Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca²⁺-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca²⁺. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca²⁺-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca²⁺-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.     

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms,the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.     

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca²⁺ concentration. However, the mutations differently affected the Ca²⁺ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca²⁺-dependent interaction of myosin heads with F-actin.     

Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca²⁺. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca²⁺ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca²⁺, R342W and Q426R were stabilized at higher Ca²⁺ concentrations than the wild-type form, and R402P was completely insensitive to Ca²⁺. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).     

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca²⁺ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.     

Correlation between crystal structure and microwave dielectric properties of two garnet-type ceramics in rare-earth-free gallates

Two garnet-type rare-earth-free ceramics, Ca₃M₂SiGa₂O₁₂ (M = Sn and Zr), were prepared through a solid-state reaction method. The relationship between crystal structure and microwave dielectric properties was investigated. The larger deviation of ε_r from ε_theo in Ca₃Zr₂SiGa₂O₁₂ could be ascribed to the rattling Zr⁴⁺. The increase in packing fraction and the decrease in FWHM enhance the Q × f value by substituting Zr⁴⁺ with Sn⁴⁺. The smaller oxygen bond valence in Ca₃Zr₂SiGa₂O₁₂ indicates a smaller τ_f value. Good microwave dielectric properties are obtained with ε_r = 9.14 ± 0.02, Q × f = 106,800 ± 1700 GHz and τ_f = -45.8 ± 1.8 ppm/°C for Ca₃Sn₂SiGa₂O₁₂ and ε_r = 11.98 ± 0.03, Q × f = 84,200 ± 1500 GHz, and τ_f = -32.8 ± 1.4 ppm/°C for Ca₃Zr₂SiGa₂O₁₂. Furthermore, near-zero τ_f values of +5.7 ± 1.9 ppm/°C and +4.5 ± 1.6 ppm/°C appear in 0.95Ca₃Sn₂SiGa₂O₁₂-0.05CaTiO₃ and 0.96Ca₃Zr₂SiGa₂O₁₂-0.04CaTiO₃, respectively.     

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca²⁺, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca²⁺ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg²⁺ alone and Mg²⁺ and Ca²⁺. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC₅₀ value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca²⁺. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca²⁺ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca²⁺ levels, which would ultimately lead to toxic accumulation of cGMP and Ca²⁺ in the photoreceptor outer segment, thus triggering cell death.     

Structural dependence of microwave dielectric performance of wolframite structured Mg1-xCaxZrNb2O8 ceramics: Crystal structure,microstructure evolution,Raman analysis and chemical bond theory

Wolframite-structured Mg_1-xCa_xZrNb₂O₈ (0?0.1) ceramics were synthesized through a solid-state procedure. Crystal refinement indicates a pure Mg_1-xCa_xZrNb₂O₈ ceramic with a wolframite structure. Ca²⁺ substitution led to the redshift of the Ag mode at approximately 896 cm^?1. Additionally, Ca²⁺ substitution could promote grain growth and contribute to microstructure evolution from a polyhedral shape to a rod shape. According to chemical bond theory, an appropriate Ca²⁺ concentration can increase NbO bond iconicity and NbO bond lattice energy, which contributed to the great improvement in the dielectric constant and Q × f value. Additionally, the τ_f value was affected by the bond valence and thermal expansion coefficient of the MgO bond. The Mg_1-xCa_xZrNb₂O₈ (x = 0.04) ceramics exhibited great improvement in the Q × f value: ε_r = 25.21, Q × f = 116,000 GHz (@7.17 GHz) and τ_f = ?24.4 ppm/°C, which provides enormous potential for future millimeter-wave applications.     

Abnormal Ca2+ Signals in Reactive Astrocytes as a Common Cause of Brain Diseases

In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca²⁺ signals in pathological brains and found that reactive astrocytes share abnormal Ca²⁺ signals, even in different types of diseases. In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca²⁺ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca²⁺ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca²⁺ signals. Because the abnormal astrocytic Ca²⁺ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP₃RII), we reanalyzed these pathological events using IP₃RII-deficient mice and found that all abnormal Ca²⁺ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca²⁺ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca²⁺ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.     

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca²⁺ content and lower frequency of spontaneous Ca²⁺ signals in SGCE MSNs. Blocking of voltage-gated Ca²⁺ channels by verapamil was less efficient in suppressing KCl-induced Ca²⁺ peaks of SGCE MSNs. Ca²⁺ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca²⁺ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.     

Intracellular Ca2+ Signaling in Protozoan Parasites: An Overview with a Focus on Mitochondria

Ca²⁺ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca²⁺ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca²⁺ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca²⁺ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca²⁺-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca²⁺ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca²⁺ uptake by pathogenic protists.     

Ultrasensitive Imaging of Ca2+ Dynamics in Pancreatic Acinar Cells of Yellow Cameleon-Nano Transgenic Mice

Yellow Cameleons are genetically encoded Ca²⁺ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca²⁺-sensor probe. To achieve ultrasensitive Ca²⁺ imaging for low resting Ca²⁺ or small Ca²⁺ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca²⁺ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca²⁺ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca²⁺ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca²⁺ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca²⁺ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from R_max and R_min values under in vivo condition. The mice will be useful for ultrasensitive Ca²⁺ imaging in vivo.     

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.     

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

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

Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells

CRAC, which plays important role in Ca²⁺-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca²⁺-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca²⁺-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca²⁺-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.     

Genetic Deletion of NOD1 Prevents Cardiac Ca2+ Mishandling Induced by Experimental Chronic Kidney Disease

Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of Nod1 or Rip2 in mice could prevent cardiac Ca²⁺ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca²⁺ dynamics in cardiomyocytes from Wild-type (Wt), Nod1^−/− and Rip2^−/− sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt-Nx cells showed an impairment in the properties and kinetics of the intracellular Ca²⁺ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca²⁺ load, together with an increase in diastolic Ca²⁺ leak. Cardiomyocytes from Nod1^−/−-Nx and Rip2^−/−-Nx mice showed a significant amelioration in Ca²⁺ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca²⁺ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.     

Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms

Mitochondrial [Ca²⁺] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca²⁺ overload. This regulation depends on Ca²⁺ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca²⁺ uniporter (MCU). Here, we have studied the mitochondrial Ca²⁺ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca²⁺ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca²⁺] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca²⁺ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca²⁺ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca²⁺ pathway.     

Influence of calcium doping concentration on the performance of Ce,Ca:LuAG scintillation ceramics

Ce,Ca:LuAG scintillation ceramics with different Ca²⁺ co-doping concentrations were prepared by the solid-state reaction method. The concentration of Ce³⁺ was fixed at 0.3 at% and the concentration of Ca²⁺ ranged from 0 to 1.2 at%. We systematically studied how the Ca²⁺ concentration affects the optical quality of Ce,Ca:LuAG ceramics by influencing the microstructure in the vacuum sintering and HIP post-treatment. Good optical transmittance could be obtained with Ca²⁺ concentrations between 0.05 and 0.8 at%, which reached 76.0–81.9 % at 520 nm. The PL and scintillation decay times decrease with increasing Ca²⁺ concentration up to 0.6 at% with no clear trend above this value. The light yield (LY) values at different shaping times decrease with increasing Ca²⁺ concentration but the fast scintillation component (LY_0.5 μs/ LY_3.0 μs) increases significantly from 79 % to 97 %. The co-doping of Ca²⁺ also reduces the afterglow level by more than one order of magnitude.     

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca²⁺ influx channels involved in eATP-induced [Ca²⁺]_cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca²⁺ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca²⁺]_cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.     

Effects of co-ion initial concentration ratio on removal of Pb<Superscript>2+</Superscript> from aqueous solution by modified sugarcane bagasse

A modified sugarcane bagasse (SCB) fixed bed column was used to remove Pb²⁺ from aqueous solution. To determine the optimal condition for Pb²⁺ separation, Ca²⁺ was chosen as the model interfering ion, and effects of Ca²⁺ and Pb²⁺ initial concentration ratio (C ₀ ^Ca : C ₀ ^Pb ) on the adsorption of Pb²⁺ were investigated. Results showed that adsorption amount ratio of Ca²⁺ and Pb²⁺ (q _e ^Ca : q _e ^Pb ) had a good linear relationship with C ₀ ^Ca : C ₀ ^Pb . Mass ratio of Pb²⁺ absorbed on the modified SCB was higher than 95% at C ₀ ^Ca : C ₀ ^Pb <1.95, illustrating that Pb²⁺ could be selectively removed from aqueous solution. To verify that, simulated waste water containing co-ions of K⁺, Na⁺, Cd²⁺ and Ca²⁺ was treated, and results showed that the equilibrium amount of Pb²⁺, K⁺, Na⁺, Cd²⁺ and Ca²⁺ adsorbed was 134.14, 0.083, 0.058, 1.28, and 1.28mg g^?1, respectively, demonstrating that the modified SCB could be used to remove Pb²⁺ from aqueous solution in the investigated range.