic dysfunction. A modest level of inhibition of Na,K-ATPase by digitalis slightly raises cardiac intracellular Na+ concentrations, which in turn decreases the driving force for Ca2+ extrusion via NCX1, the Na+:Ca2+ exchanger. In the therapeutic range, the extra cytoplasmic Ca2+ will be loaded into the sarcoplasmic reticulum, resulting in normal diastolic Ca2+ concentrations but improved systolic Ca2+ release, and thus improved contractility. β-adrenergic stimulation, on the other hand, improves the loading of the sarcoplasmic reticulum (SR) by acting on the Ca2+-ATPase, SERCA. β-adrenergic stimulation improves SR loading by phosphorylating a small regulatory membrane protein, phospholamban, which otherwise acts to reduce the …

Insulin and Inositol 1,4,5-Trisphosphate Trigger Abnormal Cytosolic Ca2+ Transients and Reveal Mitochondrial Ca2+ Handling Defects in Cardiomyocytes of ob/ob Mice

Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by approximately 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.

Similar Ca2+-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca2+-ATPase SPCA1

Mutations in the ubiquitously expressed secretory-pathway Ca2+-ATPase (SPCA1) Ca2+ pump result in Hailey–Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca2+ signaling in human keratinocytes by measuring the free Ca2+ concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca2+-ATPase (SERCA) and SPCA1 can mediate Ca2+ uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca2+ pump to load the Golgi complex with Ca2+ whereas the SERCA Ca2+ pump was mainly used in control COS-1 cells. Cytosolic Ca2+ signals in keratinocytes induced by extracellular ATP or capacitative Ca2+ entry were characterized by an unusually long latency reflecting extra Ca2+ buffering by an SPCA1-containing Ca2+ store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca2+ elicited spontaneous cytosolic Ca2+ transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca2+ signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca2+ in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey–Hailey patients.